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FISHERIES AND MARINE SERVICE Translation Series No. 3985 REF

Electrophoretic spectra of isomerases, transferases and oxidoreductases of the Patina pectinifera

LIBRARY FISHERIES AND OCEANS BIBLIOTHÈQUE PÉCHES ET OCÉANS

by G. P. Manchenko and O. L. Serov

Original title: Elektroforeticheskie spektry izomeraz, transferaz i oksidoreduktaz morskoi zvezdy Patina pectinifera

From: Biol. Morya (5): 57-60, 1976

Translated by the Translation Bureau (UDP) Multilingual Services Division Department of the Secretary of State of Canada

Department of the Environment Fisheries and Marine Service Halifax Laboratory Halifax, N.S.

1977

(

9 pages typescript (91e1)(3— 1 •

DEPARTMENTOFTHESECRETARYOFSTATE SECRÉTARIAT D'ÉTAT TRANSLATION BUREAU BUREAU DES TRADUCTIONS •

MULTILINGUAL SERVICES DIVISION DES SERVICES CANADA DIVISION MULTILINGUES

TRANSLATED FROM - TRADUCTION DE INTO - EN Russian English AUTHOR - AUTEUR G. P. Manchenko and OY, L, Serov

TITLE IN ENGLISH - TITRE ANGLAIS Electrophoretic Spectra of Isomerases, Transferases and Oxidoreductases of the Starfish -pectinifera,

TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÉRES ROMAINS) Elektroforeticheskie spektry izomraz,.transferaz i oksidoreduktaz morskoi zvezdy Patirià-Lpéctinifera.

REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATETOREIGN CHARACTERS , RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÉRES ROMAINS,

Biologiya morya,

REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS

Marine Biàlogy.

PUBLISHER- ÉDITEUR PAGE NUMBERS IN ORIGINAL DATE OF PUBLICATION NUMÉROS DES PAGES DANS DATE DE PUBLICATION L'ORI GINAL Not given. 57-60 YEAR ISSUE NO. VOLUME PLACE OF PUBLICATION ANNÉE NUMÉRO NUMBER OF TYPED PAGES. ' LIEU DE PUBLICATION NOMBRE DE PAGES DACTYLOGRAPHIÉES USSR 1976 5 9

REQUESTING DEPARTMENT Environment TRANSLATION BUREAU NO. WNISTÉRE- CLIENT NOTRE DOSSIER NCI 1089221 Fisheries/Scientific Info.

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PERSON REQUESTING Dr. P. H, Odense, Halifax Lab, P:i..A.NSLA:HON DEMANDÉ PAR i . Th Y) 0 n! ,1

YOUR NUMBER VOTRE DOSSIER NCI 5053-1 seurnDnt DATE OF REQUEST 2 March 1977 DATE DE LA DEMANDE 'APR -- 7 1971

503.200-10-6 (REV. 2/68 ) 7530-21-029-5333 Fd-)II it 3 g^s ,%cretary Secrétariat `- 'of 11,3tate d'État TRANSLATION BUREAU F3UREAU DES TRADUCTIONS

MULTILINGUAL SERVICES DIVISION DES SERVICES DIVISION MULTILINGUES

CLIENT'S NO. DEPARTMENT DIVISION/HRANCH CITY NO DU CLIENT MINISTLRE DIVISION/DIRECTION VILLE Fisheries/Scientific Info. 5053-1 Environment & Publications Branch Halifax, N. S.

SUREAU NO. LANGUAGE TRANSLATOR(INITIALS) ND DU BUREAU LANGUE TRADUCTEUR ( INITIALES;

1089221 Russian WDP APR - 7 1977

Biologiya morya (Marine Biology), No, 5, pp, 57-60? 1976 (USSR)

^^^i:ÉJ?? '3^ :.;.*' ?QÇf. ;.Nntd UDC 593,93+577.15 's°or Biochemistry

^lèctrôphôrètic Spectra Of Isonièràse$; Trà^is^èràsès and .(?xidoreductases.of .the .Starfish-Td-tirià ^ièctiriifèra ^-^-^--- By

