DEHYDROGENASES — one of the most numerous subgroups of the enzymes catalyzing reactions of biological oxidation and recovery, i.e. the reactions connected with processes of breath, glycolysis and fermentation; activity of some dehydrogenases serves as additional diagnostic test at a number of diseases in blood serum.
Are eurysynusic in the nature. They are found in all bodies and tissues of animals and plants, and also in microorganisms. Belong to the class of oxidoreductases (KF 1.1.1; 1.1.2; 1.1.99; 1.2.1; 1.2.2; 1.2.4; 1.2.99; 1.3.1-1.3.3; 1.3.99; 1.4.1; 1.5.1; 1.6.4; 1.6.99; 1.8.5) also catalyze hydrogen transfer (protons) from the oxidized substrate (RH 2 ) on connections acceptors (R1) (but not directly on oxygen) which at the same time are recovered:
RH 2 + R1 → R + R1H 2 .
Reactions of an enzymatic degidrogenirovaniye are reversible. Differ in high specificity and catalyze oxidation reactions of strictly certain substrate or group related to it connections.
Functions D. in a human body and animals are very diverse. The majority of the aerobic processes which are coming to the end with hydrogen transfer on oxygen with formation of water as an end product and allocation of a large amount of the energy spent by an organism for implementation of various vital signs begins with the reactions catalyzed by D. Such D. as alcohol dehydrogenase (KF 184.108.40.206) and a lactate dehydrogenase (KF 220.127.116.11), processes complete fermentations (see) and glycolysis (see) respectively.
In pure form D. were for the first time allocated from yeast in laboratory O. of Warburg: in 1937 — alcohol dehydrogenase, and in 1939 — a glitseraldegidfosfatdegidrogenaza (a triozofosfatdegidrogenaza; KF 1. 2.1.9). Since then a large number of different D. is allocated in a high cleaning state from the most various sources. Represent two-component enzymes and consist of a proteinaceous part (apoenzyme) and a nonprotein part, function a cut in most cases is performed by nikotinamidny coenzymes and flavin coenzymes are more rare (see. Coenzymes ). The enzymatic reaction catalyzed by D. represents direct transfer of hydrogen atoms from one molecule to another (e.g., in a case alcohol - or lactate dehydrogenases). The oxidation of substrate catalyzed by D. can be also multistage process consisting of several consecutive stages which need also other cofactors, except coenzymes, necessary for reaction. Can be an example a pyruvatedehydrogenase (KF 18.104.22.168) and an oksoglutaratdegidrogenaz (KF 22.214.171.124) which functioning, except OVER, requires presence lipoic to - you, thiamine pyrophosphate and KOA. Results from effect of these enzymes not only hydrogen transfer, but also decarboxylation (see) and transfer of the acylic rest, and primary hydrogen acceptor (proton) is lipoic to - that and only then, with the participation of a flavin coenzyme, hydrogen is transferred on OVER. At the oxidation of phosphoglyceric aldehyde catalyzed glitseraldegidfosfatdegidrogenazy (KF 1. 2.1.12 and binding of phosphate happens 126.96.36.199). This reaction is interesting that the energy which is released at oxidation of substrate accumulates in the form of high-energy phosphatic communication of ATP (i.e. there is a so-called substrate phosphorylation).
D.'s most for the action needs presence of ions of bivalent metals which, apparently, stabilize active structure of a molecule of enzyme and participate in activation of substrate.
Primary structure of D. allocated from various sources is very similar, especially concerning the amino-acid remains important for implementation of the catalytic act. The spatial relationship of a polypeptide chain in the field of an active center OVER - dependent D. has strong likeness that indicates a genetic linkage between these enzymes. A structure of active centers of FAD-and NAD - dependent D. variously.
As a rule, D. are characterized by existence of quarternary structure, i.e. molecules of individual enzymes consist of various number of subunits. Subunits have no often enzymatic activity (it is accurately shown for OVER - and NADF-zavisimykh to D.), it appears only as a result of association of subunits in oligomer — an active molecule of enzyme.
Interaction of subunits in oligomer can result in cooperativity in functioning of active centers of enzyme. Regulation of activity of D. can be carried out by interaction with allosteric effectors — the metabolites communicating on special sites of a surface of a molecule of enzyme. The most typical representatives of regulatory D. are a glutamate - and lipoamiddegidrogenaza.
For many D. the functional groups participating in a catalysis in binding of a coenzyme and substrate are characterized. Are the most studied from this point of view OVER - and NADF-zavisimye. The remains of tyrosine, a histidine and asparaginic to - you take part in binding of a coenzyme; a role of the acid-base catalyst is played by either the rest of a histidine, or an ion of metal. The majority OVER - dependent D. is inactivated at addition of reagents on sulphhydryl (SH-) groups, napr, couple-hlormerkuribenzoata. Reactivation happens at addition of cysteine or glutathione (see). The coenzyme which is in solution possesses protective action concerning complexes enzyme — a coenzyme at an inactivation SH reagents. Numerous data indicate that SH groups are located in an active center of D. or in close proximity to it and participate in the catalytic act which is carried out by enzyme.
