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What is Metabolism? Catabolism vs Anabolism, Definition,...
Continue ReadingWhat is Metabolism?
Metabolism is the sum of all metabolic pathways within a cell, tissue, or organism.The sum of all metabolic pathways within a cell, tissue, or organism.
Type of Metabolic Pathway
There are three (3) main types of pathways that reactions can occur through in the body:
1) Linear
2) Branch point
3) Cyclic
Types of Metabolism
Of the 3 types of pathways they fall into two (2) categories of metabolic pathways
I. Catabolism
Oxidizing pathways that generate energy.
Large molecules are broken and the energy stored as chemical energy within the bonds are converted into ATP or transferred into other small molecule shuttles such as NAD, FAD, and NADPH.
II. Anabolism
Biosynthetic pathways where small molecules come together to form large macromolecules.
The ATP that was generated during catabolic stages is then used generate the various molecules that are needed for the survival of cell.
"Catabolic and Anabolic pathways are linked with each other so that energy or molecules gained from catabolic reactions can be used for anabolic reactions"
Catabolic and Anabolic pathways are not the reverse of each other.
This would lead to futile cycle where in the end only energy is spent.
To avoid these futile cycle each metabolic pathways contains a committed step.
A committed step makes the pathway unidirectional.
The anabolic and catabolic pathways are usually quite different, which allows for them to be regulated separately.
Catabolic and anabolic pathways are regulated such that they usually take place in different physical environments.
Energy Source for organisms:
Light (energy from photons) => phototroph
Molecules (inorganic or organic) => chemotroph
Carbon sources for organisms:
Inorganic (CO2 or Bicarbonate) => autotroph
Organic (carbohydrates, lipids, etc.) => heterotroph
Basic Steps of Metabolism
1) Macromolecules are broken down into their constituent parts
2) Constituent parts are oxidized to acetyl CoA, pyruvate or other metabolites forming some ATP and reduced coenzymes (NADH and FADH2) in a process that doesn’t directly utilize oxygen
3) If oxygen is available and the cell is capable of using oxygen, these metabolites go into the citric acid cycle and oxidative phosphorylation to form large amounts of energy.
"Oxygen is the final electron acceptor of the electron transport chain because O2 has a high electron affinity for electrons"
The second and third stages, the energy acquiring stages, are called respiration.
If oxygen is used, the respiration is aerobic. If oxygen isn’t used, the respiration is anaerobic.
Aerobic organisms: Glycolysis is the first step and molecule generates most of the energy from the oxidation of organic oxygen to molecular oxygen.
Anaerobic organism: They only do glycolysis and fermentation
Obligatory anaerobes: can only live in enviroments without oxygen and perform only anaerobic respiration
Facultative anaerobes can use both aerobic and anaerobic respiration to survive
Substrate-level phosphorylation is a type of chemical reaction that results in the formation of adenosine triphosphate (ATP) by the direct transfer of a phosphate group to adenosine diphosphate (ADP) from a reactive intermediate.
In cells, it occurs in the cytoplasm (in glycolysis) under both aerobic and anaerobic conditions. Unlike oxidative phosphorylation, here the oxidation & phosphorylation is not coupled.
Metabolism Citations
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Enzyme Regulation: Definition, Types, and Mechanism
Continue ReadingWhat is Enzyme Regulation?
Enzyme regulation is very important. You want to control how fast an enzyme is working based on the ability of substrate so not to be wasteful.
On a fundamental level enzyme activity is controlled by regulating the amount of copies of a particular enzyme (transcriptional regulation).
Types of Enzyme Regulation
I. Irreversible Inhibition
Agents which bind covalently to enzymes and disrupt their function are irreversible inhibitors.
A few irreversible inhibitors bind noncovalently.
Irreversible inhibitors tend to be highly toxic
Penicillin is an irreversible inhibitor that binds to a bacterial enzyme that assists in the manufacturing of peptidoglycan cell walls
II. Competitive Inhibition
A molecule similar in structure to the substrate reversibly binds to the active site and inhibits the enzyme’s activity.
Competitive inhibitors raise the apparent Km but do not change the Vmax
III. Noncompetitive Inhibition (Allosteric Modulation)
Allosteric regulators are molecules that alter enzyme kinetics by noncovalently binding to the enzymes at locations far away from the active site, allosteric site.
The allosteric regulator thus alters the 3- D structure of the enzyme.
Allosteric effectors can activate or inhibit enzyme activity.
a. Homotropic activation
Substrate serves as an activator, and You achieved the typical S or sigmodical curve found in describing behavior of enzymes such as hemoglobin.
Oxygen binds to the most difficult site first.
This alters the conformation of the protein so the next subunit binds oxygen more easily.
This process is then repeated for the other two subunits.
This change in conformation caused by the progressive binding of a ligand is known as cooperativity and leads to sigmoid kinetics.
The binding of one ligand (substrate or modulator) changes the conformation of the protein.
This can increase or decrease the affinity for further ligands – Both positive and negative cooperativity exist
Fetal hemoglobin must have a higher affinity for O2 than adult hemoglobin because it steals the O2 from the adult
b. Heterotropic activation/inhibition
A different molecule other than the substrate serves as the allosteric activator/inhibitor.
Can either activate or repress enzyme activity.
Noncompetitive inhibitors do not resemble the substrate, so commonly act on more than one enzyme.
Unlike competitive inhibitors, they can’t be overcome by increasing substrate concentration the substrate.
