Isoenzymes. Some enzymes do not consist of a single protein chain, but of several subunits. Isoenzymes are a family of enzymes that catalyze the same reaction but differ in structure and physiology. chemical properties.

For example: lactate dehydrogenase (LDH) consists of 4 subunits of 2 types: the H subunit isolated from the cardiac muscle (heart - heart), the M subunit isolated from skeletal muscles (musculus - muscle). These subunits are encoded different genes. Various organs have various forms LDH with a different set of subunits. There are 5 isoenzymes of LDH:
LDH1: LDH2: LDH3: LDH4: LDH5: (H4) (H3M) (H2M2) (HM3) (M4)
LDH1 is expressed in cardiac muscle and brain, while LDH5 is expressed in skeletal muscle and liver. Other forms in other organs. The appearance of LDH in the blood indicates damage to organs (the enzyme from destroyed cells enters the blood - hyperenzymemia). An increase in the activity of the LDH1 fraction in the blood is observed with damage to the heart muscle (myocardial infarction), and an increase in the activity of LDH5 in the blood is observed with hepatitis and damage to skeletal muscles. That is, thanks to isoenzymes, it is possible to determine the localization of the damaged organ. The most sensitive test for myocardial infarction is an increase in the blood of the cardiac isoenzyme of creatine kinase.

Enzymopathies hereditary (phenylketonuria) and acquired (scurvy). The use of enzymes in the treatment of diseases.

At the heart of many diseases are violations of the functioning of enzymes in the cell - enzymopathies. There are primary (hereditary) and secondary (acquired) enzymopathies. Acquired enzymopathies, as well as proteinopathies in general, seem to be observed in all diseases.

In primary enzymopathies, defective enzymes are inherited mainly in an autosomal recessive manner. Heterozygotes, most often, do not have phenotypic abnormalities. Primary enzymopathies are usually referred to as metabolic diseases, as there is a violation of certain metabolic pathways. In this case, the development of the disease can proceed according to one of the following "scenarios". Consider the conditional scheme metabolic pathway:

Substance A as a result of successive enzymatic reactions turns into product P. With hereditary deficiency of an enzyme, for example, the E3 enzyme, various metabolic pathway disorders are possible:

Violation of the formation of end products. The lack of the end product of this metabolic pathway (P) (in the absence alternative ways synthesis) can lead to the development clinical symptoms, characteristic for this disease:

Accumulation of precursor substrates. If the enzyme E 3 is deficient, substance C will accumulate, and in many cases also the preceding compounds. An increase in the precursor substrates of a defective enzyme is a leading link in the development of many diseases:

Violation of the formation of end products and the accumulation of precursor substrates. Diseases are noted when both the lack of the product and the accumulation of the initial substrate cause clinical manifestations.

Enzyme preparations are widely used in medicine. Enzymes in medical practice are used as diagnostic (enzymodiagnostics) and therapeutic (enzymotherapy) agents. In addition, enzymes are used as specific reagents for the determination of a number of substances. For example, glucose oxidase is used to quantification glucose in urine and blood. The enzyme urease is used to determine the amount of urea in the blood and urine. With the help of various dehydrogenases, the corresponding substrates are detected, for example, pyruvate, lactate, ethanol and etc.

A. Enzymodiagnostics

Enzymodiagnostics consists in making a diagnosis of a disease (or syndrome) based on determining the activity of enzymes in biological fluids person. The principles of enzymodiagnostics are based on the following positions:

• when cells are damaged in the blood or other biological fluids (for example, in urine), the concentration of intracellular enzymes of damaged cells increases;
• the amount of released enzyme is sufficient for its detection;
• the activity of enzymes in biological fluids detected when cells are damaged is stable for a sufficiently long time AND differs from normal values;
• a number of enzymes have a predominant or absolute localization in certain organs (organ specificity);
• there are differences in the intracellular localization of a number of enzymes.

Isoenzymes are isofunctional proteins. They catalyze the same reaction but differ in some ways. functional properties due to differences in:

Amino acid composition;

electrophoretic mobility;

Molecular weight;

Kinetics of enzymatic reactions;

Way of regulation;

Stability, etc.

Isoenzymes are molecular forms of an enzyme, differences in amino acid composition are due to genetic factors.

Examples of isozymes: glucokinase and hexokinase.

Hexokinase can phosphorylate any six-membered cycle, hexokinase can only convert glucose. After eating a meal rich in glucose, glucokinase begins to work. Hexokinase is a stationary enzyme. It catalyzes the breakdown of glucose at low concentrations entering the body. They differ in localization (glucokinase - in the liver, hexokinase - in the muscles and liver), physiological significance, the Michaels constant.

