Isoenzymes origin biological role methods of determination. Introduction, brief history of biochemistry
Enzymes that catalyze the same chemical reaction, but differ in the primary structure of the protein, are called isoenzymes, or isoenzymes. They catalyze the same type of reaction with a fundamentally identical mechanism, but differ from each other in kinetic parameters, activation conditions, and features of the connection between the apoenzyme and the coenzyme.
The nature of the appearance of isoenzymes is varied, but most often due to differences in the structure of the genes encoding these isoenzymes. Consequently, isoenzymes differ in the primary structure of the protein molecule and, accordingly, in physical chemical properties. Methods for determining isoenzymes are based on differences in physicochemical properties.
In their structure, isoenzymes are mainly oligomeric proteins. Moreover, one or another tissue predominantly synthesizes certain types protomers. As a result of a certain combination of these protomers, enzymes with different structures are formed - isomeric forms. The detection of certain isoenzyme forms of enzymes allows their use for diagnosing diseases.
Isoforms of lactate dehydrogenase. The enzyme lactate dehydrogenase (LDH) catalyzes the reversible oxidation of lactate (lactic acid) to pyruvate (pyruvic acid) (see section 7).
Lactate dehydrogenase- oligomeric protein with a molecular weight of 134,000 D, consisting of 4 subunits of 2 types: M (from English, muscle - muscle) and H (from English, heart - heart). The combination of these subunits underlies the formation of 5 isoforms of lactate dehydrogenase (Fig. 2-35, A). LDH 1 and LDH 2 are most active in the heart muscle and kidneys, LDH4 and LDH5 - in skeletal muscles and liver. Other tissues contain various forms of this enzyme.
LDH isoforms differ in electrophoretic mobility, which makes it possible to determine the tissue identity of LDH isoforms (Fig. 2-35, B).
Creatine kinase isoforms. Creatine kinase (CK) catalyzes the formation of creatine phosphate:
The KK molecule is a dimer consisting of two types of subunits: M (from English, muscle) and B (from English, brain). From these subunits, 3 isoenzymes are formed - BB, MB, MM. The BB isoenzyme is found primarily in the brain, MM in skeletal muscles, and MB in cardiac muscle. KK isoforms have different electrophoretic mobilities (Fig. 2-36).
CK activity should normally not exceed 90 IU/l. Determination of CK activity in blood plasma has diagnostic value in case of myocardial infarction (there is an increase in the level of the MB isoform). The amount of the MM isoform may increase during trauma and damage to skeletal muscles. The BB isoform cannot penetrate the blood-brain barrier, therefore it is practically undetectable in the blood even during strokes and has no diagnostic value.
Isoenzymes- these are enzymes, the synthesis of which is encoded by different genes, they have different primary structures and different properties, but they catalyze the same reaction. Types of isoenzymes:
Organ - glycolysis enzymes in the liver and muscles.
Cellular - cytoplasmic and mitochondrial malate dehydrogenase (the enzymes are different, but they catalyze the same reaction).
Hybrid - enzymes with a quaternary structure, formed as a result of non-covalent binding of individual subunits (lactate dehydrogenase - 4 subunits of 2 types).
Mutant - formed as a result of a single gene mutation.
Alloenzymes are encoded by different alleles of the same gene.
10. I. Use of enzymes with therapeutic purpose in turn, is divided into two types: 1) application for replacement therapy and 2) in order to influence the enzyme on the site of the disease.
With the aim of replacement therapy most widely used digestive enzymes, when the patient is found to be deficient. Examples include gastric juice preparations or pure pepsin or acidin-pepsin, which is indispensable for gastritis with secretory insufficiency and dyspepsia in children. Pancreatin - the drug, which is a mixture of pancreatic enzymes, is used for pancreatitis, mainly of a chronic nature. Well-known drugs have the same meaning Cholenzym, panzinorm, etc.
Another area of application of replacement therapy is the treatment of diseases associated with the so-called enzymopathies. These are congenital or hereditary diseases in which the synthesis of any enzymes is impaired. These diseases are usually extremely severe; children with a hereditary lack of any enzyme do not live long, suffer from severe mental and mental disorders, physical and mental retardation. mental development. Replacement therapy can sometimes help overcome these disorders.