IV. Feedback Inhibition
When the supply of final product is sufficiently high, the 1st reaction of the pathway is slowed down (negative feedback), this type of regulation is more prevalent than positive feedback.
You can also have it when the supply of final product is low the 1st reaction of the pathway is sped up (positive feedback).
V. Reversible Covalent Modification
Many enzymes are activated or inhibited by the transfer of inorganic phosphate from ATP or modifier to an acceptor.
Most of the time the modifier is removed via hydrolysis. Ex. AMP – modifier
The combination of protein phosphorylation by kinases and dephosphorylation by phosphatases can afford a fine level of control over enzyme activity.
Hexokinase is the enzyme which phosphorylates glucose as soon as it enters the cell.
VI. Zymogen Activation
Proteins typically function extracellularly
Initially synthesized as inactive precursor, zymogen or proenzyme.
For instance pepsinogen (notice the “-ogen” at the end indicating zymogen status) is the zymogen of pepsin and is activated at low pH.
Is irreversible.
Activated by proteolysis of one or a few peptide bonds.
Example: Chymotrypsinogen
Proteolysis is the directed degradation (digestion) of proteins by cellular enzymes called proteases or by intramolecular digestion
VII. Control Proteins
Control proteins are protein subunits that associate with certain enzymes to activate or inhibit their activity.
Calmodulin or G- proteins are typical examples of control proteins Other proteins as well as enzymes undergo types of regulation.
Hemoglobin is a good example that is shown in noncompetitive inhabitance – homotropic activation.
Enzyme Regulation Citations
- Enzymes Regulated via Cystathionine β-Synthase Domains. Biochemistry (Mosc) . 2017 Oct;82(10):1079-1087.
- Regulation of enzyme synthesis in the tryptophan pathway of Acinetobacter calcoaceticus. J Bacteriol . 1976 Jul;127(1):367-79.
- Mode of Action, Properties, Production, and Application of Laccase: A Review. Recent Pat Biotechnol . 2019;13(1):19-32.
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Enzymes: Definition, Functions, Types, and Examples
Continue ReadingWhat are Enzymes?
Proteins that facilitate chemical reactions are called enzymes.
Enzymes are biological catalysts that convert a substrate to a product.
They are typically globular proteins.
Isozymes catalyze the same reaction but with different kinetic parameters and possess different subunit composition.
Properties of Enzymes
They are active at very low concentrations within a cell.
They increase the rate of reaction (by decreasing the Activation Energy [Ea] of a reaction) towards equilibrium (they catalyze both the forward and reverse rxn) but they themselves aren’t altered in the process.
They do not change the nature of the products.
"Enzymes are typically globular proteins"
Although most enzymes are composed of proteins, many enzymes possess nonprotein components called as cofactors.
Cofactors can be metal ions or organic cofactors, coenzymes (ex. Acetyl CoA), which are usually derived from vitamins.
Vitamins are essential, can’t be produced by the body, organic molecules.
Coenzymes are divided into two (2) types:
a. Prosthetic group is an organic cofactor that is covalently bonded into an enzyme.
b. Cosubstrate reversibly binds to a specific enzyme, and transfers some chemical group to another substrate then reverted back to its original form by another enzymatic reaction.
ATP is an example of a cosubstrate type of coenzyme.
"Enzymes that require a cofactor but do not have one bound are called apoenzymes or apoproteins (are completely nonfunctional)"
Recall that lipoproteins contain a lipid core surrounded by phospholipids and apoproteins.
An apoenzyme together with its cofactor(s) is called a holoenzyme (this is the active form).
The equilibrium is not changed by enzymes, just the rate at which the equilibrium is reached.
In the process of catalyzing a reaction, the enzyme is not used up or permanently altered.
Enzymes don’t affect the direction of the reaction because they catalyzed both the forward and the reverse reaction at the same time by reducing the Ea.
Although enzymatic reactions use the same substrate and yields the same product as the uncatalyzed reaction, it produces a different intermediate at the transition state.
Like other chemical reactions, enzyme reactions are reversible, proceeding through the same set of reaction intermediates.
"The position on the enzyme to where the substrate binds, usually with numerous noncovalent bonds, is called the active site"
First the enzyme (E) and the substrate (S) bind to form the ES complex.
After conversion to transition states (ES* and EP*), the final product (P) is formed and then is released by the enzyme.
S + E ←→ ES ←→ ES* ←→ EP* ←→ EP ←→ E + P
An enzymatic reaction begins with the substrate binding at a specific location called the active site.
Think of an active site as a pocket into which the substrate fits.
The enzyme can bind the substrate only if it possesses the proper configuration.
Thus enzymes generally have a very high degree of specificity.
Many enzymes act only on one particular substrate.
The equilibrium between Substrate (S) and Product (P) reflects the difference in the free energy of the ground states, ΔG.
On the other hand, the rate of the reaction depends on the height of the energy barrier between S and P.
The peak of the barrier represents the transition state of the reaction.
The difference between the energies of the ground states and the transition state is called the activation energy, ΔG‡.
To accelerate a reaction, an enzyme must lower the barrier for reaction – decrease ΔG‡
Once it binds the substrate, the enzyme induces a change in the molecular structure of the substrate, and when this occurs it makes the substrate more likely to spontaneously undergo more significant changes.
Enzyme Kinetics
They exhibit saturation kinetics; as the relative concentration of substrate increases, the rate of reaction also increases, but to a lesser and lesser degree until a maximum rate (Vmax) has been achieved.