If the enzyme is an oligomeric protein, then isoforms can be obtained as a result of various combinations of protomers. For example, lactate dehydrogenase consists of 4 subunits. H - subunits of the cardiac type, M - muscle. There may be 5 combinations of these subunits, and, consequently, 5 isoenzymes: HHHH (LDH 1 - in the heart muscle), HHHM (LDH 2), HHMM (LDH 3), HMMM (LDH 4), MMMM (LDH 5 - in the liver and muscles). [rice. these 4 letters in circles.

It is necessary to distinguish isoenzymes from plural forms enzymes. Multiple forms of enzymes are enzymes that are modified after their synthesis, such as phosphorylase A and B.

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All topics in this section:

Proteins and their biological role
Protein (proteins) - protos - preceding everything, primary, most important, determining everything else. Proteins are high molecular weight nitrogen-containing organic matter, consist

Characterization of simple proteins
The classification (created in 1908) is based on the solubility of proteins. On this basis, there are: I. histones and protamines, soluble in saline solutions. O

Chromoproteins
For them, the prosthetic part is colored (chromos - paint). Chromoproteins include hemoglobin, myoglobin, catalase, peroxidase, a number of flavin-containing enzymes (succinate dehydrogenase, aldehydeox

Lipid-protein complexes
Lipid-protein complexes are complex proteins, the prosthetic part of which consists of various lipid components. These components include: 1. limiting and unlimiting B

Nucleoproteins
Nucleoproteins are complex proteins containing nucleic acids as a small part (up to 65%). NPs consist of 2 parts: protein (contains histones and protamines, which

Carbohydrate-protein complexes
Carbohydrates act as a prosthetic group. All carbohydrate-protein complexes are divided into glycoproteins and proteoglycans. Glycoproteins (GP) - a complex of proteins with carbohydrate co

Phosphoproteins
Proteins where the prosthetic group is phosphoric acid. Accession phosphoric acid to the polypeptide chain comes with the formation of an ester bond with AK SEP or TPE.

The structure of coenzymes
Coenzymes in catalytic reactions transport various groups atoms, electrons or protons. Coenzymes bind to enzymes:- covalent bonds; - ionic

Enzyme Properties
Common features enzymes and non-biological catalysts: 1) both of them catalyze only energetically possible reactions; 2) increase the reaction rate; 3) n

Enzyme nomenclature
1) Exists trivial nomenclature- the names are random, without a system and base, for example, trypsin, pepsin, chymotrypsin. 2) Working nomenclature - the name of the enzyme is compiled from the name

Modern concepts of enzymatic catalysis
First theory enzymatic catalysis was put forward at the beginning of the 20th century by Warburg and Baylis. This theory proposed to consider that the enzyme adsorbs the substrate on itself, and was called adsorption, but

Molecular effects of enzyme action
1) The effect of concentration is the adsorption of molecules of reactants on the surface of an enzyme molecule, i.e. substrate, which leads to their better interaction. Ex: electrostatic attraction

Theory of acid-base catalysis
The active site of the enzyme contains both acidic and basic functional groups. As a result, the enzyme exhibits acid-base properties during catalysis; plays a role

Regulation of enzyme activity
Enzymes are regulated catalysts. Metabolites, poisons can act as regulators. Distinguish: - activators - substances that increase the reaction rate;

Digestion and absorption of proteins
The functions of proteins are diverse, but structural, catalytic and energy function. The energy value protein about 4.1 kcal/g. Among all the substances entering

The transformation of proteins in the digestive organs
All proteins are subject to the action of hydrolases (the third class of enzymes), namely peptidases - they are usually produced in an inactive form and then activated by partial proteolysis.

Digestion of complex proteins and their catabolism
1. Glycoproteins are hydrolyzed by glycosidases (amylolytic enzymes). 2. Lipoproteins - with the help of lipolytic enzymes. 3. Heme containing chromoprote

Rotting of proteins and neutralization of its products
Protein putrefaction is the bacterial breakdown of proteins and AAs under the action of the intestinal microflora. Goes in the large intestine, but can also be observed in the stomach - with a decrease in acidity

Amino acid metabolism
The body's AA fund is replenished due to the following processes: 1) hydrolysis of food proteins, 2) hydrolysis of tissue proteins (under the action of lysosome cathepsins). AK-fund is spent on the process

Common metabolic pathways
1. Transamination (discovered in 1937 by Braunstein and Kritzm).