A number of enzyme preparations are used in surgical practice for cleaning the wound surface from pus, microbes, excess granulation tissue; in the clinic of internal medicine they are used: to liquefy viscous secretions, exudates, blood clots, for example, in severe inflammatory diseases of the lungs and bronchi. These are mainly enzymes - hydrolases, capable of breaking down natural biopolymers - proteins, NA, polysaccharides. Due to their anti-inflammatory effect, they are also used for thrombophlebitis, inflammatory-dystrophic forms steam O dontosis, osteomyelitis, sinusitis, otitis and other inflammatory diseases.
Among them are enzymes such as trypsin, chymotrypsin, RNA-ase, DNA-ase, fibrinolysin. Fibirinolysin also used to remove intravascular thrombi. RNase and DNase are successfully used to treat some viral infections, for example to destroy herpes virus.
Enzymes such as hyaluronidase, collagenase, lidase, are used to combat excess scar formations.
Asparaginase- an enzyme produced by some strains of Escherichia coli. It has a therapeutic effect on some forms of tumors. The therapeutic effect is associated with the ability of the enzyme to disrupt the metabolism of the amino acid asparagine, which is necessary for tumor cells to grow.
The use of enzyme preparations for medicinal purposes is still a very young area of medical science. The limitation here is the complexity of the technology and the high cost of obtaining pure enzyme preparations in crystalline form, suitable for storage and use in humans. In addition, when using enzyme preparations, other circumstances must also be taken into account:
1) Enzymes are proteins, and therefore in some cases can cause an unwanted allergic reaction.
2) Rapid decomposition of the introduced enzymes (the protein drug is therefore immediately captured by “scavenger” cells - macrophages, fibroblasts, etc. Hence, large concentrations of drugs are required to achieve the desired effect.
3) However, with increasing concentrations, enzyme preparations may turn out to be toxic.
And yet, in cases where these obstacles can be overcome, enzyme preparations have an excellent therapeutic effect.
For example, these disadvantages are partially eliminated by converting enzymes into the so-called “immobilized” form.
You will read more about methods of enzyme immobilization and methods of their use in your teaching aids.
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 to form glucose and . In particular, after isolating malate dehydrogenase from different sources (rat liver, E. coli), it was discovered that the enzymes from the liver and the enzyme from E. coli, which catalyze the same reaction, differ in many respects in their physical and chemical properties. Physically distinct forms of enzymes possessing the same type of catalytic activity may be present in different tissues of the same organism, in different types cells of the same tissue and even in a prokaryotic organism, such as E. coli. This discovery was made through the use of electrophoretic methods for separating proteins, resulting in the discovery of electrophoretic different shapes certain enzymatic activity.
The term "isoenzyme" ("isozyme") covers all of the above-mentioned physically distinguishable proteins with a given catalytic activity, but in practice, and especially in clinical medicine, it is used in a narrower sense, meaning physically distinguishable and separable forms of a given 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 single-celled organisms. However, the number of enzymes and their content vary greatly. Isoenzyme 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 the discovery that human serum contains several lactate dehydrogenase isozymes and that their relative abundance varies significantly under certain pathological conditions. Subsequently, many other cases of changes in the relative content of isozymes in various diseases were identified.
Isozymes of serum lactate dehydrogenase are detected after electrophoresis on starch, agar or polyacrylamide gels. At the indicated value, isozymes carry different charges and are distributed on the electropherogram in five different places. Further, isozymes can be discovered due to their ability to catalyze the reduction of colorless dyes into 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 transporter from NADH to the dye [for example, phenazine metasulfate (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 β-lactate dehydrogenase.
(Fig. 7.8). If the electropherogram is sprayed with the above mixture and then incubated, the coupled electron transfer reaction will occur only in those places where lactate dehydrogenase is present (Fig. 7.9). Relative density The stripe colors can be further quantified using a scanning photometer (Figure 7.10). The isozyme with the greatest negative charge is designated .
Physical nature of isozymes
Oligomeric enzymes, formed by different protomers, can be presented 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 of 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 coupled reaction system.
If the order of the protomers is not important, then the protomers can be arranged in five ways:
Markert selected the conditions for the destruction and reconstruction of the quaternary structure and was able to clarify the relationships between the isozymes of lactate dehydrogenase. 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 subunit composition given below:
The synthesis of H and M subunits is determined by different genetic loci, and they are expressed differently in different tissues (for example, in cardiac and skeletal muscle).
