From Big Medical Encyclopedia

PHOSPHATASES — the enzymes catalyzing splitting of ester groups in monoesters of phosphoric acid with formation of free ortho-phosphate; belong to the class of hydrolases, a subclass of hydrolases of phosphoric monoesters (KF 3.1.3).

T. are present at all animals and vegetable organisms and take the important place in cellular metabolism; biol. role F. it is connected with their participation in exchange of carbohydrates (see. Carbohydrate metabolism), nucleotides (see. Nucleic acids) and phospholipids (see Phosphatides), and also with an osteogenesis (see the Bone). Change of activity nek-ry F. in blood is a valuable diagnostic character at a number of diseases. Genetically caused disturbance of synthesis or enzymatic full value

nek-ry F. is the reason of a serious hereditary illness (see the Hypophosphatasia).

On the nature of catalytic action all F. represent the phosphomonoesterases splitting an ester group gidrolitiche-

we sleep in the way. The systematic name of these enzymes always includes the term «hydrolase» (naimenovanpz «phosphatases» is the worker formed from the name of substrate). T. can be considered as phospho-transferases (see) as are capable to catalyze transfer of the phosphatic rest on molecules of other acceptors, except water, but since water is physiologically main and activest acceptor, phosphatases rank as hydrolases (see).

Substrate specificity. Majority F. is among the enzymes (see) having rather wide substrate specificity. However nek-ry F. differ in a limited circle of the turned substrates. These are, first of all, the enzymes operating on phosphoric derivatives of sugars, and also the nucleotidases (see) splitting mononucleotides. In many fabrics F. are presented by the multiple forms differing on the catalytic and physical properties (see Isoenzymes). At phosphatases from different biol. sources also observe distinctions in substrate specificity and catalytic activity. Nek-rye F. find looking alike the enzymes relating to other groups. So, exist F., capable to catalyze reactions of rephosphorylation (see) or to split acid angidridnuyu pyrophosphatic communication (see Pi ~ rofosfataza). E.g., glyukozo-6-fos-fataza (D - glyukozo-@-phosphate phospho-hydrolase; KF on substrate specificity and catalytic properties very reminds fos-fotransferaza (KF and, and also inorganic pyrophosphatase (KF

Mechanism of action. For many F. three-dimensional structure of their molecules is established and detailed chemical mechanisms are offered rolled itichesky action. Assume that in the course of the catalytic act several various groups localized on a surface of a molecule of enzyme in an active center take the cooperative (combined) part. One of such F. glyukozo-6-phosphatase is. This enzyme connected with mikrosomny fraction of cells along with hydrolysis of glyukozo-6-phosphate catalyzes transfer of phosphatic group from an inorganic pyrophosphate (see Phosphorus) on glucose (see), and also exchange reaction between glucose and glyu-kozo-6-phosphate. Researches of kinetics of hydrolytic, transfe-different and exchange reactions (see Kinetics of biological processes) showed that their mechanism has character of two-phasic transfer, in Krom as intermediate compound (intermediat) phosphoenzyme, or phosphoryl-enzyme is formed. At the same time the transferable phosphatic group in a molecule of enzyme contacts the rest of a histidine (see). The ion of bivalent metal is necessary for glyukozo-@-phosphatase for manifestation of activity. According to assumed (from shares of the known simplification) the mechanism of reaction the ion of metal contacts negatively charged phosphatic group of substrate, and the reactive rest of a histidine having nucleophilic properties — atom of phosphorus, as fosfofermen-that leads to education. The last then either is exposed to hydrolysis, or interacts with nucleophilic groups of acceptor molecules (e.g., with hydroxylic groups of sugars) with formation of end products of reaction and release of enzyme, free from phosphate.

Not all fosfatazny reactions proceed with formation of intermediate phosphoenzyme, in Krom fosfo-rilirovan the rest of a histidine. When reaction is catalyzed by an alkaline phosphatase (KF allocated from tissues of mammals or from bacteria, the rest of serine is exposed to phosphorylation in a molecule of enzyme (see). Enzyme represents a tsinksoderzhashchy metalloprotein (see Metalloproteins), in Krom 2 — 3 gram atoms of zinc fall on 1 mol of protein. Ions of zinc or other metal are necessary for manifestation of catalytic activity of an alkaline phosphatase and, perhaps, for stabilization of native structure of a molecule of enzyme. Bivalent cations of So2+, Mg2 + and Mp2 + activate F., allocated from various fabrics while ions of Ve2 + and complexing agents (e.g., EDTA) are inhibitors of these enzymes. The mechanism of action of an alkaline phosphatase is similar to that mechanism, to-ry is postulated for glyukozo - 6 - phospha - Tazy, but atom of phosphorus interacts not with gistidinovy, and with the serino-vy rest of a molecule of enzyme.