Temporary neutralization of ammonia
Ammonia is toxic (50 mg of ammonia kills a rabbit, while = 0.4-0.7 mg / l). Therefore, in the tissues, ammonia is neutralized in temporary ways: 1) mainly - images

Ornithine urea cycle
Urea contains 80-90% of all urine nitrogen. 25-30 g of urea NH2-CO-NH2 is formed per day. 1. NH3 + CO

Synthesis and breakdown of nucleotides
Features of nucleotide exchange: 1. Neither the nucleotides themselves nor nitrogenous bases, coming from food, are not included in the synthesis nucleic acids and body nucleotides. i.e., food nucleotides

Purine nucleoside oxidation
Adenosine® (adenosine deaminase, +H2O, -NH4+) inosine® (purine nucleoside phosphorylase, + Fn -ribosyl-1-P) hypoxanthine (6-oxopurine) ® (xanthineoxy

DC functioning
Substrate H2 → NAD → FMN → CoQ → 2b → 2c1 → 2c → 2a → 2a3 → O

Replication (self-doubling, biosynthesis) of DNA
In 1953, Watson and Crick discovered the principle of complementarity (complementarity). So, A \u003d T, and GºC. Conditions required for replication: 1. page

Transcription (transfer of information from DNA to RNA) or RNA biosynthesis
Transcription, unlike replication, transfers information from a small section of DNA. elementary unit transcription is an operon (transcripton) - a section of DNA that undergoes trans

Regulation of protein biosynthesis
Cells multicellular organism contain the same set of DNA, but different proteins are synthesized. For example, connective tissue actively synthesizes collagen, but in muscle cells there is no such protein. AT

Mechanisms of development of a cancerous tumor
Cancer - genetic disease, i.e. gene damage. Types of gene damage: 1) gene loss, 2) gene damage itself, 3) gene activation,

Lipid digestion
When taken with food, lipids in oral cavity are only mechanically processed. Lipolytic enzymes are not formed in the oral cavity. Lipid digestion will take place in those departments

Mechanism of fat resynthesis
The resynthesis of fat in the intestinal wall occurs as follows: 1. first, the products of hydrolysis (glycerol, HFA) are activated with using ATP. Next comes the sequential acylation

Transport forms of lipids in the body
Lipids are water-insoluble compounds, so special water-soluble carriers are needed for their transport in the blood. Such transport forms are plasma lipoproteins

The transformation of lipids in tissues
In tissues, the processes of disintegration and synthesis of lipids are constantly going on. The bulk of the lipids of the human body are TG, which are present in the cell in the form of inclusions. TG renewal period in different tissues

Biosynthesis of glycerol and fatty acids in tissues
The biosynthesis of glycerol in tissues is closely related to the metabolism of glucose, which, as a result of catabolism, goes through the stages of triosis formation. Glyceraldehyde-3-phosphate in the cytoplasm

Pathology of lipid metabolism
At the stage of intake with food. Abundant fatty foods against the background of physical inactivity leads to the development of alimentary obesity. Metabolic disorders may be associated with insufficient intake of fat

Ca2+ ions
They form a compound with a protein - calmodulin. The Ca2+-calmodulin complex activates enzymes (adenylate cyclase, phosphodiesterase, Ca2+-dependent protein kinase). There is a group

Parathyroid hormones
Parathormone, consists of 84 AA, regulates the level of Ca2 +, stimulates the release of calcium (and phosphorus) from the bones into the blood; Increase the reabsorption of calcium in the kidneys, but stimulate the release of phosphorus; With

The role of vitamins in metabolism
1.(!) vitamins are precursors of coenzymes and prosthetic groups of enzymes. For example, B1 - thiamine - is part of the coenzyme of ketoacid decarboxylases in the form of TPP (TDF), B2 - riboflavin -

The concept of hypovitaminosis, avitaminosis and hypervitaminosis
Hypovitaminosis is a pathological condition associated with a lack of a vitamin in the body. Avitaminosis is a pathological condition caused by the lack of a vitamin in the body.

Causes of hypovitaminosis
1. Primary: lack of vitamin in food. 2. Secondary: a) loss of appetite; b) increased consumption of vitamins; c) malabsorption and disposal disorders, e.g., entero

Vitamin A
Vitamers: A1 - retinol and A2 - retinal. Clinical name: antixerophthalmic vitamin. By chemical nature: cyclic unlimited monohydric alcohol based on ring b-

Vitamin D
Antirachitic vitamin. There are two vitamers: D2 - ergocalciferol and D3 - cholecalciferol. Vitamin D2 is found in mushrooms. Vitamin D3 is synthesized in the body

Vitamin E
Obsolete: anti-sterile vitamin, antioxidant enzyme. AT chemical terms these are alpha, beta, gamma and delta tocopherols, but alpha tocopherol is predominant. Vitamin E stable