As we can see from this graph, once we have achieved saturation of the free enzymes the rate of reaction levels off and it cant go any faster.
"Vmax is proportional to enzyme concentration"
Turnover number: The number of substrate molecules one enzyme active site can convert to product in a given unit of time when an enzyme solution is saturated with substrate.
Km is the substrate concentration at which the reaction rate is equal to 1/2Vmax.
Km does not vary when the enzyme concentration is changed.
Is a good indicator of an enzyme’s affinity for its substrate.
Lower the Km the higher the affinity the enzyme has for a substrate.
Factors Affecting Enzyme Kinetics
1) pH:
Example: Pepsin prefers pH of below 2 while trypsin, active in the small intestine prefers pH between 6 and 7
2) Temperature: At first, as the temperature increases, the reaction rate goes up, but at some point, the enzyme denatures, and the rate of reaction drops off.
3) Salt Concentration
4) Hydrostatic Pressure
5) Substrate Concentration
First, environmental conditions can produce changes in weak bonds that can alter the 3-D structure of the enzyme.
For example: Warm temperatures can break bonds that are necessary to form the active site.
Second, environmental conditions can alter the ionization state of critical amino acids within the active site.
Third, environmental conditions can alter the ability of the enzyme to undergo structural changes necessary for catalysis.
Enzymes Citations
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Nucleotides: Definition, Functions, Types, and Examples
Continue ReadingWhat are Nucleotides?
Nucleotides are composed of 3 units;
1) a Five Carbon Sugar (Pentose)
Deoxyribose in DNA – In DNA the 2′ OH of ribose is replaced by a hydrogen atom
Ribose in RNA – In RNA the there is a hydroxyl group on both the 2′ and 3′ position.
This contributes to RNA’s inherent poor half- life.
2) a Nitrogenous Base
a) Purine:
Adenine (found in DNA and RNA)
Guanine (found in DNA and RNA)
b) Pyrimidines:
Cytosine (found in DNA and RNA)
Thymine (found in DNA only)
Uracil (found in RNA only)
3) a Phosphate Group
In nucleic acids, nucleotides are joined together by phosphodiester bonds.
A phosphodiester bond is a group of strong covalent bonds between the phosphorus atom in a phosphate group and two other molecules over two ester bonds.
Nucleotides are written 5’ → 3’
RNA and DNA are nucleotide polymers, both of which are created in the nucleus.
In DNA, two strands are joined by the hydrogen bonds to make the structure called the double helix.
This model was proposed by Watson and Crick.
The members of each base pair can fit together within the double helix only if the two strands of the helix are antiparallel.
By convention the top DNA strand goes 5’ → 3’ and the bottom 3’ → 5’.
Going in the 5’ → 3’ Direction is referred to as going downstream.
Going in the 3’ → 5’ Direction is referred to as going upstream.
Adenine (A) and Thymine (T) form two (2) hydrogen bonds, while cytosine (C) and guanine (G) form three (3) hydrogen bonds.
The more G-C%, the higher the melting temperature because of this additional hydrogen bond.
Differences Between DNA and RNA
Structurally, DNA and RNA are nearly identical.
However, there are three fundamental differences that account for the very different functions of the two molecules.
1. RNA is a single stranded nucleic acid and no double helix is formed.
2. RNA has a ribose sugar instead of a deoxyribose sugar like DNA.
3. RNA nucleotides has a uracil base instead of thymine.
Other than these differences, DNA and RNA are the same.
Their phosphates, sugars, and bases show the same bonding patterns to form nucleotides and their nucleotides bind to form nucleic acids in the same way.
Other Important Nucleotides
1) ATP (adenosine triphosphate)
2) Cyclic AMP – an important component in many secondary messenger systems
3) NADH and FADH, the coenzymes involved in the Krebs cycle.
Nucleotides Citations
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Carbohydrates: Definition, Functions, Types, and Examples
Continue ReadingWhat are Carbohydrates?
Carbohydrates (also called sugars or saccharides) are made from carbon and water.
They have the formula CnH2nOn.
"Most saccharides (Carbohydrates) are almost always more abundant in nature as the "D" form"
Five and six carbon carbohydrates (pentoses and hexoses) are the most common in nature.
The six carbon carbohydrate called glucose is the most commonly occurring six carbon carbohydrate.
All digested carbohydrates reaching body cells have been converted to glucose by the liver and enterocytes.
The ring form has two anomers.
It is a stereoisomer of a cyclic saccharide that differs only in its configuration at the anomeric carbon.
The anomeric carbon is important to the reactivity of carbohydrates because it is the site at which ring opening occurs, becoming the carbonyl group, the important functional group.
Anomers are identified as “α” or “β” based on the relation between the stereochemistry of the anomeric carbon and the furthest chiral centre in the ring.
The α anomer is the one in which these two positions have the same configuration; they are opposite in the β anomer.
Opposite carbon 1 and 6 = alpha; Same carbon 1 and 6 = beta
"The cell can oxidize glucose (Carbohydrates) transferring its chemical energy to a more readily useable form, ATP"
If the cell has sufficient ATP, glucose (a monosaccharide) is polymerized to the polysaccharide, glycogen or converted to fat.
Glycogen is a short term store of energy in animal cells and this process is called glycogenesis.
Glycogen contains alpha linkages and is found in all animal cells, but especially large amounts are found in the muscle and liver cells.