G. P. Manchenko and 0, L. Serov, Genetics Laboratory, Institute of Marine Biologÿ, Far Eastern Science Center, Academy of Sciences USSR, Vladivostok 690022, and the Laboratory of Genetic Fundamentals of Ontogeny, Institute of Cytology and Genetics, Siberian Branch, Academy of Sciences, USSR, Novosibirsk 630090

Using the method of vertical starch gel electrophoresis we have /57* derived and described the spectra of 10 enzymes from different organs of the starfish-Patiria pectinifera: glucosophosphatisomerase, phosphoglucomutase, adenineat^ cinâse, hexokinase, oXidase of tetrazolium, xanthindehydrogenase, a - glycerophosphatedehydrogenase, 6 = phosphogluconatedehydrogenase, glucoso-6-phosphatedehydrogenase and malate dehydrogenase

In pôpulation genetics wide use is made of biochemical features found

by the methdd of electrophoresis in gels (Habby and Lewontfin, 1966 ; Altukhov,

1973, and others) The Mendelizing, as a rule monogenetic, features of a

protein nature,are-a direct result of the activity of individual genes.

Numbers in the right-hand margin indicate the correspondzng pages in the original.

-Trarislàtôr's-'note. The foregoing names have all been takenndirectly from the or'iginal Russiâ.n; hence spellings may in individual cases vary some- what from the standard orthography both here and elsewhere in this translation.

SEC 5-25T (6/76) 2

Polymorphic and monomorphic proteins are used not ânly in population, but also in taxonomic, research on different groups of . Dhanani and

Kitto (1970). as well as Schopf and Murphy (1973)y showed that it is advisable to use biochemical features when resolving problems involving the macro- an.dd microevolution of . Moore and Villéé (1963) have studied the electrophoretic spectra of multiple forms of malate dehydrogenase in embryos of the starfish Asteria.s ^forbesi. Dhanani and Kitto (1970) and Schopf and

Murphy (1973) have described a series of enzymes found with starch gel electrophoresis in some of starfish, This paper describes the electro- phoretic spectra of 10 enzymes of the starfish Patiria pectinifera.

Materials and Methods

In our study we used 26 starfish of Patiniâ. Tectinifèra (Mûller et , .3 .. Troschel), collected in November in Ussuri Gulf of the Sëà of Japan (Lazurnaya

Bay). We studied the ambulacral feet, stomachs,lliver processes and gonads of both live animals and specimens stored at a temperature of -55°C for 60 days.

We got extracts of the organs by homogenizing them in an equal amount of deionized water and centrifuging the homogenates at 18,000 g for 1 hour.

We used the supernatant for electrophoresis, All of the operations involved in deriving the extracts, were carried out in the cold.

We performed electrophoresis in a 14% starch gel in a discontinuous ^e buffer system ( BS-1): gel buffer - 0.05M tris-0.002M EDTA -Na2 - boric acid, pH 7.9; electrode7-,buffer - 0.3M boric acid - NaOH, pH 8.1, after Serov (Serov,

1973), as well as in continuous buffer systems:

1) gel buffer - 0.01M phosphate, pH 6.8; electrode buffer - 0.2M phosphate, pH 6,,^J BS-II);

-Tràrislàtor's note. Expands to ethylenédiaminetetraacetic acid. I • 3

2) gel buffer - 0.01M tris - 0.0029M citric acid, pH 7.0; electrode

buffer - 0.155M tris - 0.043M citric acid, pH 7,0 (BS-III), after Shaw and

Prasad (1970).

When we finished the electrophoresis we cut the gel into plates 2 mm

thick and found the zones of enzyme activity using the standard histochemical

colors after Shaw and Prasad (1970); we found tetrazolium oxidase after

Breyer (1967).

Results and Discussion

We got electrophoretic spectra of the following enzymes: /5 8

glucosophosphatisomerase, phosphoglucomutase, adenylatkinase, hexokinase,

oxidase of tetrazolium, xanthindehydrogenase, a - glycerophosphatedehydro-

genase, 6 - phosphogluconatedehydrogenase, glucoso-6-phosphatedehydrogenase

and malate dehydrogenase.