Vitamin K
Antihemorrhagic vitamin. Vitamers: K1 - phylloquinone and K2 - menaquinone. The role of vitamin K in metabolism It is a cofactor for carboxylation of glutamino

Vitamin C
Ascorbic acid, anti-scurvy vitamin (scorbut = scurvy). It is a lactone. Easily oxidized: O=C─┐ O=C─┐ | │ | │ NO-S

Vitamin B1
Thiamine, an anti-neuritic vitamin. Thiamine is stable in acidic environment(up to 140ºС), and in an alkaline environment would

Vitamin B2
Riboflavin is stable in an acidic environment, but is destroyed in a neutral and alkaline environment. Easily oxidized by two

Vitamin PP
Antipellagric vitamin. Vitamers: nicotinic acid, nicotinamide, niacin.

Vitamin B6
Antidermatitis vitamin. Pyridoxine → pyridoxal → pyridoxamine [draw formulas]

Vitamin B12
Cobalamin. Antianemic vitamin. Has a red color. Decomposes in the world. The role of cobalamin in metabolism - transport of methyl groups; - participates in

Vitamin B3
pantothenic acid. [rice. formulas HOCH2-C((CH3)2)-CH(OH)-CO-NH-CH2-CH2-COOH] Consists of butyric acid with b-alanine.

Hydroxylation of xenobiotics with the participation of the microsomal monooxygenase system
1. benzene: [fig. benzene + O2 + NADPH2® (hydroxylase, cytochrome P450) phenol + NADP + H2O] 2. indole: [Fig. indole + O2 + H

The role of the liver in pigment metabolism
Pigment metabolism is a set of complex interconversions of colored substances in tissues and fluids of the human body. Pigments include 4 groups of substances: 1. heme

Heme biosynthesis
Heme biosynthesis occurs in most tissues, with the exception of erythrocytes, which do not have mitochondria. In the human body, heme is synthesized from glycine and succinyl-CoA formed as a result of

Heme breakdown
Most of gemchromogenic pigments in the human body is formed during the breakdown of heme. The main source of heme is hemoglobin. In erythrocytes, the hemoglobin content is 80%, the lifetime

Pathology of pigment metabolism
As a rule, it is associated with a violation of the processes of heme catabolism and is expressed by hyperbilirubinemia and manifests itself in jaundice of the skin and visible mucous membranes. Accumulating in the CNS, bilirubin causes

Types of changes in the biochemical composition of blood
I. Absolute and relative. Absolute ones are due to a violation of the synthesis, decay, excretion of a particular compound. Relative ones are due to a change in the volume of c

The protein composition of the blood
Functions of blood proteins: 1. support oncotic pressure (mainly due to albumins); 2. determine the viscosity of blood plasma (mainly due to albumin);

total protein
Normally, total blood protein is 65-85 g/l. Total protein is the sum of all proteins in the blood. Hypoproteinemia - a decrease in albumin. Causes:

Globulins are normal 20-30 g/l
I. α1-globulins α-antitrypsin - inhibits trypsin, pepsin, elastase, and some other blood proteases. Performs anti-inflammatory

Residual nitrogen
Residual nitrogen is the sum of the nitrogen of all non-protein nitrogen-containing substances in the blood. Normally 14-28 mmol / l. 1. Metabolites: 1.1. amino acids (25%); 1.2. creat

carbohydrate metabolism
Glucose in capillary blood on an empty stomach 3.3-5.5 mmol / l. 1. Hyperglycemia (increased glucose): 1.1. pancreatic hyperglycemia - in the absence of insulin

lipid metabolism
Cholesterol is normal 3-5.2 mmol / l. In plasma, it is part of LDL, VLDL (atherogenic fractions) and HDL (anti-atherogenic fraction). The likelihood of developing atherosclerosis

Mineral exchange
Sodium is the main extracellular ion. The level of Na + in the blood is influenced by mineralocorticoids (aldosterone retains sodium in the kidneys). Sodium levels are increased by heme

Plasma enzymes
Classified: 1. Functioning enzymes (actual plasma). For example, renin (increases blood pressure through angiotensin II), cholinesterase (breaks down acetylcholine). Their activity is higher in

Physical properties of the urine of a healthy person, their changes in pathology
I. The amount of urine is normally 1.2-1.5 liters. Polyuria - an increase in the amount of urine due to: 1) an increase in filtration (under the action of adrenaline, fi

Indicators of the chemical composition of urine
Total nitrogen is the total nitrogen of all nitrogen-containing substances in the urine. Normal - 10-16 g / day. With pathologies total nitrogen may: ü increase - hyperazoturia

Features of metabolism in the nervous tissue
Energy exchange. Increased in brain tissue cellular respiration(aerobic processes predominate). The brain consumes more oxygen than a constantly working brain.

