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Glycolysis vs Fermentation: Definition, Function, and Steps
Continue ReadingGlycolysis
Glycolysis: (anaerobic, occurs in the cytoplasm) Can be performed in all cells.
Phosphorylated molecules cannot diffuse through the membrane.
The process of ATP production in glycolysis is called Substrate-level phosphorylation.
Net Glycolysis Equation
Glucose + 2ATP + 2NAD+ + 4ADP + 2Pi = 2pyruvate + 2ADP + 2 NADH + 2H+ + 4ATP + 2H2O
Reduced Glycolysis Equation
Glucose + 2NAD+ + 2ADP + 2Pi = 2pyruvate + 2NADH + 2H+ + 2ATP + 2 H2O
"In Glycolysis, 2 net ATP (4 total made, but 2 needed to complete this stage)"
2 NADH produced (making 4 ATP in ETC for eukaryotes and 6 ATP for prokaryotes) via reduction of NAD+
2 pyruvate (3 carbons) produced Pyruvate can then be converted into various products:
1. It can be oxidized to give the acetyl group of acetyl- coenzyme A, which is then oxidized completely to give CO2.
2. It can be reduced to lactate (a type of fermentation).
3. It can be converted to ethanol and CO2 (another type of fermentation)
ADP & AMP is a activator for glycolysis
ATP and Citrate are inhibitors for glycolysis In order for glycolysis to continue operating the produced NADH must be oxidized back into NAD+
Fructose can enter glycolysis as fructose 6-phosphate!!
Fermentation
Fermentation is anaerobic, occurs in the cytoplasm.
It includes the process of glycolysis
Animal tissues reduce pyruvate to lactate under anaerobic conditions, at the same time oxidizing NADH back to NAD+, which is needed for continued glycolysis.
Reaction is catalyzed by lactate dehydrogenase. Lactate can be recycled; it is carried in blood to the liver where it is converted back to glucose (in a process requiring the input of energy).
The acidification of muscle explain why continued exercise can cross cramping, aka Acidosis.
Yeast produce ethanol and CO2
0 ATP is produced.
Low [NAD+]/High [NADH] is a driver for fermentation
Pyruvate Decarboxylation
Aerobic, occurs in the cytoplasm for prokaryotes, mitochondrial matrix for eukaryotes).
Both pyruvate and NADH move through the mitochondrial membrane through a large membrane protein called porin.
The inner mitochondrial membrane, however, is less permeable.
Although pyruvate moves into the matrix via facilitated diffusion, each NADH (depending upon the mechanism used for transport) may or may not require the hydrolysis of ATP (this is why the NADH in glycolysis only produce 4 ATP instead of 6).
Pyruvate decarboxylation is the biochemical reaction that uses pyruvate to form acetyl-CoA, releasing NADH, a reducing equivalent, and carbon dioxide
Total:
2 CO2
0 ATP produced
2 NADH produced, for the decarboxylation of both pyruvate (making 6 ATP in ETC)
Glycolysis vs Fermentation Citations
- Glucose, glycolysis, and neurodegenerative diseases. J Cell Physiol . 2020 Nov;235(11):7653-7662.
- Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Curr Opin Plant Biol . 2004 Jun;7(3):254-61.
- Mini-review on glycolysis and cancer. J Cancer Educ . 2013 Sep;28(3):454-7.
- Fermentation, fermented foods and lactose intolerance. Eur J Clin Nutr . 2002 Dec;56 Suppl 4:S50-5.
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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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Western Blot: Overview, Technique, Theory, and Trouble...
Continue ReadingObjective of Western Blot
Transfer of proteins from SDS-PAGE to solid supports and detection of immobilized proteins by antigen-antibody reaction by Western blotting.
Principle of Western Blot
Western blot process is to proteins what southern blotting is to DNA. In both techniques, electrophoretically separated components are transferred from Polyacrylamide gel electrophoresis (PAGE) to a solid support (nitrocellulose membrane) and probed with reagents that are specific for particular sequences of amino acids (western blot) or nucleotides (southern hybridization).
In the case of proteins probes usually are antibodies that react specifically with the antigenic epitopes displayed by the target protein attached to the solid support (nitrocellulose membrane).
Therefore, Western blot technique is extremely useful for the identification and quantitation of specific proteins in the complex mixtures of various proteins that are not radiolabeled.
Created with BioRender
A variety of different solid supports have been used for western blotting such nitrocellulose membrane and Polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane.
These include cyanogen-bromide-activated paper, PVDF (polyvinyledene difluoride) membrane, nitrocellulose filter etc.
Nowadays, most western blotting is carried out by direct electrophoretic transfer of proteins from the gel to a nitrocellulose filter.
Two types of electrophoretic apparatuses are available for electroblotting.
In the first one, one side of the gel is placed in contact with a piece of nitrocellulose filter and the polyacrylamide gel and its attached filter are then sandwiched between whatmann 3 mm paper, two porous pads and two plastic supports.