Isoenzymes- these are multiple forms of one enzyme that catalyze the same reaction, but differ in physical and chemical properties (affinity for the substrate, maximum speed of the catalyzed reaction, electrophoretic mobility, different sensitivity to inhibitors and activators, pH optimum and thermal stability). Isoenzymes have a quaternary structure, which is formed by an even number of subunits (2, 4, 6, etc.). Enzyme isoforms are formed by different combinations of subunits.
As an example, consider lactate dehydrogenase (LDH), an enzyme that catalyzes a reversible reaction:
NADH 2 NAD +
pyruvate ← LDH → lactate
LDH exists in the form of 5 isoforms, each of which consists of 4 protomers (subunits) of 2 types M (muscle) and H (heart). The synthesis of M and H type protomers is encoded by two different genetic loci. LDH isoenzymes differ at the level of quaternary structure: LDH 1 (NNNN), LDH 2 (NNMM), LDH 3 (NNMM), LDH 4 (NMMM), LDH 5 (MMMM).
Polypeptide chains of the H and M types have the same molecular weight, but the former are dominated by carboxylic amino acids, the latter by diamino acids, so they carry different charges and can be separated by electrophoresis.
Oxygen metabolism in tissues affects the isoenzyme composition of LDH. Where aerobic metabolism dominates, LDH 1, LDH 2 predominate (myocardium, adrenal glands), where anaerobic metabolism - LDH 4, LDH 5 (skeletal muscles, liver). During the individual development of the organism, changes in oxygen content and LDH isoforms occur in tissues. In the embryo, LDH 4 and LDH 5 predominate. After birth, the content of LDH 1 and LDH 2 increases in some tissues.
The existence of isoforms increases the adaptive capacity of tissues, organs, and the body as a whole to changing conditions. The metabolic state of organs and tissues is assessed by changes in isoenzyme composition.
Creatine kinase isoforms. Creatine kinase (CK) catalyzes the formation of creatine phosphate:
The KK molecule is a dimer consisting of two types of subunits: M (from English, muscle- muscle) and B (from English, brain - brain). From these subunits, 3 isoenzymes are formed - BB, MB, MM. The BB isoenzyme is found primarily in the brain, MM in skeletal muscles, and MB in cardiac muscle. KK isoforms have different electrophoretic mobilities (Fig. 2-36).
CK activity should normally not exceed 90 IU/l. Determination of CK activity in blood plasma has diagnostic value in case of myocardial infarction (there is an increase in the level of the MB isoform). The amount of the MM isoform may increase during trauma and damage to skeletal muscles. The BB isoform cannot penetrate the blood-brain barrier, therefore it is practically undetectable in the blood even during strokes and has no diagnostic value.
6. Localization and compartmentalization of enzymes in cells and tissues: general-purpose enzymes, organ-specific and organelle-specific (marker) enzymes.
Enzymes are divided into 3 groups based on localization:
I – general enzymes (universal)
II- organ-specific
III-organelle-specific
Common enzymes found in almost all cells, they ensure the vital activity of the cell by catalyzing the reactions of protein and nucleic acid biosynthesis, the formation of biomembranes and main cellular organelles, and energy exchange. Common enzymes of different tissues and organs, however, differ in activity.
Organ-specific enzymes characteristic only of a specific organ or tissue. For example: For the liver - arginase. For kidneys and bone tissue - alkaline phosphatase. For the prostate gland – AF (acid phosphatase). For the pancreas – α-amylase, lipase. For the myocardium – CPK (creatine phosphokinase), LDH, AST, etc.
Enzymes are also unevenly distributed inside cells. Some enzymes are in a colloidal dissolved state in the cytosol, others are embedded in cellular organelles (structured state).
Organelle-specific enzymes . Different organelles have a specific set of enzymes, which determines their functions.
Organelle-specific enzymes are markers of intracellular formations, organelles:
Cell membrane: ALP (alkaline phosphatase), AC (adenylate cyclase), K-Na-ATPase
Cytoplasm: enzymes of glycolysis, pentose cycle.
ER: enzymes providing hydroxylation (microsomal oxidation).
Ribosomes: enzymes that provide protein synthesis.