Chemical transmission of nervous excitation
The transfer of excitation from one cell to another occurs with the help of neurotransmitters: - neuropeptides; - AK; - acetylcholine; - biogenic amines (adrenaline,

Warburg found that yeast aldolases from various animal tissues differ in a number of properties. Pepsin, trypsin, chymotrypsin also differed in solubility, pH, temperature optimum.

In the late fifties, the biochemists Wieland and Pfleiderer, as well as other researchers, isolated pure crystalline preparations of the enzyme from animal tissues. lactate dehydrogenase and subjected to electrophoresis. As a result of electrophoresis, the enzyme was divided, as a rule, into 5 factions having different electrophoretic mobility. All these fractions had lactate dehydrogenase activity. Thus, it was found that the enzyme lactate dehydrogenase is present in tissues in several forms. These forms, in accordance with their electrophoretic mobility, were designated LDH1, LDH2, and LDH3. LDG4, LDG5. (LDH - short for lactate dehydrogenase), with the number 1 designating the component with the highest electrophoretic mobility.

Studies of lactate dehydrogenase ioenzymes isolated from various organs animals showed that they differ both in electrophoretic and chromatographic properties, and in chemical composition, thermal stability, sensitivity to the action of inhibitors, K m and other properties. When analyzing lactate dehydrogenase from different animal species, very large interspecies differences were revealed, however, within a given species, the distribution of isoenzymes is characterized by great constancy.

Lactate dehydrogenase was the first enzyme whose individual components were subjected to detailed study. Somewhat later, data were obtained on the multiple forms and molecular heterogeneity of a number of other fermeates, and in 1959 it was proposed to call such forms isoenzymes or isoenzymes. The Enzyme Commission of the International Biochemical Union has officially recommended this term to refer to multiple forms of enzymes from the same biological species.

So, isoenzymes - is a group of enzymes from the same source, with the same type of substrate specificity, catalyzing the same chemical reaction, but differing in a number of physicochemical properties.

The presence of multiple forms of enzymes, or isoenzymes, has been established by more than for100 enzymes, isolated from various kinds animals, plants and microorganisms. Isoenzymes do not always consist of two or more subunits. In a number of enzymes, individual isoenzymes are different in chemical structure proteins that have the same catalytic activity but consist of only one subunit.

Currently, the main criterion for the nomenclature of isoenzymes is their electrophoretic mobility. This is explained by the fact that, compared with other methods of characterizing enzymes, electrophoresis gives the highest resolution.

To date, as a result of the study of plant isoenzymes, it has been established that many enzymes are present in plants in the form of multiple forms. Let's take a look at some of these enzymes.

Malate dehydrogenase (1.1.1.37) has a rather complex isoenzyme composition. In cotton seeds and spinach leaves, 4 malate dehydrogenase isoenzymes were found, differing in electrophoretic mobility, and the molecular weight of each of the four spinach isoenzymes was approximately 60 thousand. Different plants contain an unequal number of malate dehydrogenase isoenzymes. For example, 7–10 isoenzymes were found in the seeds of different wheat varieties, 4–5 in corn roots, and 9–12 malate dehydrogenase isoenzymes were found in various organs of the mountain (root, cotyledons, subcotyledonous and supracotyledonous knee), and the number of isoenzymes varied depending on from the phase of plant development.

It was noted that the molecular weights of isoenzyme malate dehydrogenase sometimes differed significantly. For example, cotton leaves contain 7 isoenzymes of malate dehydrogenase, of which 4 isoenzymes are isoforms with different electrical charges, but the same molecular weight, equal to approximately 60 thousand. The fifth isoenzyme had a molecular weight of about 500 thousand and was an oligomer according to at least one of the isoforms of malate dehydrogenase with a molecular weight of 60 thousand. Since in these studies the molecular weights were determined approximately, it can be assumed that this isoenzyme consists of 8 subunits of the isoenzyme with a molecular weight of 60 thousand.

The resistance and susceptibility of plants to diseases is often associated with the regulation of the synthesis of isoenzymes. As a response to the introduction of infection in plants, the intensity of the exchange of centuries, primarily redox, is increased. Therefore, the activity of OB enzymes and the number of their isoenzymes increase when plants are damaged.

An increase in activity and an increase in the number of peroxidase and o-diphenol oxidase isoenzymes are observed in various diseases of corn, beans, tobacco, clover, potatoes, flax, oats and other plants. Figure 22 schematically shows the change in the number of peroxidase isoenzymes and their activity when tomatoes are damaged by phytophthora. If the leaves of healthy plants contained four isoenzymes of peroxidase, then in the affected leaves their number increased to nine, and the activity of all isoenzymes increased significantly.