The entire set-up is then immersed in an electrophoresis tank containing Tris-glycine electrophoresis buffer at pH 8.3 and equipped with standard platinum electrodes.
The nitrocellulose paper is placed towards the anode.
An electric current is then passed for around 12 h during which the proteins get transferred from the gel to nitrocellulose filter.
In order to prevent over-heating and consequent formation of air-bubbles in the sandwich the transfer is carried out in cold conditions.
In a newer type of apparatus, the gel and its attached nitrocellulose filter are sandwiched between pieces of whatmann 3 mm paper that have been soaked in the transfer buffer containing Tris, glycine, SDS and methanol.
In this case since there is very little requirement of the electrophoresis buffer, the system is termed as semi-dry system resulting in semi-dry blotting.)
The sandwich is then placed between plate electrodes, with nitrocellulose filter on the anodic side.
Transfer of proteins can be carried out at room temperature and the time required is minimized to 1.5-2 h for complete transfer.
Western Blot Requirements
Transfer buffer: 39 mM glycine, 48 mM Tris base, 0.037% SDS and 20% methanol.
- Make the volume with distilled water.
- pH should be 8.3.
Tris Buffered saline (TBS) with containing 0.1% (v/v) Tween-20 (TBST)
Western Blot Procedure
1. When the SDS-PAGE is approaching the end of its run, rinse the plates with laboratory grade distilled water followed by wipe off any beads of liquid that adhere to them with nonabsorbent tissues using methanol.
2. Wearing gloves cut around 4 pieces of whatmann 1 mm paper and one piece of PVDF membrane to the exact size of the SDS-PAGE gel (if the whatmann paper or filter is of size larger than the polyacrylamide gel there is a good chance that the overhanging edges of the paper and the filter will touch causing a short circuit that will prevent the transfer of protein from the polyacrylamide gel to the filter). Mark one end of the filter with a soft lead pencil or cut the filter slightly on the lower left side.
3. To activate the PVDF membrane, soak it in absolute methanol for 1 minute. Thereafter, soak the PVDF membrane in transfer buffer.
4. Soak the pieces of whatmann paper in a shallow tray containing a small amount of transfer buffer.
5. Wearing gloves, set up the transfer apparatus as follows:
I) Place around 2 pieces of soaked whatmann paper on the bottom electrode.
II) Now very carefully place the soaked PVDF membrane on top of the whatmann paper carefully.
III) On top of the PVDF memberane carefully place the gel preventing entrapment of any air bubble.
IV) Place 2 pieces of soaked whatmann papers on top of the gel (At each step, stack the sheets on top of the other so that they are exactly aligned.) Using a spacer or a glass pipette as a roller, squeeze out air bubbles if any.
V) Place the upper electrode on top of the stack. Connect the electrical leads. Apply a constant current of 2.5 A for 7 min.
VI) After 7 min turn off the power supply and disconnect the leads. Disassemble the transfer apparatus from the top to downward, peeling off each layer in turn. Place the memberane in a tray containing Ponceau S for staining.
VII) Wash the membrane with distilled water. Block the non-specific binding by incubating the membrane with 1xTBS containing 2% BSA w/v for 1 h at room temperature.
VIII) After blocking, wash once with 1xTBS containing Tween 20 and incubate with primary antibody overnight at 4ᵒC. The antibody should be diluted with 1xTBS with 0.5% BSA (w/v) according to the supplier’s instructions. Once the incubation time is over, remove the primary antibody and wash the membrane three times with 1 x TBST
IX) After washing, incubate the membrane with secondary antibody which is conjugated with HRP (Horse Radish Peroxidase) and diluted in 01xTBS with 0.5% BSA for 1 hr at room temperature. Following the incubation, remove the antibody solution and wash three more times with 1 x TBST
X) Visualize protein expressions using the chemiluminescence detection substrate (Millipore) using chemidocumentation system.
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Cytoplasm: Function, Definition, Types, and Examples
Continue ReadingWhat is Cytoplasm?
The cytoplasm is the ground substance present between the plasma film and core of all cells with the exception of viral cells.
It is a shapeless, clear, homogenous, colloidal substance, separated into the cytoplasmic lattice and cytoplasmic designs.
Cytoplasmic lattice, hyaloplasm, or cytosol is separated into fringe denser and non-granular parts called cortex or ectoplasm and an internal less thick and granular part called medulla or endoplasm.
It is a genuine arrangement of nucleotides, nutrients, RNAs, minerals, sugars, amino acids, and a colloidal arrangement of proteins.
Cytoplasmic designs incorporate cell organelles.
Composition of Cytoplasm
The cytoplasm is made out of a thick and liquid part, the cytosol, comprised of water (which addresses 75-85% of the complete load of the cell), of inorganic substances separated in ionic structure (particularly particles K+, Na+, Ca++ and Mg++) and from different natural atoms (counting proteins with enzymatic or primary capacity).