Glucosophosphatisomerase (GPhI). The enzyme spectrum from the stomach,

the liver processes and the ambulacral feet during electrophoresis in BS-1

was represented by one zone (Fig. 1, 2, see inset I), which had the same

mobility for all organs. The most intensively colored GPhI zones were

[those of] the stomach and ambulacral feet. When we stored the organs in a

frozen state, and the extracts at a temperature of 3°C, the activity of the

enzyme dropped considerably or disappeared altogether,

Phosphoglucomutase (PhGM), During electrophoresis of freshly prepared

extracts of the stomach, ambulacral feet and gonads in BS-III we found one

zone of activity of PhGM (Fig. 2, 5; see inset I) with the same mobility for

all organs. We found no enzyme activity in the liver processes. We noted a

conSiderable reduction in PhGM activity for organs that had been stored in a

frozen state. 4

Adenylatkinase (AK), The enzyme spectrum of the stomach, obtained in BS-III, was represented by one zone of activity (Fig. 2, 1). The same spectrum was characteristic of the ambulacral feet and livér processes, but the AK activity of the liver processes was much lower than in the other organs.

Storage of the organs in a frozen state led to a reduction in the enzyme activity, while a double freezing/thawing procedure brought about the complete disappearance of such activity,

Hexokinase (HK). The electroîshoretic HIC spectrum of different organs was represented by two zones, of which one (the fast-migrating) was monomorphic for all organs, while the second (the slow-migrating) varied in different organs with regard to mobility and intensity of coloring (Fig. 2, 2 and 3).

Enzyme activity did not drop when the organs were stored in a frozen state, but it was completely lost after a double freezing and thawing prôcedure.

--Oxidase of tetrazolium.--(0T), The OT spectrum of liver processes found during electrophoresis in BS-1 was represented by two zones (Fig. 1, 4). In the stomach and the ambulacral feet under these conditions we found two analogous zones, but the activity of the fast-migrating OT was much lower than in the case of the liver processes. We found high OT activity in freshly prepared extracts. The storagé of organs in a frozen state, and of extracts at a temperature of 3 °C, reduced enzyme activity.

-Xanthindehydrogenase (XDH), In each of the organs that we investigated we found one zone of enzyme activity with the same mobility. We noted the greatest XDH activity in the stomach and liver processes (Fig. 1, 3), Enzyme acitivity was much lower ih the gonads and in th ë ambulacral feet. The storage of the organs in a frozen state did not reduce the activity of the enzyme.

XDH quickly lost its activity in an extracted state, 5

-a - Glycerophosphatedehydrogenase (GPhD). The spectrum of the stomach enzyme that we got in BS-1 was characterized by fast-migrating and slow- migrating zones of activity (Fig. 1, 1). In the liver processes we found only a fast-migrating zone, and in the ambulacral feet, only a slow-migrating zone. When we performed electrophoresis of a stomach extract in BS-III no division of the enzyme into two fractions occurred. Freezing of the organà did not significantly affect the activity of GPhD,

6 - Phosphogluconatedehydrogenase (6-PHGD). The spectrum of 6-PhGD /59 was represented by one zone with the same mobility for all of the organs that we studied with electrophoresis in BS-III (Fig, 2, 6), as well as in BS-II.

We noted the greatest enzyme activity in the àmbulacral feet and in the stomach, but found no 6-PhGD activity in the liver processes. Only extracts prepared from the organs of live starfish are suitable for electrophoresis.

Glucoso-6-phosphatedehydrogenase (G-6-PhD). In BS-II we found one zone of activity (Fig. 1, 5) whose mobility was the same for all organs. An exception was the G-6-PhD of the ambulacral feet, where we found two zones, of which one (the slow-migrating) colored intensively, while the second coin- cided in mobility with a zone found in other organs, bût colored much more weakly. In the same way as 6-PhGD, ,er:6-PhD was found only with the electro- phoresis of extracts derived from the organs of live starfish. When we increased the time of the electrophoresis, the enzyme activity decreased considerably, which, as Manwell and Baker (1963) have shown, is also chàracteristic of the

G-6-PhD of holothurians.