The liver regulates the blood glucose level, so liver cells are one of the few cell types capable of reforming glycogen back to glucose and releasing it to the bloodstream.
The breakdown of glycogen into glucose is called glycogenolysis.
Glucose → Glycogen = glycogenesis
Glycogen → Glucose = glycogenolysis
The method of glucose uptake differs throughout tissues depending on two factors; the metabolic needs of the tissue and availability of glucose.
The two ways in which glucose uptake can take place are facilitated diffusion (a passive process) and secondary active transport (an active process which indirectly requires the hydrolysis of ATP).
Only certain epithelial cells (enterocytes) in the digestive tract and the proximal tube of the kidney are capable of absorbing glucose against a concentration gradient.
This is done via a secondary active transport mechanism down the concentration gradient of sodium.
"Cells absorb glucose via facilitated diffusion"
Insulin increases the rate of facilitated diffusion for glucose and other monosaccharides.
In the absence of insulin, only neural and hepatic (liver) cells are capable of absorbing sufficient amounts of glucose via the facilitated transport system.
Plants form starch and cellulose from glucose.
a) Starch comes in two forms: amylose and amylopectin
1) Starch has alpha linkages
2) is a straight chain of glucose molecules
b) Cellulose has beta linkages Animals have the enzymes to digest alpha linkages but not beta linkages.
Some animals have bacteria in their digestive tracts that release an enzyme to digest the beta linkages in cellulose.
"Animals eat alpha linkages, bacteria break beta linkages"
Carbohydrates Citations
- “One-Pot” Protection, Glycosylation, and Protection-Glycosylation Strategies of Carbohydrates. Chem Rev . 2018 Sep 12;118(17):8025-8104.
- Halogenated carbohydrates. Adv Carbohydr Chem Biochem . 1967;22:177-227.
- Carbohydrates in therapeutics. Cardiovasc Hematol Agents Med Chem . 2007 Jul;5(3):186-97.
- Carbohydrates. Sci Am . 1980 Nov;243(5):90-116.
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Lipids: Definition, Examples, and Structures
Continue ReadingWhat are Lipids?
Lipids are any biological molecule that has low solubility in water and high solubility in nonpolar organic solvents.
Type of Lipids
There are six major groups of lipids:
I. Fatty Acids
Building blocks for most, but not all, complex lipids
Usually an even number of carbons, maximum number of carbons in humans is 24
Can be saturated or unsaturated
i. Saturated fatty acids contain only single carbon-carbon bonds
ii. Unsaturated fatty acids contain one or more carbon-carbon double bonds
Oxidation of fatty acids liberates large amounts of chemical energy for the cell, and most lipids reach the cell in the form of fatty acids and NOT as triacylglycerols.
II. Triacylglycerols
Commonly called triglycerides or simply fats and oils, are constructed from a three carbon backbone called glycerol, which is attached to 3 fatty acids.
Their function is to store energy and may also provide thermal insulation and padding to an organism.
Adipocytes, also called fat cells, are specialized cells whose cytoplasm contains almost nothing but triglycerides.
Lypolysis of triacylglycerols take place inside the adipose cells when blood levels of epinephrine, norepinephrine, glucagon or ACTH are high.
III. Phospholipids
Are built from a glycerol backbone as well, but a polar phosphate group replaces one of the fatty acids.
The phosphate group lies on the opposite side of the glycerol from the fatty acids making this lipid polar on one end and nonpolar on the other end.
This condition is called amphipathic, and makes phospholipids especially well suited as the major component of membranes
"Lipids are also used as intracellular messengers"
IV. Glycolipids
Are similar to phospholipids, except that glycolipids have one or more carbohydrates attached to the 3-carbon glycerol backbone instead of the phosphate group.
Are also amphipathic
They are found in abundance in the membranes of myelinated cells composing the nervous system.
Also serve as markers for cellular recognition.
V. Steroids
Are four ringed structures, which regulate metabolic activities.
Include some hormones, vitamin D, and cholesterol, an important membrane component.
VI. Terpenes
Include vitamin A, a vitamin important for vision.
Their building block is the hydrocarbon isoprene, CH2=C(CH3)-CH=CH2.
Terpene hydrocarbons therefore have molecular formulas (C5H8)n
Eicosanoids
Another class of lipids is the 20 carbon eicosanoids.
Eicoanoids include prostaglandins, thromboxanes, and leukotrienes
Eicosanoids are released from cell membranes as local hormones that regulate, among other things, blood pressure, body temperature, and smooth muscle contraction.
Aspirin is a commonly used inhibitor in prostaglandin synthesis
Lipoproteins
Since lipids are insoluble in aqueous solution, they are transported in the blood via lipoproteins.
A lipoprotein is a biochemical assembly that contains both proteins and lipids.
It contains a lipid core surrounded by phospholipids and apoproteins.
Thus lipoproteins are able to dissolve lipids in its hydrophobic core, and then move freely through the aqueous solution due to its hydrophilic shell
Are classified according to density.
The greater the ratio of lipid to protein, the lower the density.
The major classes of lipoproteins are chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL – “bad cholesterol”), and high density lipoproteins (HDL – “good cholesterol”) – is arranged according to density.
Lipids Citations
- Far from Inert: Membrane Lipids Possess Intrinsic Reactivity That Has Consequences for Cell Biology. Bioessays . 2020 Mar;42(3):e1900147
- Amino acid-containing membrane lipids in bacteria. Prog Lipid Res . 2010 Jan;49(1):46-60.