When studying changes in the isoenzyme composition of mitochondrial peroxidase and polyphenol oxidase during viral pathogenesis of tobacco species resistant and resistant to tobacco mosaic virus, it was found that a viral infection causes qualitatively different changes in the isoenzyme composition of tobacco species of different resistance. In a resistant species, the activity of a number of isoenzymes increases to a greater extent than in a susceptible one. Thus, depending on the potential ability of the plant to biosynthesis of isoenzymes, the susceptibility of the plant to infectious diseases changes.

Glutamate dehydrogenase

Esterases

Sucrase

The biological role of isoenzymes in plants.

IF testify to the great lability of the enzymatic apparatus of plants, makes it possible to carry out the necessary metabolic processes in the centuries. in the cell when the environmental conditions change, provides the specifics of the exchange of centuries. for a given plant organ or tissue. Promotes the adaptability of plants to changing conditions. environment.

The simultaneous presence in cells of multiple forms of the same enzyme, along with other regulatory mechanisms, contributes to the consistency of metabolic processes in the centuries. in the cell and rapid adaptation of plants to changing environmental conditions.

Indeed, we have noted that individual isoenzymes differ in temperature optima, pH optima, attitudes toward inhibitors, and other properties. Hence it follows that if, for example, the temperature conditions change sharply, which become unfavorable for the manifestation of the catalytic activity of some isoenzymes, then their activity is suppressed. However, this enzymatic process in plants does not stop completely, since other isoenzymes of the same enzyme, for which this temperature is favorable, begin to exhibit catalytic activity. If, for any reason, the pH of the reaction medium changes, then the activity of some isoenzymes also weakens, but instead of them, isoenzymes with a different pH optimum begin to show catalytic activity. High salt concentrations inhibit the activity of many enzymes, which is one of the reasons for the deterioration of plant growth on saline soils. However, even at high salt concentrations in cells, enzymatic processes do not stop completely, since individual isoenzymes are not equally related to an increase in salt concentration: the activity of some isoenzymes decreases, while others increase ..

Resistance and susceptibility to disease is often based on the regulation of IF synthesis.

The biosynthesis of isoenzymes is determined by genetic factors, and each plant species is characterized by a specific set of isoenzymes for this species, i.e. species specificity is manifested in isoenzyme composition.

Different organs of the same plant differ in IF. The study of the properties of lactate dehydrogenase isoenzymes isolated from various animal tissues showed that all isoenzymes have approximately the same molecular weight (about 140 thousand) under conditions, for example, under the action of treatment with 42 M urea, each of the isoenzymes dissociates into 4 subunits with with a molecular weight of about 35 thousand. Thus, each of the five isoenzymes of lactate degttdrogenase is a tetramer. It has been established that all lactate dehydrogenase isoenzymes are possible combinations of only two types of subunits, conventionally denoted by the letters A and B. Different combinations of these types of subunits form all five lactate dehydrogenase isoenzymes (Fig. 18). This shows that lactate dehydrogenase isoenzymes have a strictly ordered structure, and individual subunits in the molecule of this enzyme protein are connected. hydrogen bonds, which can be broken by the action of a concentrated solution of urea.

The question arises, how do the individual subunits of lactate dehydrogease differ from each other and what is the reason for the different electrophoretic mobility of individual isoenzymes? This question has now received quite definite answers. It turned out that subunits A and B are t-c amino acids. Subunit B contains more acidic small amino acids compared to subunit A. In this regard, all isoenzymes of lactate dehydrogenase (LDH1 - LDH2) differ in the number of these amino acids, their molecules have different sizes electric charge and different electrophoretic mobility. Lactate dehydrogeaase isoenzymes also differ in a number of other properties, in particular, Michaelis constants Km, relation to a number of inhibitors, and thermal stability.

At the heart of many pathological and prepathological conditions of the body are violations of the functioning of enzyme systems. Many enzymes are localized inside cells, and therefore their activity in the blood serum (plasma) is low or absent altogether. That is why, by analyzing extracellular fluids (blood), by the activity of certain enzymes, it is possible to identify changes occurring inside the cells. various bodies and body tissues. other enzymes are constantly contained in the blood, in known quantities and have certain function(for example, enzymes of the blood coagulation system).

The activity of enzymes in the blood serum reflects the balance of the rate of enzyme synthesis inside the cells and their release from the cells. An increase in the activity of blood enzymes may be the result of an acceleration of synthesis processes, a decrease in the rate of excretion, an increase in permeability cell membranes, the action of activators, cell necrosis. A decrease in enzyme activity is caused by an increase in the rate of enzyme excretion, the action of inhibitors, and inhibition of synthesis.