For other phosphatases, napr, for a fruktozo-bisfosfataza (KF, there are no data on formation of phosphoenzyme yet. It is possible that the enzymatic reaction catalyzed by it proceeds on the single-stage coordinated mechanism, but not by two-phasic transfer.

Methods of definition. Majority of ways of definition of activity F. it is based on measurement of amount of the inorganic phosphate (which is formed as a result of the reaction catalyzed by these enzymes) by means of various colorimetric methods (see Colorimetry), to-rye are connected with recovery phosphomolybdenic to - you. In the classical way of definition of activity F. Bo-dansky's method with use as substrate (3 glycerophosphates is (see Vodansky a method). Often in practice it is more convenient to measure amount of the phenol released from aryl phosphomonoester. So, in blood serum widely apply King method to definition of activity of an alkaline phosphatase — Armstrong (cm of King — • Armstrong a method), based on the same principle мето^ Jenner — Kay or their modification. The most sensitive method of definition of activity of an alkaline phosphatase in blood serum is Bessey's method (see Bessey methods). Widely apply a method to definition of activity of acid phosphatase Guttman — Guttman. These standard methods of definition of activity F. provide use as substrates of mono-phosphorus phenol ethers in blood serum, gs-trofenola, phenolphthalein or thymolphthalein. The free phenols formed as a result of reaction (see) define spektrofotometrichesk (see the Range about photometry). Big sensitivity methods of measurement of fosfatazny activity using fluorescent substrates, such as (3-naftilfosfat and Z-O - metilflyuores-tseinfosfat (see Flyuorokhroma) have. Trace quantities of a pyrophosphate, marked 32P, it is possible to determine by sedimentation by its ammonium molybdate and triethylamine in the presence of not marked carrier. Feelings the ptelnost of this tracer technique makes apprx. 3 ng.

Acid and alkaline phosphatases. Among F. two groups of enzymes — alkaline and acid phosphatases are most eurysynusic and studied. Having wide substrate specificity, these enzymes considerably differ on the properties depending on a source, from to-rogo they are emitted. Various monoesters orthophosphoric to - you — as aliphatic, napr, glitserol-1-and glitserol-2-fos-veils, and aromatic, e.g. 4 nitrophenyl phosphate can be their substrates; at the same time these enzymes are inactive concerning di - both three ef and a ditch of phosphoric acids (see). Big difference between acid and alkaline F. observe at their action on sulfur-containing ethers. The alkaline phosphatase hydrolyzes the S-replaced monoesters thiophosphoric to - you, e.g. tspsteamin-S-phosphate; oxygen of the split radio communication, apparently, is necessary for effect of acid phosphatase: acid phosphatase hydrolyzes O-za-

paved monoesters thiophosphoric to - you, e.g. O-4-nitrofeniltnofos-veils.

Alkaline phosphatase (phosphomono-esterase; KF shows the maximum activity at pH 8,4 — 9,4 and catalyzes hydrolysis of almost all phosphomonoesters with formation of inorganic phosphate and corresponding alcohol, phenol, sugar, etc. The alkaline phosphatase contains in the majority of fabrics and liquids of a human body and animals, and also in plants and microorganisms. At the person especially high activity of this enzyme is noted in an epithelium of a small bowel, kidneys, bones, a liver, leukocytes, etc. Widely used source of an alkaline phosphatase is the ossifying cartilage that indicates a possible role of this enzyme in processes of calcification of a bone tissue. Existence of an active alkaline phosphatase is characteristic of the fabrics connected with transport of nutrients, it often is present at the developing fabrics and secretory bodies. The alkaline phosphatase is practically absent in muscles, mature connecting fabric and erythrocytes, in this enzyme also walls of vessels and a hyaline cartilage are poor.