- Fatty Acids and Bioactive Lipids of Potato Cultivars: An Overview. J Oleo Sci . 2016;65(6):459-70.
- Lipids and Surfactants: The Inside Story of Lipid-Based Drug Delivery Systems. Crit Rev Ther Drug Carrier Syst . 2018;35(2):99-155.
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Plant Cells: Labelled Diagram, Definitions, and Structure
Continue ReadingStructure of Plant Cells
Cell Wall
Plant cells are eukaryotic cells, but unlike animal cells which have a cell membrane, plant cells have cell walls.
Plants have a rigid cell wall that surrounds the plasma membrane.
The cell wall is made of cellulose and lignin, which are strong and tough compounds.
Plant Cells Labelled
Plastids and Chloroplasts
Plants make their own food through photosynthesis.
Plant cells have plastids, which animal cells don’t. Plastids are organelles used to make and store needed compounds.
Chloroplasts are the most important of plastids.
They convert light energy from the sun into sugar and oxygen.
The most exposed parts of the plants to the sun, tending to be the most green, are filled with chloroplasts, which are making food and oxygen for the plant.
Central Vacuole
Vacuoles have a very important role in plant cells compared to others, which is why only some animal cells have vacuoles, and even then, their vacuoles don’t have as big a role.
Plant cells can push water into vacuoles. They provide turgor pressure inside the cell, which reinforces the plant.
When the plant loses water, the turgor pressure drops, and the plant wilts.
The vacuole is a storage container not only for water for other compounds. It can contain and export things that the cell doesn’t need.
Endoplasmic Reticulum
The endoplasmic reticulum(s) are organelles that create a network of membranes that transport substances around the cell.
They have phospholipid bilayers. There are two types: the rough ER, and the smooth ER The rough endoplasmic reticulum is rough because it has ribosomes (which I will explain later) attached to it.
It helps in the synthesis and packaging of proteins. The smooth endoplasmic reticulum doesn’t have ribosomes attached.
It contains enzymes that help with the creation of important lipids.
It has a role in the process of cell detox.
The smooth ER adds a carboxyl group to noxious substances, making them soluble in water.
Ribosomes
Ribosomes create proteins.
They can float freely in the cytoplasm or can be attached to the nuclear envelope.
They create proteins by assembling amino acids into polypeptides.
As the ribosomes build an amino acid chain, the chain is pushed into the endoplasmic reticulum.
When the protein chain is complete, the endoplasmic reticulum pinches it off and sends it to the Golgi apparatus.
Golgi Apparatus
The Golgi apparatus focuses on protein processing and packaging.
Golgi bodies are the Golgi apparatus’s layers.
Golgi bodies cut up large proteins into smaller hormones.
They can combine proteins with carbohydrates to make various molecules.
They then package these products into sacs called vesicles, which will ship the products of the Golgi body to other parts of the cell, and outside the cell as well.
Lysosomes
Lysosomes are enzyme sacs that break down cellular waste – they process cell digestion.
They can take substances from outside of the cell and cellular waste and turn them into simple compounds.
The compounds are then transferred into the cytoplasm where they can be used as a cell building material
Nucleus
The nucleus is a highly specialized organelle that lives in its own double membrane with the nucleolus.
The nucleus stores the cell’s DNA and holds all the information the cell needs to do its job.
Chromatin is a web-like substance that holds the nucleus’s DNA.
Chromatin gathers into rod-shaped chromosomes that hold DNA molecules when the cell is ready to split during cell division.
The nucleolus lives inside the nucleus and is the only organelle that is not enveloped by its own membrane.
It makes ribosomal RNA, rRNA, which is important during protein synthesis.
Ribosomal RNA or rRNA combines with proteins to form the basic units of ribosomes.
When the units are done, the nucleus spits them out of the nuclear envelope, where they are assembled into ribosomes.
The nucleus sends orders in the form of messenger RNA, or mRNA.
The messages are sent to ribosomes, which carry out the orders in the rest of the cell.
Mitochondria
The mitochondria is the “power plant” of the cell.
This is where cellular respiration takes place.
During this, energy is derived and converted into ATP from fats, carbohydrates, and other fuels.
Mitochondria almost act as their own organism and have their very own DNA which is an exact replication of the mother’s DNA
Plant Cells Citations
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Gelatin Hydrolysis Test: Definition, Principle, Procedure, and...
Continue ReadingGelatin Hydrolysis Test
Generally, gelatin is used to define an animal protein or a collagen which is a component of a vertebrate in a connective tissue. Gelatin is used as a solidifying agent in a food for a long day ago, Organisms that produces proteolytic enzyme, specifically gelatinases help in hydrolysing the gelatin into a polypeptide and individual amino acids.
During this process, gelatin losses its structure and converts in to liquid form. Robert Koch used gelatin in his culture in the form of a nutrient gelatin which is one of the oldest solid culture media.
Gelatin usually dissolves in a water at 50 degree Celsius and it exists as the liquid at a temperature of above 25 degree Celsius and it further solidifies and forms a gel like substance when it is cooled below 25ºC.
What is Gelatin Hydrolysis Test?
Gelatin hydrolysis test is also called as Gelatin liquefication test, as this test involves the process of liquefication of gelatin in the presence of an enzyme gelatinase.
Gelatinase is considered as one of the most important enzymes in various pathogenic organisms as it is produced extracellularly, and hydrolysis gelatin which is derived from the connective tissues of the vertebrates in the form of collagen.