Malate dehydroenase (MDR>. The MDH spectrum of the different organs derived in BS-III was represented by two zones, of which one migrated faster towards the anode and was equally mobile in all of the organs that we analyzed, while the second, slow-migrating, zone of the MDH of different organs varied 6 in its electrophoretic parameters (Fig. 2, 4). The slow-migrating zone of the

MDH of the liver processes migrated faster than the corresponding zone in other organs, and in our phoregrams it appeared in an intermediate position in relation to the two zones of the MDH of the gonads, the stomach or the ambulacral feet. When we stored the organs in a frozen state, the enzyme activity decreased insignificantly.

Our research on the intraspecies variability of the electrophoretic spectra of MDH during electrophoresis in BS-III has shown that the fast- migrating zone of MDH, which is probably of mitochondrial origin (Dhanani and

Kitto, (1970), is electrophoretically monomorphic, while the slow-migrating zone, which results from the activity of soluble MDH, is polymorphic. We found sevenrelectrophoretic variants of soluble MDH in 20 starfish that we investigated (Fig. 3, 1-4, 6-8, see inset I). These variants differoeither in their number of zones or in their mobility and are divided into two groups: variants with one zone of activity, and variants with three zones of activity of soluble MDH. The nature of their relative distribution, as well as of the relative intensity of coloring of their zones, permits us to assume that these variants are genetically controlled by soluble MDH. Probably the locus of the soluble MDH of P. pectinifera is represented by a series of alleles that inter- act in accordance with the codominant type, while the molecule of the enzyme consists of two subunits. The authors (Serov and Manchenko, 1974) have pro.1= posed and demonstrated an analogous genetic model for similar electrophoretic variants of 6-PhGD of erythrocytes in rats.

Final conclusions as to the nature qf the piilymorphism of soluble

MDH of P, pectinifera require additional population/genetic research.

Thus the number of zones of activity of the enzymes that we have analyzed, as well - as the pattern of distribution of the zones among the organs, 7

indicate that one locus each of GPhI, PhGM, AK, XDH, and 6-PhGD, and two

loci,each of HK and MDH are present in P. ,pectiriifera. Probably GPhD and

G-6-PhD each correspond to one locus, but we have not ruled out the possibility

of the manifestation of differential activity by two non-allele genes of these

enzymes in different organs.

References

1. Altukhov, Yu. P., 1973 - Local stocks of fish as.genetically stable popu- /60 lation systems. In the collection of papers entitled Biokhimicheskaya genetika tyb (The Biochemical Genetics of Fish), Leningrad, Nauka (Science) Press, 43-53.

2. Serov, 0. L, and Manchenko, G. P., 1974 - Genetic control of electro- phoretic variants of 6-phosphogluconatedehydrogenase in wildeand+' laboratory rats. Genetika, 10, 3:40-43.