Fig. Electrophoretic mobility of the alkaline phosphatase allocated from a liver, a bone tissue, a placenta and intestines of the person: «+» and «—» — position of the anode and cathode, circles represented sites of application of solution of enzyme, hulls — location of enzyme after fixing and coloring an electrophorus - grams.

The alkaline phosphatase possesses extremely wide an isoenzyme-nym a range. By means of immunochemical and electrophoretic methods it was shown that between its isoenzymes (see) there are expressed physical and chemical and catalytic distinctions. At an electrophoresis in polyacrylamide gel the alkaline phosphatase received from a mucous membrane of intestines remains near the place of entering of solution of enzyme into gel (the line of start), and the alkaline phosphatase allocated from a liver moves towards the anode together with fraction and \-or a2-globu-

lin (fig). Electrophoretic division of a serumal alkaline phosphatase at increase in its activity gives the chance to establish a bone or hepatic origin of enzyme, emission to-rogo caused the increased activity of an alkaline phosphatase in blood. In blood serum normal main source of an alkaline phosphatase is, apparently, the liver. Emergence of an isoenzyme characteristic of a mucous membrane of a small bowel is under genetic control: there are data that its presence at blood is characteristic of people with a zero group of blood.

Distribution of activity of enzyme even in one morphological features negomogenno. So, activity of an alkaline phosphatase is various in different departments of intestines, in cortical substance of a kidney it is much higher, than in brain. Activity of an alkaline phosphatase is influenced by hormonal factors: the asset

a nost of enzyme in blood decreases after a hypophysectomy, castration, and also as a result of use of corticosteroid drugs. After administration of thyroxine activity of enzyme increases. The person has various factors causing a stress, promote increase in activity of an alkaline phosphatase in leukocytes.

Activity of an alkaline phosphatase in blood in a nek-swarm of degree depends on age and a floor. At men activity of enzyme in blood is 20 — 30% higher, than at women, however during pregnancy women have considerable (by 2 — 3 times) an increase in activity of this phosphatase that can speak growth of an embryo, especially process of bone formation of a fruit.

Functions of an alkaline phosphatase in each fabric are definitely not established so far. In a bone tissue she, apparently, participates in processes of calcification. In a cell the alkaline phosphatase is usually connected with a lipoprote-idny membrane, and at nek-ry microorganisms as showed to gistokhy. researches, it is between a membrane and a cell wall. Localization of enzyme on absorption surfaces indicates its possible role in transmembrane transfer.

Pier. the weight (weight) of the alkaline phosphatase allocated from different sources varies within 70 000 — 200 000; the enzyme from a placenta of the person received in a crystal look has a pier. weight 125 000. Believe that its molecule consists of two subunits equal a pier. weight, but not identical each other. Results of genetic researches indicate existence of three types of subunits shche-

lochny phosphatase, various combinations to-rykh give six phenotypical options differing on electrophoretic mobility and representing the main multiple forms (isoforms) of enzyme. Assume that the difference as a part of subunits is caused by existence in molecules of nek-ry alkaline phosphatases of the carbohydrate part covalently connected with protein.

The alkaline phosphatase is stable at neutral and alkaline pH values, but is sensitive to soured - laziness of the environment. In the field of pH 7,0 — 8,0 and at ion concentration of Zn2 + higher than 10-5M enzyme form the active tetramer connecting 16 ions of Zn2+. The microbic alkaline phosphatase allocated from different sources is capable to form active hybrids with use of monomers of different enzymes that indicates proximity of secondary structure of microbic phosphatases, despite distinctions in structure and to them-munol. properties of subunits.

Substrate specificity of alkaline phosphatases from different sources is not identical. So, enzyme from a bone tissue hydrolyzes a number of phosphoric connections, including geksozofosfata, glycerophosphates, ethyl phosphate, adenylate and phenyl phosphate. Enzyme from Escherichia coli is capable to hydrolyze various on-lifosfaty, including metaphosphates with various chain length, and also phosphoserine, phosphothreonine, pyridoxal phosphate and phosphosincaline. A number of alkaline phosphatases from tissues of mammals at pH 8,5 shows ii-rofosfatazny activity, and enzyme from a mucous membrane of intestines of a chicken hydrolyzes to a cyst - amine - in - phosphate and other S-phosphates with formation of inorganic phosphate and corresponding thiol. Nek-ry alkaline phosphatases have also transferazny activity and in reactions of a perefosforili-rovaniye can catalyze transfer of phosphate from phosphoether on spirit group of an acceptor.