This enzyme also works as a virulent factor which dissolves the connective tissues of the host and aids in producing invasive infections.
Gelatin is a protein that hydrolyses in the presence of an enzyme gelatinase by breaking down the complex structure into monomeric amino acids.
This test is being followed from early days in the form of presumptive test to identify the pathogenic organisms like Serratia, Pseudomonas, Flavobacterium and Clostridium.
Gelatin Hydrolysis Test Principle
Gelatin is a type of protein derived from the animal tissues, collagen and it forms a solid structure at low temperature. This protein is metabolised or degraded by a group of enzymes known as gelatinase.
Gelatinases are the proteolytic enzymes which liquifies the gelatin into polypeptides and individual amino acids.
The degradation of the gelatin into polypeptides, is followed by covering the polypeptides into amino acids.
Gelatinase is very important in bacteria as gelatin is comparatively a large polymer and thus it cannot be transported into the cell membranes, as gelatinase breaks this compound into the smaller peptides it can easily be transported into the cell and it is utilised by the bacteria.
In hydrolysis test, media containing gelatin is used and its hydrolysis is detected either by liquification of the media or by flooding the media with mercuric chloride, as mercuric chloride helps in precipitating the gelatin and make the hydrolysed area to look clear.
This test is commonly used to determine the ability of an organism to produce the extracellular proteolytic enzymes, gelatinases, which hydrolyses the gelatin, a component of the vertebrate connective tissue.
This reaction usually occurs in a series of two steps, in first reaction process, Gelatinases hydrolyses gelatin into polypeptides and further the polypeptides are converted into amino acids.
The amino acids are taken up by the cells and they are used for their own metabolic purposes. Generally, the presence of gelatinases can be detected using a nutrient gelatin medium.
When an organism produces gelatinase, the enzymes liquifies the growth medium by liquifying the gelatin that is present in the medium.
Gelatin Hydrolysis Test and Microorganism
Gelatin hydrolysis test is generally used to detect the micro-organisms Gram negative rods require gelatine for the identification of specific fluorescent pseudomonas. Such as Pseudomonas putida (negative) from pseudomonas fluorescens (positive). Whereas the gram-positive rods are needed for identifying the pathogens in species level.
Gelatin Hydrolysis Test Materials
Nutrient Gelatin media is generally used for the purpose of demonstrating the hydrolysis of gelatin by adding mercuric chloride or by using liquification of gelatin.
Ingredients Gram/litre Tryptose 20.0 Gelatin 10.0 Magnesium sulphate 0.01 Beef extract 3.0 Agar 15.0 Reagents:
• Mercuric chloride
Supplies:
• Inoculating needle
• Incubator
• Pipettes
Gelatin Hydrolysis Test Procedure
1. Preparation of the media
First, the media is prepared. 128 grams of dehydrated medium mixed with 1000 millilitres of warm distilled water in a beaker.
The prepared solution is heated with agitation to bring about the boiling point and the media is let to dissolve completely.
Then the prepared medium is dispensed into series of test tubes and it is autoclaved at 15lbs pressure for about 15 minutes. If agar plate method is used, the medium is autoclaved in the beaker.
The tubes are cooled after autoclaving to about 49 to 50 degree Celsius by keeping it in an upright position.
2. Gelatin Hydrolysis
Gelatin hydrolysis can be identified by nutrient gelatin slab method or by agar plate flooding method by using mercuric chloride.
a. Stab Method of Gelatin Hydrolysis Test
Gelatin medium in a tube is inoculated with 4 to 5 drops of a 24-hour broth medium.The inoculated tubes are then incubated at a temperature of about 37 ºC in the air for 24 to 48 hours, If the organisms have the capability to grow at 25ºC then the incubation should be done at 25ºC. After the first incubation, the tubes are placed at 4ºC for one 24 hours.
b. Plate Method of Gelatin Hydrolysis Test
In plate method, heavy inoculum of a culture is taken using an inoculating loop and it is inoculating using nutrient gelatin medium. And the plates are kept in an incubator, by setting a temperature of 37ºC for about 24 to 48 hours.
Gelatin Hydrolysis Test Result
1. Tube Method: In positive result, either partial or total liquefication of the gelatin can be observed in the tubes. In negative result, complete solidification of gelatin is seen at a temperature of 4ºC.
2. Plate Method: In positive result, clear zone around the colonies is noted after adding mercuric chloride. If there is no clear zone after adding mercuric chloride, it indicates the negative result.
Gelatin Hydrolysis Test Uses
Gelatin hydrolysis test is usually used to test the capability of an organism to produce gelatinase.
This test also helps in identification of Serratia, Pseudomonas, Flavobacterium and clostridium.
Gelatin hydrolysis test also helps in distinguishing the gelatinase positive staphylococcus aureus from the gelatinase negative non-pathogenic S. epidermidis.
Using this test; genera of bacteria such as Serratia and proteus are differentiated from other members of Enterobacteriaceae family
Gelatin Hydrolysis Test Citations
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Hippurate Hydrolysis Test: Definition, Principle, Procedure, and...
Continue ReadingHippurate Hydrolysis Test
Hippurate hydrolysis test is one of the biochemical tests, which is used to differentiate the micro-organisms on the basis of their ability to hydrolyze Hippurate into benzoic acid and glycine by the action of the enzyme hippuricase, present in the bacteria.