Eukaryote Cytoplasmic Structures
The endoplasmic reticulum is a mind-boggling organization of imparting tubules, packs, and channels, which open up at the level of the atomic film.
The endoplasmic reticulum is of two sorts; the harsh kind is a continuation of the atomic layer and conveys a large number of little granules, called ribosomes.
On the outside surface, they are connected to the blend of proteins; the smooth sort is liberated from ribosomes and is liable for lipid combination. At last, the space between the collapsed layers of the two kinds of endoplasmic reticulum is utilized to store and move particles starting with one point then onto the next in the cell.
Ribosomes are the site of protein amalgamation, i.e., the get together of amino acids to shape proteins.
They comprise of two subunits of inconsistent size, comprised of ribonucleic corrosive (RNA) and proteins.
They can be related with the harsh endoplasmic reticulum (for this situation, they combine proteins bound to be emitted outside the cell) or free in the cytoplasm (they blend proteins that the cell holds inside).
The Golgi contraption comprises of tubules smoothed in the middle and swollen at the closures, stacked on one another and finishing at the lower part of the visually impaired, where different materials (chemicals, proteins, lipids) are changed and collected, which will be shipped to different pieces of the cell or ousted.
For this reason, little vesicles disengage from the finishes of the leveled sacs and relocate towards the plasma film and converge with it; the substance of the vesicles are accordingly spilled out.
Lysosomes are vesicles that get from the Golgi contraption and contain hydrolytic compounds (fit for annihilating proteins and lipids).
The cell utilizes lysosomes to reuse worn pieces of organelles or to “digest” a whole cell (for instance, a bacterium).
Mitochondria are roundish or ovoid organelles, delimited by a twofold film; the inner one is collapsed on itself to frame septa (mitochondrial peaks), which increment the inside surface of the organelle.
The space encased by the inside film is the network, while the space between the two layers is the between layer space.
Cell breath happens in the mitochondria, through which energy is removed from supplements (sugars and fats) following their oxidation and ensuing destruction up to carbon dioxide and water.
The energy got from this cycle is put away as ATP atoms; at the suitable time, the hydrolysis of ATP makes accessible the energy fundamental for the cell to complete its exercises.
Thus, cells with an elevated capacity to burn calories (for instance, muscle cells) have a more prominent number of mitochondria; all things being equal, the red platelets are free.
A characteristic of mitochondria is simply the capacity repeat, permitted by the presence of a mitochondrial DNA, ribosomes, and every one of the particles essential for the duplication of the hereditary data.
Mitochondria, truth be told, live a couple of days and must, along these lines, be consistently created (this happens by separating vesicles with a twofold layer containing mitochondrial DNA from the crude mitochondria).
The organelles are wrapped and upheld by protein-like filaments that structure an organization, the cytoskeleton. The cytoskeleton likewise offers help for cells without an inflexible divider and has a functioning part in cell division and in the developments of the organelles and the whole cell.
The cytoskeleton is certainly not an inflexible and lasting construction since the filaments that establish it are constantly collected and dismantled.
These are partitioned into three gatherings dependent on their measurements, microfilaments (5-7 nm in width), middle of the road fibers (8-10 nm in distance across) and microtubules, empty tubules with a breadth of around 25 nm, which additionally comprise the centrioles, eyelashes, and flagella.
The centrioles are empty tube shaped constructions, comprising of 9 trios melded with microtubules, which are found in all creature cells (two for every cell) and in a couple of plant cells. The centrioles intercede during cell division to effectively disseminate the chromosomes in the two girl cells.
The eyelashes and flagella are filiform and versatile members with indistinguishable construction, 9 sets of microtubules welded to shape a ring around two focal microtubules.
They are recognized by the length and the number wherein they are available on the plasma film. The eyelashes are various and short (10-25 µm), the flagella are not many and long (50-75 µm).
Their planned developments move the cell into the general climate or make flows in the extracellular fluid that cause a consistent progression of the suspended particles.
The centrioles are organelles as an empty chamber that plays out the capacity of getting sorted out focuses of the inward design of eyelashes and flagella, and for this situation, they are called basal bodies.
Likewise, all phones have a construction called the centrosome, comprising of a couple of centrioles, which plays out a significant capacity when a phone recreates itself.
Eyelashes and flagella are exceptionally flimsy portable expansions present on the outside of numerous sorts of cells; they comprise of explicitly coordinated microtubules.
Eyelashes are hair-like limbs that have the capacity of moving extracellular liquids, however, can likewise furnish a few cells with development.
Every eyelash independently plays out a whiplash-like development, and in general, the lashes move in a state of harmony, making a wave development on the cell surface. Protozoa utilize their eyelashes both to move and to acquire food particles cytoplasm, making the particles that are found in the fluids outside the cell advance with their beat.
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