An increase in the activity in the blood of one or another enzyme is a very early diagnostic test. An additional definition of the isoenzyme spectrum allows you to clarify the localization of the pathological process, since each organ has its own specific isoenzyme spectrum.

In clinical biochemistry great importance has an indicator of activity of aspartate aminotraisferase and alanine aminotransferase. These transaminases are found in mitochondria and in the soluble fraction of the cytoplasm of cells. The role of transaminases is reduced to the transfer of amino groups of amino acids to keto acid. The coenzyme of transaminases is pyridoxal phosphate, a derivative of vitamin B6. In the blood of animals, the activity of both enzymes is very low compared to their activity in other tissues. However, in pathologies accompanied by cell destruction, transaminases exit through cell membranes into the blood, where their activity is significantly increased compared to the norm. Despite the lack of strict organ specificity of these enzymes, an increase in their activity is observed in hepatitis, muscular dystrophy, trauma, and excessive physical activity on the body, in particular, in sports horses.

Lactate dehydrogenase (LDH), a glycolytic enzyme that catalyzes reversible reaction reduction of pyruvic acid to lactic acid. LdG consists of four subunits and includes five isoenzymes. Moreover, the LdG5 isoenzyme predominates in muscle tissue, LdG1 and LdG2 in the heart muscle. In acute myocardial infarction in patients in the blood serum, the activity of LDH1 and LDH2 isoenzymes increases. In parenchymal hepatitis, the activity of the LdG4 and LdG5 isoenzymes significantly increases in the blood serum, while the activity of LdG1 and LdG2 decreases. The activity of LdG in whole blood is significantly higher than the activity of the enzyme in blood plasma. Therefore, even minimal blood hemolysis significantly changes the activity of the enzyme in plasma, which should be taken into account in laboratory work.

Creatine phosphokinase (CPK), important role play in energy exchange. Creatine phosphokinase is required for ATP resynthesis by transphosphorylation of AdP with creatine phosphate. Creatine phosphate refers to energy-rich phosphate compounds that provide muscle fiber contraction, relaxation, and transport of metabolites into muscle tissue.

Creatine-P + AdP CPK > Creatine + ATP.

Creatine phosphokinase consists of two subunits - M and B, forming three isoenzymes: MM (muscle type), MB (heart type), BB (brain type).

Tissue analysis shows that significant activity CPK occurs in skeletal muscle, myocardium, and brain. The heart muscle contains mainly the MM and MB isoenzymes. An increase in the activity of the MB isoenzyme in the patient's blood serum indicates damage to the heart muscle. The definition of CPK isoenzymes is best method diagnosis of hereditary muscular dystrophy in chickens, with a lack of selenium in cattle, with paralytic myoglobinuria in horses.

Alkaline phosphatase (AP) is a hydrolytic enzyme synthesized mainly in the liver and excreted from the body as part of bile. Its activity optimum is at pH = 8-9. It is a non-specific enzyme that catalyzes the hydrolysis of many phosphate esters and is present in plasma in the form of isoenzymes. The main source of alkaline phosphatase in young growing animals is bone tissue. The activity of alkaline phosphatase is significantly increased in diseases of the liver and bones, in particular, in osteomalacia. The main role of alkaline phosphatase is probably associated with the deposition of calcium phosphates in bone tissue. An increase in the activity of alkaline phosphatase in the blood serum in bone neoplasms was established.

Cholinesterase - an enzyme involved in the transmission process nerve impulse, hydrolysis of acetylcholine to acetate and choline. Serum cholinesterase includes two types of body cholinesterases, the main substrate of which is acetylcholine. Acetylcholinesterase (AChE), which hydrolyzes acetylcholine in synapses, is called true acetylcholinesterase. It is present in the liver, erythrocytes, and only a small amount is localized in the plasma. Plasma cholinesterase is a pseudocholinesterase, it hydrolyzes butyrylcholine 4 times faster than acetylcholine. This enzyme is also found in the liver, pancreas, and intestinal mucosa. The synthesis of AChE in blood serum occurs in the liver, and therefore, in the pathology of this organ, a decrease in the activity of the enzyme is observed.

Irreversible inhibitors of AChE are toxic organophosphorus compounds (OPs). Thus, FOS insecticides (chlorophos, phosphamide, karbofos, octamethyl) selectively bind the active centers of the AChE molecule and thereby block its activity. Due to the high lipotropy of FOS, they are able to penetrate into the body of an animal through intact skin and mucous membranes. In case of FOS poisoning, the animal's anxiety, a feeling of fear, agitation, convulsions, which develop against the background of asthma attacks and coughing due to bronchospasm, are noted. In this case, changes in the eyes are characteristic: the pupil narrows sharply, lacrimation begins, and accommodation is disturbed. Often direct cause The death of an animal poisoned with FOS is paralysis of the respiratory center.