Hippuricase is one of the constitutive enzymes which helps in hydrolyzing the Hippurate and helps in producing amino acid, glycine.
Glycine can be detected by the oxidizing the Ninhydrin reagent, that results in the production of a deep purple color.
Hippurate hydrolysis test is used in the identification of Gardnerella vaginalis, campylobacter jejuni, Listeria monocytogenes and group B streptococci; to detect the ability of the organism to hydrolyze Hippurate.
What is Hippurate Hydrolysis Test?
In olden days this test was performed using a ferric chloride indicator to identify the benzoic acid, but those traditional methods would take a longer time to resolve.
But now a days with the modern techniques, this test has been modified and it is used as a rapid test by detecting the glycine by adding ninhydrin as an indicator.
This test can also be used to distinguish the Group B streptococci from other groups A, C, F and G which cannot hydrolyze sodium Hippurate.
Other group of viridians like Group D can be also detected by using Hippurate sodium hydrolyze.
This test is considered as one of the class of test which differentiates the bovine-β-hemolytic Group B streptococci from human β-hemolytic Group B species of streptococcus.
Hippurate Hydrolysis Test Objective
Hippurate hydrolysis test is generally used to detect the production of the enzyme hippuricase; for identifying the presumptive of different microorganisms. Its main aim is to differentiate bacteria based on their ability to produce hydrolyzed Hippurate.
Hippurate Hydrolysis Test Principle
Hippurate test is based on the ability of the organism to hydrolyze sodium Hippurate into glycine and benzoic acid with the action of an enzyme hippuricase.
This test is primarily used in identifying the campylobacter jejuni, Gardnerella vaginalis, Listeria monocytogenes and streptococci’s agalactiae.
This ability of the few bacterial species to hydrolyze the Hippurate was classically tested using a ferric chloride indicator which helps in detecting the benzoic acid.
However, now a days a 2-hour rapid method is opposed to 48-hour tractional method, for detecting the Hippurate hydrolysis.
The rapid is done by using ninhydrin as an indicator, which on reaction with protein or amino acids detects glycine.
As glycine is deaminated by oxidizing action of ninhydrin, which results in the reduction of ninhydrin and the substance resembles in purple color.
The test medium used here should contain only Hippurate as a source of protein as ninhydrin acts with the free amino acids that is present.
Thus, rapid Hippurate hydrolysis test helps in detecting the by- product of the benzoic acid which is seen as a sensitive and classical method.
Hippurate → Glycine + Benzoic acid
Glycine + Ninhydrin → Purple colored complex
The two important reagents used in this test are Hippurate solution and Ninhydrin.
1. Hippurate Solution
Hippurate reagent can be found commercially in the form of dehydrated tubes or in the form of disks or tablets. This can also be prepared in the laboratory in the form of 1% of Hippurate solution, for preparing in the laboratory, one gram of sodium Hippurate is added into 100 ml of distilled water.
2. Ninhydrin
Ninhydrin can also to purchased commercially. But mostly it is prepared in the laboratories while performing the diagnosis, for laboratory preparation, of the ninhydrin, 50ml of 1-butanol is added into a dark colored glass bottle. Then 3.5 grams of ninhydrin is added to the bottle and it is mixed well.
Hippurate Hydrolysis Test Materials
Sterilised wooden stick and inoculating loops
Incubator
Test tubes
Distilled water
Hippurate Hydrolysis Test Procedure
1. Preparation of Hippurate Solution
If dehydrated Hippurate is used, 0.2ml of distilled water is added at a pH of 6.8 to 7.2 is added with the test reagent. Then the two drops of distilled water are added to an empty tube for disk or tablets If Hippurate solution is prepared in the laboratory, 0.4ml of the reagent is added to the tube for each test.
2. Hippurate Hydrolysis Test
A heavy suspension is prepared in a tube from an 18 to 24-hour culture. The colony is then picked up carefully where the agar which contains protein should not be taken. The tube is then incubated for 30 minutes, at 35ºC to 37ºC.
Then the tube is observed for every for 10 minutes intervals till the deep blue colour; The change in colour appears usually appears within 15 minutes, after the addition of ninhydrin solution.
Hippurate Hydrolysis Test Result
A positive Hippurate hydrolysis test results in the appearance deep blue which almost looks like a crystal violet within 30 minutes.
A negative Hippurate hydrolysis test, results in the appearance of a faint blue colour or there will be no colour change.
Control Organism in Hippurate Hydrolysis Test
Streptococcus agalactiae: it indicates Hippurate positive
Streptococcus pyogenes: It indicates Hippurate negative
Hippurate Hydrolysis Test Citations
- Rapid, colorimetric test for the determination of hippurate hydrolysis by group B Streptococcus. J Clin Microbiol . 1976 Jan;3(1):49-50.
- A rapid hippurate hydrolysis test for the presumptive identification of group B streptococci. Pathology . 1983 Jul;15(3):251-2.
- Evaluation of the rapid hippurate hydrolysis test with enterococcal group D streptococci. J Clin Microbiol . 1977 Mar;5(3):290-2.
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Esculin Hydrolysis Test: Principle, Procedure, Purpose, and...
Continue ReadingEsculin Hydrolysis Test
Biochemical tests are considered as the most important method to detect the micro-organisms and other pathogens. One such biochemical test is Esculin hydrolysis test.