Thus, the alkaline phosphatase is capable to hydrolyze the connections containing communication P — F, P — About — With, P — About — P, P — S and P — N, and the catalyzed reaction consists in transfer of phosphate from the donor of type

(where to X it can be presented by fluorine, oxygen, sulfur, nitrogen, and R — hydrogen atom, the alkyl deputy or absolutely to be absent) on an acceptor like R' — IT (where R' is presented by hydrogen atom or the alkyl deputy) from times -

ryvy bonds of P — X. As enzyme catalyzes also back reaction, acceptor specificity extends to all connections of R type — HN. The alkaline phosphatase catalyzes transfer only of trailer phosphate, characteristic feature of enzyme is that relative speeds of hydrolysis of various substrates are very close.

Definition of activity of an alkaline phosphatase in blood has diagnostic value at diseases of a liver and bone system. So, the giperfosfatazemiya is noted at hron. diseases of a liver, a sarcoidosis (see), tuberculosis (see), an amyloidosis (see) and a lymphogranulomatosis (see). At rickets (see) increase in activity (sometimes by 2 — 4 times) an alkaline phosphatase it is noted in 65% of cases. Pedzhet's disease (see Ped-zhet a disease), and also an osteosarcoma (see), phosphate diabetes (see) are followed by substantial increase of activity of an alkaline phosphatase in blood serum.

Genetically caused low activity of an alkaline phosphatase in blood (hypophosphatasia) is the reason of the serious hereditary disease which is followed by anomalies of a skeleton owing to disturbance of processes of ossification; defect of enzyme is inherited on an autosomal re-tsessivnomu to type.

Acid phosphatase (fosfomonoeste-time; KF is also eurysynusic in the nature. It is found in yeast, plesenyakh, bacteria, vegetable and animal fabrics and biol. liquids. At the person activity of acid phosphatase in a prostate is especially high. Erythrocytes also contain a lot of acid phosphatase. Extract from tissue of a prostate gland shows fosfatazny activity in the subacidic environment, edges are nearly 1000 times higher, than fosfatazny activity of extracts from a liver or kidneys. Gistokhim. researches show that enzyme contains hl. obr. in a ferruterous epithelium of a prostate; large amounts of enzyme are found in sperm. There is close connection between synthesis of acid phosphatase in a prostate and the content of sex hormones (see). At low concentration of androgens (see) in urine note low activity of acid phosphatase in sperm. The same is observed at kr a ptorkhizm (see) and a hypogonadism (see).

The optimum of pH for acid phosphatase is in an interval of pH values between 4,7 and 6,0 (however the maximum of activity of the acid phosphatase received from a spleen is observed at pH values from 3,0 to 4,8). The substrate range and speeds of hydrolysis of various substrates acid phosphatase and an alkaline phosphatase are very excellent. So, acid phosphatase is not capable to hydrolyze the S-replaced monoesters thiophosphoric to - you while the O-replaced monoesters in the same conditions actively are hydrolyzed by it (in case of an alkaline phosphatase the return is observed).

By electrophoretic division of the acid phosphatase emitted from various fabrics existence at this enzyme of four components — And, In, was installed by C and D. The combination of the ABD components dominates in kidneys; BD — in a liver, intestines, heart and skeletal muscles; the component B prevails in skin, a D — in a pancreas; the component C is present at a placenta and does not meet in one body of an adult organism. In general the combination of BD is characteristic of acid phosphatase of the majority of tissues of the person except for skin, kidneys and a pancreas. In total

4 electrophoretic components represent genetically determined isoforms of acid phosphatase. Characteristic feature of acid phosphatase is sensitivity to an inactivation on an interface of phases; addition of surfactants (see Detergents) to solution of enzyme protects acid phosphatase from an inactivation.

Pier. the weight of acid phosphatase is various at the enzymes received from different sources, napr, two immunological various molecular an isoenzyme of acid phosphatase from a prostate of the person have a pier. weight 47 000 and 84 000.

Definition of activity of acid phosphatase serves as important diagnostic test at detection of a prostate cancer in blood serum (see the Prostate, pathology). Without metastasises increase in activity of acid phosphatase in blood is found in patients with cancer of a prostate in 25% of cases, and at a prostate cancer with metastasises of a tumor in other bodies — in 80 — 90% of cases. Dynamics of indicators of activity of this enzyme at a prostate cancer can serve in blood as criterion of efficiency pr about the Vod mo y those r ap and.