Mitochondria: enzymes of oxidative phosphorylation, TCA cycle (cytochrome oxidase, succinate dehydrogenase), β-oxidation of fatty acids.
Cell nucleus: enzymes ensuring the synthesis of RNA, DNA (RNA polymerase, NAD synthetase).
Nucleolus: DNA-dependent RNA polymerase
As a result, compartments are formed in the cell, which differ in the set of enzymes and metabolism (compartmentalization of metabolism).
Among the enzymes there is a small group R regulatory enzymes, which are capable of responding to specific regulatory influences by changing activity. These enzymes are present in all organs and tissues and are localized at the beginning or at the branching points of metabolic pathways.
The strict localization of all enzymes is encoded in genes.
Determination of the activity of organelle-specific enzymes in plasma or serum is widely used in clinical diagnostics.
Enzymes that catalyze the same chemical reaction, but differing in the primary structure of the protein, are called isoenzymes, or isoenzymes. They catalyze the same type of reaction with a fundamentally identical mechanism, but differ from each other in kinetic parameters, activation conditions, and features of the connection between the apoenzyme and the coenzyme. The nature of the appearance of isoenzymes is varied, but most often due to differences in the structure of the genes encoding these isoenzymes. Consequently, isoenzymes differ in the primary structure of the protein molecule and, accordingly, in physicochemical properties. Methods for determining isoenzymes are based on differences in physicochemical properties.
In their structure, isoenzymes are mainly oligomeric proteins. Moreover, one or another tissue preferentially synthesizes certain types of protomers. As a result of a certain combination of these protomers, enzymes with different structures are formed - isomeric forms. The detection of certain isoenzyme forms of enzymes allows their use for diagnosing diseases.
The enzyme lactate dehydrogenase (LDH) catalyzes the reversible oxidation reaction of lactate (lactic acid) to pyruvate (pyruvic acid). Increased activity is observed in acute lesions of the heart, liver, kidneys, as well as in megaloblastic and hemolytic anemia. However, this indicates damage to only one of the listed tissues.
Creatine kinase (CK) catalyzes the formation of creatine phosphate. Determination of CK activity in blood plasma has diagnostic value in case of myocardial infarction (there is an increase in the level of the MB isoform). The amount of the MM isoform may increase during trauma and damage to skeletal muscles. The BB isoform cannot penetrate the blood-brain barrier, therefore it is practically undetectable in the blood even during strokes and has no diagnostic value.
10. Organ specificity of LDH isoenzymes. Physiological values of the total activity of lactate dehydrogenase and its isoenzymes in blood plasma. Diagnostic significance of determining the activity of LDH and its isoenzymes.
Lactate dehydrogenase is a glycolytic enzyme and catalyzes the following reaction: Lactate + NAD Lactate dehydrogenase Pyruvate + NADH
The LDH molecule is a tetramer consisting of one or two types of subunits, designated M (muscle) and H (heart). In blood serum, the enzyme exists in five molecular forms, differing in primary structure, kinetic properties, and electrophoretic mobility (LDG-1 moves faster to the anode compared to LDH-5, that is, it is more electrophoretically mobile). Each form has a characteristic polypeptide composition: LDH-1 consists of 4 H-subunits, LDH-2 - of 3 H-subunits and 1 M-subunit, LDH-3 is a tetramer of 2 H-subunits and 2 M-subunits, LDH -4 contains 1 H-subunit and 3 M-subunits, LDH-5 consists only of M-subunits. According to the degree of decrease in the overall catalytic activity of the enzyme, all organs and tissues are arranged in the following order: kidneys, heart, skeletal muscles, pancreas, spleen, liver, lungs, blood serum.
The preferred method of glucose oxidation in tissue depends on which isoenzyme is most represented: aerobic (to CO2 and H2O) or anaerobic (to lactic acid). This difference is due to varying degrees affinity of isoenzymes for pyruvic acid. Isoenzymes containing mainly H subunits (LDH-1 and LDH-2) have low affinity for pyruvate and are therefore unable to effectively compete for substrate with the pyruvate dehydrogenase complex. As a result, pyruvate undergoes oxidative decarboxylation and enters the Krebs cycle in the form of acetyl-CoA.