It is one of the differential tests, which helps in differentiating the bacteria depending on the ability of an organism to hydrolyse esculin. This test is generally based on the hydrolysis of the esculin, a glucoside into a glucose and esculetin by a micro-organism which has constitutive β-glucosidase or esculinase enzyme.
What is Esculin Hydrolysis Test?
Esculin hydrolysis test is used to differentiate the type of bacterial species like the gram-positive and gram-negative bacteria, which also includes along with it a a broad spectrum of aerobes, anaerobes and facultative anaerobes.
This test is generally used as a taxonomic tool in identifying the variety of microbes, including Enterobacteriaceae family, streptococcus genera and Listeria, non-fermentative gram negative-bacilli and anaerobes.
Esculin hydrolysis test thus helps in differentiating the bacteria based on their ability to hydrolyse esculin.
This test is usually done selectively by adding bile in the medium, and it is commonly called as bile esculin test.
Hydrolysis of esculin by bacteria can be done by detecting the growth in the supporting media such as Vaugh-Levine, bile-esculin, or Pfizer selective enterococcus media or by non-growth supporting media like Patho Tech or Rapid spot tests.
This test fully depends upon the hydrolysis of esculin into glucose and esculetin with the help of microorganism which constitutes β-glucosidase or an enzyme esculin’s.
Objective of Esculin Hydrolysis Test
• This test helps in detecting the ability of an organism to hydrolyse esculin by producing the enzyme esculinase.
• This test also helps in differentiating the members of the family Enterobacteriaceae.
Esculin Hydrolysis Test Principle
The basis of esculin test is to hydrolyse the esculin during the presence of bile salts, by the enzyme esculinase.
Esculin is a glucosidase consisting of glucose and the hydroxycoumarin which is linked together by an ester bond through oxygen.
The bile esculin test selects an organism on the basis of their ability to grow in a medium containing 4% of salts which is followed by selection on the basis of their ability to hydrolysis of the esculin, which results in the formation of the compound called esculetin.
After the degradation of esculetin it reacts with the iron ions in the medium to form phenolic iron complex which results in a dark brown or black colour.
In the other sense, esculin is a fluorescent compound and on hydrolysis can be observed by loss of fluorescence.
When bile is added to the medium, micro-organisms grow in order to hydrolyse esculin. Bile inhibits the growth of other gram-positive organisms and makes the medium selective.
Esculin Hydrolysis Test Materials
Media: Esculin agar is used for detecting the hydrolysis of esculin. This medium is a differential medium and they can be made selective by adding bile.
Composition of Esculin agar is listed below;
Ingredients Gram/Liter Casein enzymic hydrolysate 13.0 Yeast extract 5.0 Beef heart infusion in a solid state 2.0 Sodium chloride 5.0 Ferric chloride 0.5 Agar 15.0 Reagents: In esculin spot test, 0.02% of esculin solution is prepared using distilled water.
Supplies:
• Long wave UV light up to 360nm
• Sterilised sticks, needles, or inoculating loops
• Pasteur pipettes
• Boiling heat block
Esculin Hydrolysis Test Procedure
1. Preparation of the media
About 41.5 grams of dehydrated powder is added in 1000 millilitres of distilled or deionised water and it is mixed thoroughly, after mixing the solution is heated till it reaches the boiling point and the medium to let to dissolve completely.
The solution is then dispensed into a tube having screw caps and it is sterilised in the autoclave at 121 degree Celsius for about 15 minutes.
Then the tubes are taken out from the autoclave and they are cooled by placing them in a slanted position up to a temperature of about 40 to 45º.
This position is maintained at the same angle to achieve the butts of about 1.5 to 2,0 cm depth.
2. Esculin Hydrolysis Test
Esculin hydrolysis test is usually observed in two methods namely tube test or esculin spot test. The spot test is often known as rapid test.
I. Esculin Hydrolysis Tube Test
In tube test, first the light inoculum is taken from the 18 to 24-hour culture using a sterile inoculating needle from the centre of the well-isolated colony.
Then the esculin agar tubes are inoculating by streaking a slant on the surface using the light inoculum which has been picked up from the culture.
Then the caps of the tube are checked for adequate aeration. Then further the tubes are incubated in the air at 35 to 37ºC for about 24 hours and any change is colour is observed up to 7 days of incubating.
In case, if esculin broths are used the tubes are observed daily and checked whether there is any loss in fluorescence of UV light. If there is loss of fluorescence, 1% of ferric ammonium id added in drops into the tube and colour changes are observed.
II. Esculin Hydrolysis Spot Test
In rapid spot test, the prepared 0.2% of esculin solution is autoclaved and it is placed on the filter paper; then the filter paper is placed on a standard microscopic slide and they are positioned on the supporting rods.
Esculin solution is pipetted over the filter paper and over saturation must be avoided. Then the inoculum is taken from a 24-hour colony and streaked in middle of the filter paper.
Then the prepared filter paper along with a slide is placed in an inoculum at 37ºC for about 10 to 15 minutes.
Klebsiella generally gives a positive result within 15 minutes, but it should be holded for 30 minutes to check the proper result.
Hand-held wood lamp is used in the subdued light to observe the loss of fluorescence.
Esculin Hydrolysis Test Result
Esculin Hydrolysis TubeTest: Blackening of the medium detects the positive result and lack of colour change detects the negative result.
Esculin Hydrolysis Spot Test: Loss of fluorescence and black colour under a UV light demonstrates a positive result and bright fluorescence indicates the negative result.
Esculin Hydrolysis Test Citations
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