Definition of acid phosphatase has essential value as well in forensic medicine. High activity of enzyme in sperm gives the chance with big reliability to identify suspicious spots at with at - a chemical research of material evidences.

Histochemical methods of detection of phosphatases. The alkaline phosphatase in a histochemistry is revealed by means of Gomori's method, methods using a tetrazoliya, azoindoxyl and method of azocoupling. At use of a tetrazoliyevy method and method of azocoupling use of the cryostately cuts processed atsetonokhm and also cryostately unstable cuts is recommended. Methods using salts of metals demand use of the cryostately cuts fixed in formaldehyde or frozen sections after fixing of fabric blocks in formaldehyde or glutaric dialdehyde. Gomori's method, then methods using a tetrazoliya and azoindoxyl is the most recommended. At a tetrazoliyevy method of definition of an alkaline phosphatase use 5-brom-4-hlor-

3-indoksilfosfat, toluidine salt, nitrotetrazoly blue, 0,1 — 0,2 M the tris-HC1-bufer or veronal - the acetate pH 9,2 — 9,4 buffer. Reactions of azocoupling and a tetrazoliyevy method at gistokhy. identification of an alkaline phosphatase are more sensitive, than Gomori's method, however the diffusion of enzyme occurring during the use of naphthols and salts a tetrazoliya can interfere with establishment of its exact localization.

Gomori's method with p r and m e N of e-N it salts was thrown by l and Incubating medium:

3% P ~ P and - glycerophosphate of 10 ml

of 2 — 10% solution of medinal of 10 ml

of 2% solution of Calcium chloratum Sas12 (anhydrous) 15 ml

of 2% solution of MgS04 10 epsom salt of ml a distilled water of 5 ml

the Total amount of 50 ml

carefully mix Incubating medium and in case of opacification filter. Incubate 1 — 60 min. at 37 ° or at the room temperature, then merge incubating medium, wash out cuts in running water, transfer to 1 — 2% solution of Sos12 cobaltichloride or other soluble salt of cobalt (acetate or nitrate of cobalt) for 5 min. Then wash out in running water of 2 — 5 min. At an incubation of unstable cuts it is necessary to carry out post-fixing at the room temperature to 4% solution of paraformaldehyde within 2 — 5 min. and to rinse in running water of 2 min. Cuts process solutions of ammonium sulfate of the ascending concentration (0,1 — 1%)

of 2 min. and wash out in running water of 10 min. then conclude in glyceric gel or Apati's syrup or (after dehydration) in entellan or similar to it Wednesday. Places of localization of an alkaline phosphatase are painted in black color. Control reactions carry out without addition of substrate to incubating medium.

Method of simultaneous azocoupling on Barstona Incubating medium: AS, AS-MX, AS-D, AS-B1 naphthol or phosphate of AS-TR naphthol

of 10 — 25 mg to dissolve in stable salt of diazonium I (N, N '-dimethyl formamide or a dimethyl sulfoxide) 0,5 ll

0,1 — 0,2 M veronal - acetate or the tris-HC1-bufer, pH 8,2 — 9,2 50 of ml

solid blue B, BB, RR, TR fast red, VRT solid blue (variamin blue, (gol RT),

VB solid blue (variamin blue In) or solid violet In 50 mg

carefully mix Incubating medium and filter. Instead of stable diazonium salt it is possible to use 0,5 ml freshly cooked geksazotirovanno-go new fuchsin. In this case the necessary size pH is established, adding caustic soda on drops. 5 — 60 min. at 37 incubate ° or at the room temperature. Merge incubating medium, cuts rinse in a distilled water, place in 4% of R_R of formaldehyde at several o'clock at the room temperature, then wash out in running water, if necessary dokrashivat kernels fast red or hematoxylin and conclude in glyceric gel or Apati's syrup. Depending on a type of the diazonium salt included in the incubating medium, structures having enzymatic activity alkaline фос^ fataza are painted in blue-violet or red color.