On the contrary, isoenzymes possessing mainly M subunits (LDH-4 and LDH-5) have a higher affinity for pyruvate and, as a result, convert it into lactic acid. The most typical isoenzymes have been identified for each tissue. For the myocardium and brain tissue, the main isoenzyme is LDH-1, for erythrocytes, platelets, and kidney tissue - LDH-1 and LDH-2. In the lungs, spleen, thyroid and pancreas, adrenal glands, lymphocytes, LDH-3 predominates. LDH-4 is found in all tissues with LDH-3, as well as in granulocytes and male germ cells, in the latter LDH-5 is additionally found. In skeletal muscles, isoenzyme activity is arranged in descending order in the series: LDH-5, LDH-4, LDH-3. The most characteristic isoenzyme for the liver is LDH-5; LDH-4 is also detected.
Normally, the main source of LDH activity in the blood plasma is destroyed blood cells. In serum, the activity of isoenzymes is distributed as follows: LDH-2 > LDH-1 > LDH-3 > LDH-4 > LDH-5. When electrophoresis between fractions LDH-3 and LDH-4, an additional band of the LDH-X isoenzyme is sometimes detected; this isoenzyme is localized in the same organs as LDH-5.
All diseases that occur with cell destruction are accompanied by a sharp increase in LDH activity in the blood serum. An increase in the overall activity of the enzyme is found in diseases such as myocardial infarction, necrotic kidney damage, hepatitis, pancreatitis, inflammation and infarction of the lung, tumors of various locations, injuries, muscle dystrophy and atrophy, hemolytic anemia and physiological jaundice of newborns, lymphogranulomatosis, leukemia. During myocardial infarction, the beginning of an increase in enzyme activity in the blood serum is observed at 8-10 hours from the moment of the attack, the maximum increase occurs at 24-48 hours, often 15-20 times higher than normal. Increased activity LDH persists up to 10-12 days from the onset of the disease. The degree of increase in enzyme activity does not always correlate with the size of the damage to the heart muscle and can only be an indicative factor for predicting the outcome of the disease. In patients with angina pectoris, the enzyme activity does not change, which allows the test to be used for differential diagnosis within 2-3 days after a heart attack. The presence of organ specificity of enzymes makes it possible to use the study of their activity for the purpose of topical diagnosis.
11. Physiological values of the total activity of creatinine kinase (CK) and its isoenzymes in blood plasma. Diagnostic significance of determining the activity of CK and its isoenzymes.
Creatine kinase (CK) is an enzyme, a natural catalyst for chemical reactions, significantly increasing the rate of conversion of creatine and ATP (adenosine triphosphate) into the high-energy compound creatine phosphate, which is consumed during intense muscle contractions. This enzyme is found in the cytoplasm of various muscle cells (cardiac, skeletal), as well as in the cells of the brain, lungs, and thyroid gland.
The creatine kinase molecule can be divided into two parts, each of which is realized as a separate subunit: M (muscle) and B (brain). These subunits in the human body can combine together in three ways, forming, respectively, three isoforms of creatine kinase: MM, MB and BB. These isoenzymes differ in their localization in the human body: creatine kinase MM is located in the myocardium and skeletal muscles; creatine kinase MB is localized to a greater extent in the myocardium; creatine kinase BB is found in cells of the placenta, brain, urinary tract, some tumors and other places.
The normal concentration of the enzyme directly depends on the age and gender of the person. Due to the active development of muscles and nervous system, in children the activity of the natural catalyst is increased relative to the activity in adults. Women have lower creatine kinase levels than men.
The level of the MM isoenzyme is increased to a greater extent as a result of muscle damage, and rarely with heart damage. MC CK content is associated with myocardial damage. A significant increase in the activity of this form is observed during myocardial infarction. Its level increases sharply within two to four hours after the first symptoms. Therefore, the concentration of this enzyme in the blood is actively used to determine myocardial infarction. However, it is worth noting that the content of QC MV returns to normal level after three to six days, which causes low diagnostic efficiency in later. The concentration of CC explosives increases with oncological diseases. A decrease in the level of isoenzymes does not have any diagnostic value, since the minimum threshold for CK levels in a healthy person is zero.