Remaining references in English. -- .,.-.. . JIHTepâTypa

Ji T y Y 0 B IO. I-I, 19]3, ,JIOKaJibHble T pbi6 xaK 'reHeTHYeCKH CTaiiJIbHbIQ nOnyJFft- 4FlOHHbie CHCTeMbi. B CO.: bHOYNNnn4ecICaH reHeTnKa pbig, ,ÎI„ «HayxaN: 43-53. 2p C e p o s 0. JI, n A'I a,H ti e H x o I. ri. 1974. I'eaeTH.veoTaTiiF xOHTponb sneKnpocpopeTir- 9eciQHJC BapHa•HTOB 6•cpoccporn K)ICOHa•,^eitHmporeHa3bi y j[FIQ4HY H JIaSOpaTOpHbI;C KpbFC., I'eHeT^TFKa, 10,3: 40-4t3. B r e. v e r G. J. 1^1jtr7. Achr.omat.ic reg.ions of tetrasolium stained starch electrophoretic varûatiôn.. Am, j. human gen., 19: 67.4. D h a n a n T Z. and K i t t o G. B. 1a70. Comparative studies of - asparta-- te artiinotrancfaracwc T„F h;....t,.,.,, + o. - . H u b b y J. L. and L e w ô n t i n R. C.. 1966. A molecula•r a genic heterozy.gosity in.'natural populations. I. The numbeprpofa llelestof different loci in Drosophila psetidoobscura. Genetics, 54:.577-594, -.. . - ivl a n w è l l C. and . B a k e r C. Al. A. 1963: A sibling species of -sea cucumber dis- ' covered by starch gel electrophore5,is. Cômp. biochem. physiol., : 10: 39-53. M o o r e R. O. and V i 11 e e C. A. 1963. Multiple molecular forms of malic dehydroge- nase in echinoderm embryos. Comp, bi.6chem. physiol:, 9: 81-94. S c h o p f T. J. M. and M u r p h y L. S.. 1973. Protein , polymorphism. of the hybridi= zing seastars Asterias forbesi and Asterias vulgar.is., and ''implications for their evolution. Biol. bull., 1.45: 589-597, S e r o v O. L. 1973. Genetic control of two esterases of rat plasma ( Eiochem: gen., 9: 1,17--130. Rattus norvegicus). S h a w C. R. and P r a s a d R. 1070. Starch rni electrophoresis of enzymes. A compi- ,•.; `_. lation of recipes. Biocherp. gen., 4: 297-320. TlôcTynFrna 18 IV 1975

E.LECTROPHORETIC PATTERNS OF SOME ISONÎERASES ,TRANSFERASES AND OXYDOREDUCTASES FROM THE STARFISH PATIRIA PECTINIFERA _ G. P. Manchenko and O. L. Serov

Laboratory of Genetics Institute of Marine Biology, Far East Science Cen tre, • Acadèmy of Sciences of the USSR, V l a d i v o s t o k 690022-.,. and; Laboratory of, Deaelopmental Biology Institute of Cytology and 'Genetics, ;: Srberian Branch of the :4cademy of Sciences of the USSR, N o v ô s i b-i r s Iz 630090

SUibIL!NIARY

Ten enzymes of different organs from the starfish Patiria pectinifera (Müller'. et Troschel) have been invesi+igated by V^èrtical starch gel electrophoresis. These enzy- , . mes Are glucosephosphate isomerase, phosphoglucomutase, adenilate kinase, hexokinase; tetrasolium oxydase, sant'ine dehydrogenase, 6-phosphogluconate dehydrogenase, gluco= - se 6-phosphate dehyr?rogenase and malate dehydrogenase. Electrophoretdc patterns of the- se enzymes have been obtained and described for different organs, of the starfish. The ir.traspecific,ivâriability,of soluble :°'dndér thë..genëticcontï•ol: malate dehydrogenase was found and supposed to be 8 •

InetI 'paper by Ci•.e.iMenchenIto . and 041" aerov I ; CT. 17. Ala/vie/4,K° .u' O. ‘17.. Ceposa (K éTp. 5 (p ,5e)

1 2 3 4 5 Ô-

Pile. 2 Fig. 2

Figure 1 - Electrophoregrams of Figure 2 - Electrophoregrams of enzymes enzymes of the starfish-Patiria of the starfish Patina pectinifera, pectinifera derived in borate (1-4) derived in a tris-citrate buffer system. and phosphate (5) buffer systems, 1 - adenylatkinase of the stomach; 1 - a - glycerophosphatedeherogenase 2 - hexokinase of the liver processes; of the stomach; 3 - hexokinase of the stomach; 2 - glucosophosphatisomerase of the 4 - malate dehydrogenase of the liver processes; ambulacral feet; 3 - xanthindehydrogenase of the liver 5 - phosphoglucomutase of the stomach; processes; 6 - 6 - phosphogluconatedehydrogenase 4 - oxidase of the tetrazolium of the of the ambulacral feet. liver processes; 5 - glucoso-6-phosphatedehydrogenase of the stomach. -4'

Figure 3 - E1ectrophoregram of ma1ate dehydrogenase of the stomachs of dif- ferent specimens (1-10) of the starfish'Patiria « pectinifera.