For gistokhy. identifications of acid phosphatase recommend to use cryo-stately or frozen sections after preliminary fixing in formaldehyde, and also the cryostately cuts subjected to freezing and drying and covered with photoxylin and the cryostately cuts subjected to substitution in the frozen state and covered with photoxylin. The best results are achieved during the fixing of fabrics by glutaric dialdehyde or formaldehyde. For identification of enzyme use reactions of azocoupling, Gomori's method and indigogenny reactions. The method of simultaneous azocoupling with phosphates of naphthol and gekeazotirovanny is considered universal? rosaniline or new fuchsin. The second for the frequency of use is the indigogen-ny method using 5-bromine-4-chlorine-3-indoksilfosfata as substrate. Gomori's method gives the chance to precisely identify lysosomes


Gomori's method with salts of metals (in m about d and f and to and c and i)

Incubating medium:

0,1 M the acetate buffer,

pH 5,0 or 6,0 50 «

0,24% solution of lead nitrate of 50 ml

of 3% P“ P and - glycerophosphate of sodium

or 0,1% solution tsitidinmonofos-

a veil of sodium of 10 ml

the Total amount of 110 ml

well mix Incubating medium and leave to stand for 15 — 30 min. at a temperature of an incubation, then filter. The incubation is carried out in ditches at 37 ° within 10 — 60 min. or at the room temperature to 2 hours, it is possible to incubate free-floating sections. Incubating medium is merged, cuts rinse in two changes of a distilled water 1 min. in everyone and place in 0,5 — 1% solution of yellow ammonium sulfide for 1 — 2 min. Again rinse in a distilled water and conclude in glyceric gel or Apati's syrup. The structures having activity of acid phosphatase are painted in brown color.

Method of simultaneous azocoupling with ethers of AS naphthol

Incubating medium:

phosphate of AS-BI naphthol or AS-TR naphthol of 20 — 25 mg dissolve in] \t, To' - dimethyl formamide

of 1 ml

Buffering geksazotirovanny n-rosaniline or new fuchsin (1,5 — 4,5 ml of geksazotirovanny n-rosaniline or 1,25 ml of new fuchsin dissolve in 45,5 — 48,5 ml 1,36 — 2,72% of solution of SN3SOGCHTA sodium acetate • ZN20 or 48,75 ml 0,1 M of the seronal-adetatny buffer, pH apprx. 6,0, bring to pH 5,0 — 5,5)

50 ml

the Total

amount of 51 ml

carefully mix Incubating medium and filter. 30 — 60 min. at 37 incubate ° or 1 — 2 hour at the room temperature or several hours (days) in the refrigerator at +4 °. Incubating medium is merged, cuts rinse in a distilled water and place in 4% solution of formaldehyde at several o'clock at the room temperature. Rinse in running water, if necessary dokrashivat kernels hematoxylin and conclude in glyceric gel or Apati's syrup. The structures having activity of acid phosphatase are painted in red color.

And z about and N d about to with and l N y y a method till about with with r and at

Incubating medium: toluidine salt 5-bromine-4-chlorine-3-indoksilfosfata

of 1,5 — 3 mg dissolve in 0,075 — 0,15 ml Т^^ '-dimethyl formamide of 0,1 M the acetate buffer, pH 5,0 10 of ml

Geksazotirovanny new fuchsin of 0,25 ml

or solid blue In 5 — 10 mg _

Total amount ~ 10 ml

carefully mix Incubating medium and filter, the attached or free-floating sections incubate 15 — 60 min. at 37 °. Incubating medium is merged, cuts rinse in a distilled water and place in 4% solution of formaldehyde at several o'clock at the room temperature, then rinse in running water and place in a distilled water then conclude in glyceric gel or Apati's syrup. The structures having activity of acid phosphatase are painted in bluish-brown color.

Bibliography: Dickson M. and Webb E. Enzymes, the lane with English, page 364, 458, M., 1982; Lille R. Patogistologicheskaya of the technician and a practical histochemistry, the lane with English, M., 1969; Lloyd 3., about since Wednesday and at R. and Shibler T. Gistokhimiya of enzymes, the lane with English, M., 1982; The Nomenclature of enzymes, the lane with English, under the editorship of A. E. Braunstein, M., 1979; Pearce A. Gistokhimiya, the lane with English, M., 1962; Enzymes, ed. by P. D. Boyer, v. 7, N. Y. — L., 1972. P. L. Ivanov (biochemical),

A. G. Ufimtseva (gist.).