Amylase is produced salivary glands and in large quantities pancreas. Amylase has a specific effect on the c-1,4-glucosidic bonds of polysaccharides. An increase in serum amylase activity indicates the development of acute pancreatitis. A moderate increase in enzyme activity is noted with inflammation of the salivary glands.

When we say “malate dehydrogenase” or “glucose-6-phosphatase”, we usually mean a specific protein with formative activity, but in reality these names cover all proteins that catalyze the oxidation of malate to oxaloacetate or the hydrolysis of glucose-6-phosphate with the formation glucose and. In particular, after isolation of malate dehydrogenase from various sources(rat liver, E. coli) it was found that enzymes from the liver and an enzyme from E. coli, catalyzing the same reaction, differ in many respects in their physical and chemical properties. Physically distinguishable forms of enzymes with the same type of catalytic activity may be present in different tissues of the same organism, in different types cells of one tissue and even in a prokaryotic organism, for example, in E. coli. This discovery was made through the use of electrophoretic methods for separating proteins, as a result of which electrophoretic different forms certain enzymatic activity.

The term "isoenzyme" ("isozyme") encompasses all of the above physically distinguishable proteins with a given catalytic activity, but in practice, and especially in clinical medicine, it is used in more narrow sense, meaning the physically distinguishable and separable forms of the enzyme present in various types cells of a given eukaryotic organism, such as a human. Isozymes are invariably found in the serum and tissues of all vertebrates, insects, and unicellular organisms. The number of enzymes and their content vary greatly. Isozyme forms of dehydrogenases, oxidases, transaminases, phosphatases, transphosphorylases, and proteolytic enzymes are known. Different tissues may contain different isoenzymes, and these isoenzymes may have different affinities for substrates.

Diagnostic value of isozymes

Medical interest in isozymes arose after it was discovered that human serum contains several lactate dehydrogenase isozymes and that their relative content varies significantly under certain pathological conditions. Subsequently, many other cases of changes in the relative content of isozymes in various diseases were identified.

Serum lactate dehydrogenase isozymes are detected after electrophoresis on starch, agar, or polyacrylamide gels. At the indicated value, the isozymes carry a different charge and are distributed on the electrophoregram in five different places. Further, isozymes can be detected by their ability to catalyze the reduction of colorless dyes to an insoluble colored form.

A typical set of reagents for the detection of dehydrogenase isozymes includes:

1) reduced substrate (for example, lactate);

2) coenzyme;

3) dye in oxidized form (for example, blue nitrotetrazolium salt);

4) an electron carrier from NADH to the dye [eg phenazine methasulfate (PMS)];

5) buffer; activating ions (if required).

Lactate dehydrogenase catalyzes the transfer of two electrons and one ion from lactate to

Rice. 7.8. Reaction catalyzed by α-lactate dehydrogenase.

(Fig. 7.8). If the electrophoregram is sprayed with the above mixture and then incubated, then the coupled electron transfer reaction will proceed only in those places where lactate dehydrogenase is present (Fig. 7.9). Relative density banding colors can be further quantified using a scanning photometer (Figure 7.10). Isozymes with the highest negative charge denote .

The physical nature of isozymes

Oligomeric enzymes formed by different protomers can be represented in several forms. Often, a particular tissue produces predominantly one of the protomers. If an active oligomeric enzyme (for example, a tetramer) can be built from such protomers in various combinations, then isozymes are formed.

Lactate dehydrogenase isozymes differ at the level quaternary structure. The oligomeric lactate dehydrogenase molecule (molecular weight 130,000) consists of four protomers of two types, H and M (both with a molecular weight of about 34,000). Only the tetrameric molecule has catalytic activity.

Rice. 7.9. Localization of lactate dehydrogenase on electrophoregrams using a system of coupled reactions.

If the order in which the protomers are connected does not matter, then the protomers can be arranged in five ways:

Markert chose the conditions for the destruction and reconstruction of the quaternary structure and was able to elucidate the relationship between lactate dehydrogenase isozymes. Cleavage and reconstruction of lactate dehydrogenases I and 15 do not lead to the formation of new isozymes. Therefore, these two isozymes contain only one type of protomer. When a mixture of lactate dehydrogenases 1 and 15 was subjected to the same procedure, forms 12, 13 and 14 also appeared. The ratio of isozymes corresponds to the following subunit composition:

The synthesis of the H and M subunits is determined by different genetic loci, and they are expressed differently in different tissues (for example, in cardiac and skeletal muscles).