12. Blood plasma lipases. Diagnostic significance of determining lipase activity. Lipase is a water-soluble enzyme synthesized by the human body that catalyzes the hydrolysis of insoluble esters (lipid substrates) and promotes the digestion, dissolution and fractionation of neutral fats. Together with bile, lipase stimulates the digestion of fats, fatty acids, and fat-soluble vitamins A, E, D, K, transforming them into energy and heat. The purpose of lipoprotein lipase is to break down triglycerides (lipids) in blood lipoproteins, thereby ensuring the delivery of fatty acids to tissues. Lipase is produced by: pancreas; liver; lungs; intestines special glands located in oral cavity infants. In the latter case, the so-called lingual lipase is synthesized. Each of these enzymes promotes the breakdown of a specific group of fats.
In terms of significance in making a diagnosis important role plays lipase produced by the pancreas. An increase in the level of the enzyme is observed with: pancreatitis occurring in acute form, or during exacerbation of a chronic process; biliary colic; pancreas injury; the presence of neoplasms in the pancreas; chronic pathologies of the gallbladder; formation of a cyst or pseudocyst in the pancreas; blockage of the pancreatic duct with scar or stone; intrahepatic cholestasis; acute intestinal obstruction; intestinal infarction; peritonitis; perforation of a stomach ulcer; perforation of an internal (hollow) organ; acute or chronic renal pathology; mumps, in which the pancreas is damaged; metabolic disorders that occur during diabetes mellitus, obesity or gout; liver cirrhosis; long-term use of medications - in particular, barbiturates, narcotic analgesics, heparin, indomethacin; organ transplant operations. In rare cases, the process of lipase activation is associated with certain injuries - for example, fractures of long bones. But in this case, fluctuations in the level of the enzyme in the blood cannot be considered a specific indicator of the presence of physical damage. For this reason, lipase tests are not taken into account when diagnosing injuries of various origins.
Determination of serum lipase levels is of particular importance in any pancreatic lesion. In this case, a blood test for the content of this enzyme together with an analysis for amylase (an enzyme that promotes the breakdown of starch into oligosaccharides) with high degree reliability indicates the presence of a pathological process in the tissues of the pancreas: both indicators are above normal). In the process of normalizing the patient's condition, these enzymes do not return to adequate levels simultaneously: as a rule, the lipase level remains at a high level longer than the amylase level.
High level lipase persists from 3 to 7 days from the onset of inflammation. A downward trend is recorded only after 7-14 days.
Low level lipase is fixed: in the presence of a malignant neoplasm in any part of the body except the pancreas itself; due to decreased pancreatic function; for cystic fibrosis (cystic fibrosis) - a genetic disease with a severe course that occurs as a result of pathological damage to the exocrine glands (gastrointestinal tract, lungs). after surgery to remove the pancreas; with excess triglycerides in the blood due to poor nutrition with an abundance of fatty foods in the diet or due to hereditary hyperlipidemia. In some cases, a decrease in lipase levels is a marker of the transition of pancreatitis to a chronic form.
Warburg found that yeast aldolases from various animal tissues differ in a number of properties. Pepsin, trypsin, and chymotrypsin also differed in solubility, pH, and temperature optimum.
At the end of the fifties, biochemists Wieland and Pfleiderer, as well as other researchers, isolated pure crystalline preparations of the enzyme from animal tissues lactate dehydrogenase and subjected them 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 established that the enzyme lactate dehydrogenase is present in tissues in several forms. These forms, in accordance with their electrophoretic mobility, were designated LDH1, LDH2, LDH3. LDH4, LDH5. (LDH is an abbreviation for lactate dehydrogenase), with number 1 denoting the component with the highest electrophoretic mobility.
Studies of lactate dehydrogenase enzymes isolated from different organs of animals have shown that they differ both in electrophoretic and chromatographic properties, as well as in chemical composition, thermal stability, sensitivity to the action of inhibitors, K m and other properties. Analyzes of lactate dehydrogenase from different animal species revealed very large interspecies differences, but within a given species the distribution of isoenzymes is characterized by great constancy.
Lactate dehydrogenase was the first enzyme whose individual components were studied in detail. 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 designate multiple forms of enzymes of the same biological species.
So, isoenzymes - this is a group of enzymes from the same source, possessing 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, extracted from various types animals, plants and microorganisms. Isoenzymes do not always consist of two or more subunits. For a number of enzymes, individual isofermsates are different in chemical structure proteins that have the same catalytic activity but consist of only one subunit.