The majority OVER - and NADF-zavisimykh D. are specific in relation to the cofactors; at the same time all studied D. were stereospecific, and one of them at enzymatic recovery of NAD (NADF) chip off and attach hydrogen on one side of a pyridinic ring of a molecule of a coenzyme, and others — on another, opposite. The source, from to-rogo is allocated enzyme, in this case does not play a role, i.e. the same enzymes emitted from different bodies or fabrics have the same stereospecificity. If enzyme is active with OVER and with NADF, then stereospecificity is identical in relation to both coenzymes. Existence of stereospecificity can have fiziol, value, providing a possibility of effective hydrogen transfer from one substrate on another due to consecutive action of two functionally connected D.
Methods of measurement of activity
there are several methods of measurement of activity
of 1. On the speed of decolouration of methylene blue. The fact that the oxidized form of methylene blue is painted is the cornerstone of this method, and recovered — it is colourless. Measuring the speed of decolouration of the indicator in presence of the corresponding substrate, determine the size of degidrogenazny activity. Researches are usually conducted in anaerobic conditions since methylene blue easily is oxidized oxygen of air (at measurement in aerobic conditions the speed of degidrogenazny reaction is determined manometrical by amount of absorbed oxygen). The method with methylene blue is applicable for a research of activity of D. in gistol, drugs and activity of the purified enzymes, but in the latter case it is necessary to add the flavin enzyme capable to catalyze electron transfer and protons from D. on methylene blue to reaction mixture.
2. A method using a trifeniltetrazoliya, used as hydrogen acceptor, colourless in the oxidized form and painted — in recovered. D.'s activity is determined in this case by intensity of the appearing coloring. Advantage of this method is that trifeniltetrazoliya, contrary to methylene blue, are not oxidized on air and carrying out reaction does not demand creation of anaerobic conditions.
3. Spectrophotometric method. Recovery of nikotinamidny coenzymes in the course of enzymatic reaction is followed by emergence of a new maximum of absorption in an ultra-violet part of a range. Both coenzymes before accession of a proton have a strip of absorption with a maximum at 260 nanometers, after recovery of a coenzyme in a range OVER • H appears the second strip of absorption with a maximum at 340 nanometers. Often use it both for quantitative definition of NAD and NADF, and for measurement of activity of NAD (NADF) - dependent D. Intensivnost of a strip of absorption is proportional to quantity formed got into condition a coenzyme. Oxidation rate got into condition by NAD and NADF can be determined by reduction of size of optical density at 340 nanometers. This method is specific, easily reproduced and exact. Usually it is used during the work with pure enzymes.
Absence or decrease of the activity of D. in an organism can cause a number of diseases. So, reason inborn hemolitic anemia (see) there can be a decrease in erythrocytes of activity NADF-zavisimoy of glyukozo-6-fos-veils — a dehydrogenase (G6FDG) catalyzing oxidation of glyukozo-6-phosphate with formation of the recovered NADF. The last is necessary for maintenance of normal concentration of the recovered glutathione in erythrocytes. An essential role at low activity of G6FDG is played by the reduction of formation of ATP leading to a delay of ions of Na + and the related water in erythrocytes, to their swelling and hemolysis. At total absence of enzyme hemolitic anemia is shown spontaneously already at early children's age. If enzyme is available, but its activity is lowered, then the disease can be provoked by receipt in an organism of such medicinal substances as Primachinum, streptocides, acetphenetidiene, etc., consumption of horse beans (Vicia fava) and so forth. For diagnosis of the diseases connected with disturbance of normal activity of G6FDG in erythrocytes use Motulsky's method — Campbell. For this purpose take gemolizirovanny blood, to a cut add glyukozo-6-phosphate, NADF and diamond - cresyl blue. In the presence of G6FDG there is a formation of the got into condition NADF, edges are recovered in turn by dye, causing its decolouration. The content of enzyme is higher, the less time is required for full decolouration of the indicator. Normal solution of the indicator becomes colourless in 40 — 55 min., as much as possible — in 90 min. If time of decolouration is longer, it testifies to insufficiency of G6FDG.
Also decrease of the activity other D. — a glutationreduktaza (KF188.8.131.52) can be the cause of hemolitic anemia at the normal maintenance of the recovered NADF in erythrocytes. This enzyme catalyzes recovery of glutathione at the expense of the recovered NADF. At its shortcoming concentration of the recovered glutathione patholologically decreases.