The main criterion for the nomenclature of isoenzymes is currently their electrophoretic mobility. This is explained by the fact that, compared with other methods of characterizing enzymes, electrophoresis provides 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 various varieties of wheat, 4-5 in the roots of corn, and 9-12 isoenzymes of malate dehydrogenase were found in various organs (root, cotyledon, subcotyledon and epicotyledon), and the number of isoenzymes varied depending on from the phase of plant development.
It was noted that the molecular weights of the malate dehydrogenase isoenzyme sometimes varied significantly. For example, cotton leaves contain 7 isoenzymes of malate dehydrogenase, of which 4 isoenzymes are isoforms having 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 of at least one of the isoenzymes forms 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.
Plant resistance and susceptibility to diseases is often associated with the regulation of isoenzyme synthesis. As a response to the introduction of infection in plants, the intensity of metabolism of chemicals, primarily redox ones, is increased. Therefore, the activity of OM enzymes and the number of their isoenzymes increase when plants are damaged.
Increased activity and an increase in the number of peroxidase and o-diphenoloxidase isoenzymes are observed in various diseases of corn, beans, tobacco, clover, flax potatoes, oats and other plants. Figure 22 schematically shows the change in the number of peroxidase isoenzymes and their activity when tomatoes are affected by late blight. If the leaves of healthy plants contained four peroxidase isoenzymes, 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 polyphenoloxidase during the viral pathogenesis of tobacco mosaic virus-resistant and non-resistant tobacco mosaic virus species, it was found that viral infection causes qualitatively different changes in the enzyme composition of tobacco types of different stability. U sustainable type the activity of a number of isoenzymes increases to a greater extent than in the susceptible one. Thus, depending on the plant’s potential ability to biosynthesize enzymes, the plant’s susceptibility to infectious diseases changes.
Glutamate dehydrogenase
Esterases
Saharaza
Biological role of isoenzymes in plants.
IF indicates the great lability of the enzymatic apparatus of plants, making it possible to carry out the necessary metabolic processes. in a cell when environmental conditions change, ensures the specificity of the exchange of chemicals. for a given plant organ or tissue. Promotes plant adaptability to changing indoor 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 cell and rapid adaptation of plants to changing environmental conditions.
In fact, we noted that individual isozerments differ in temperature optima, pH optima, relation to inhibitors, and other properties. It follows that if, for example, temperature conditions change sharply and become unfavorable for the manifestation of the catalytic activity of some isoenzymes, then their activity is suppressed. However, this fermeatative 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 some reason, the pH of the reaction medium changes, then the activity of some isoenzymes is also weakened, but isoenzymes that have a different pH optimum begin to exhibit catalytic activity instead. High concentrations of salts 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 concentrations of salts in cells, enzymatic processes do not stop completely, since individual isoenzymes respond differently to increased salt concentrations: the activity of some isoenzymes decreases, while others increase.
Resistance and susceptibility to diseases 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 set of isoenzymes specific to this species, i.e. species specificity in isoenzyme composition is manifested.
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 influence of treatment with 42 M urea, each of the isoenzymes dissociates into 4 subunits with molecular weight of about 35 thousand. Thus, each of the five isoenzymes of lactate dehydrogenase is a tetramer. It has been established that all lactate dehydrogenase isoenzymes are possible combinations of only two types of subunits, conventionally designated by the letters A and B. Different combinations of these types of subunits form all five lactate dehydrogenase isoenzymes (Fig. 18). This shows that the isoenzymes of lactate dehydrogenase have a strictly ordered structure, and the individual subunits in the molecule of this enzyme protein are connected by hydrogen bonds, which can be broken under the influence of a concentrated solution of urea.
The question arises: how do the individual subunits of lactate dehydrogenase differ from each other and what is associated with the different electrophoretic mobility of individual isoenzymes? Quite definite answers have now been received to this question. It turned out that subunits A and B are t-c amino acids. Subunit B contains a larger number of acidic small amino acids compared to subunit A. In this regard, all lactate dehydrogenase isoenzymes (LDH1 - LDH2) differ in the amount of these amino acids, their molecules have different electrical charge values and different electrophoretic mobility. Lactate dehydrogenase isoenzymes also differ in a number of other properties, in particular Michaelis constants Km, relation to a number of inhibitors, and thermostability.