At megaloblastichesky anemias activity of G6FDG in erythrocytes increases. Activity of G6FDG in erythrocytes is determined spektrofotometrichesk by quantity of the recovered NADF. Activity of enzyme is expressed number of units on 10 9 erythrocytes (take amount of enzyme for unit, a cut at 25 ° and the final volume of reaction mixture of 3 ml changes the optical density of NADF-N at 340 nanometers on 0,001 in 1 min. to 1 ml of a hemolysate of blood). Activity of G6FDG is equal in erythrocytes of healthy people to 272±26/10 9 erythrocytes. Definition of activity of D. in biol, liquids of an organism can serve as important diagnostic test. Significant increase in activity of a row D. (a lactate - hydroxybutyrate - izotsitrat-, malate - glitseraldegidfosfatdegidrogenaz) in blood serum can demonstrate, as a rule, heavy injuries of vitals, death of a large number of cells or essential disturbance of permeability of cellular membranes and gistogematichesky barriers. At some forms of anemias in blood activity of a malate dehydrogenase (KF 184.108.40.206) and isocitrate dehydrogenase (KF 220.127.116.11) increases. At acute and hron, nephrites, at tumors in urinary tract activity increases lactate dehydrogenases (see). At meningitis and tumors of a brain activity of a lactate dehydrogenase in cerebrospinal liquid increases. At some forms of jaundice in blood activity of a glutamatdegidrogenaza (KF 18.104.22.168) increases; significant increase in its activity at acute hepatitises usually is the evidence of heavy damage of cells of a liver.
Sorbitoldegidrogenaza contains preferential (or it is exclusive) in cells of a liver. This enzyme is practically not in normal blood serum, and its emergence indicates damage of a parenchyma of a liver (an infection, toxic damage, a hypoxia of body). Activity of a sorbitoldegidrogenaza in blood serum of healthy people makes less than 1 unit on 1 ml (1 unit is equal to amount of the enzyme which is contained in 1 ml of blood serum and causing change of optical density on 0,001 in 1 min. at 360 nanometers and 24 ° at the final volume of reaction mixture of 3 ml and thickness of its layer in spektrofotometrichesky a ditch of 1 cm).
Big distribution in clinic was gained by determination not of the general content of enzyme, but absolute or fraction of its multiple forms, so-called isoenzymes (see). E.g., one of such forms of a lactate dehydrogenase is preferential in heart, and another — in a liver (LDG-1 and LDG-5). Therefore increase in blood serum of relative and absolute quantity LDG-1 is observed at a myocardial infarction, and LDG-5 — at different types of hepatitis.
Histochemical methods of identification of dehydrogenases in fabrics
In gistokhy, reactions at D.'s definition are used the salts a tetrazoliya serving as electron sinks which donors is the oxidized substrate, and which are recovered to a water-insoluble formazan. Nitro-blue tetrazoly (NBT) is especially widely used. This salt is insoluble in fats, is rather steady against effect of light and has the expressed affinity to protein that prevents crystallization of a formazan of NST in an incubation period. The most high speed of reaction since the long incubation of drug leads to damage of mitochondrions is necessary for D.'s identification and also because at a small speed other electronic acceptors which are available in fabrics can intercept electrons, leaving salt a tetrazoliya not recovered. Products of degidrogenazny reactions often are ketones and aldehydes; for shift of balance of enzymatic reaction towards formation of these products it is necessary to increase concentration of substrate and to delete reaction product by means of the reagents connecting carbonyl groups. For this purpose enter cyanide, dimedone, phenylhydrazine, semicarbazide into incubating medium.
Incubating medium prepares just before the use. The cuts of freshly frozen fabric prepared in the cryostat paste on cover or slide plates and place in incubating medium for 5 — 30 min. at 37 °. After an incubation cuts wash out a dist, water and fix in 10% formalin - calcium solution within 10 min., then again wash out in a dist, to water and conclude in glycerin-gelatin. The dark deposit of a formazan is found in the structures having degidrogenazny activity. The system of a succinatedehydrogenase for gistokhy, definition does not demand coenzymes; the product of enzymatic reaction does not slow down reaction and therefore there is no need to enter the agent connecting it. Gistokhim, the equipment same, as well as for other. For check of specificity of reactions it is necessary to exclude substrate, specific to D., from incubating medium or to heat cuts at 80 ° within 1 hour completely to inactivate enzymes. During the definition of specificity of reaction to a succinatedehydrogenase it is possible to use, besides, low-new to - that (in concentration of 3 — 4 mg/ml), edges is braked by activity of a succinatedehydrogenase.
The diagnostic value of definition of activity of D. is undoubted therefore the list of D. which activity in biol, liquids can serve as direct or auxiliary diagnostic test extends.
Bibliography: Allosteric enzymes, under the editorship of V. L. Kretovich, Results of science and technology, a Series biol, chemical, t. 3, M., 1975; Coenzymes, under the editorship of V. A. Yakovlev, M., 1973: Lenindzher A. Biochemistry, the lane with English, M., 1976; L and l of l and R. Patogistologicheskaya of the technician and a practical histochemistry, the lane with English, M., 1969; Pearce E. A histochemistry, the lane with English, M., 1962; Cytology of enzymes, the lane with English, under the editorship of A. A. Pokrovsky, M., 1971.
G. A. Kochetov; A. G. Ufimtseva (to gistokhy.).