Comp Biochem Phl, slol

1976 Vol 54B pp 111 to 115 Pergamon Press Printed in Great Britain

KINETIC CHANGES OF LACTATE DEHYDROGENASE IN HEPATOPANCREAS AND ABDOMINAL MUSCLE OF THE CRAYFISH ORCONECTES LIMOSUS RAF. (CRUSTACEA" DECAPODA) AFTER EYESTALK REMOVAL HANNA TILGNER AND ANDRZEJ LESICKI Department of Animal Physiology. Biology Institute, A Mlckiewlcz University, ul Fredry 10, 61-701 Poznafi, Poland (Recewed 3 May 1975) Abstract--1 LDH kinetic studies of hepatopancreas and abdominal muscle in Orconectes hmosus Raf crayfish have demonstrated a kinetic resemblance of this enzyme in both tissues to the "muscle" type LDH of vertebrates 2 Decrease of KronA) and Km~LA~values as well as an increase of enzyme activity inhibition by higher pyruvate concentration after eyestatk removal was observed This indicates the LDH kinetic change of both examlnated crayfish tissues In the direction characteristic for LDH of "heart" type in vertebrates 3 Results obtained in this work suggest a LDH adaptation of both investigated tissues to the changed respiratory metabohsm in crustaceans after eyestalk removal

INTRODUCTION

influence of neurohormones synthetlsed in eyestalks on the lactate dehydrogenase kinetics m hepatopancreas and abdominal muscle of Orconectes hmosus Raf crayfish were taken up

Lactate dehydrogenase (LDH, E C 1 1 1 27) occurs usually m the form of five lsozymes which are tetramers resulting from the combination of two polypeptlde subumts of heart (H) and muscle (M) types (Cahn et al, 1962, Markert, 1963) Proportions of L D H lsozymes occurring in particular tissues depend upon the degree of their oxygenation, what is connected with the &fferent sensibility of both subunlts (H and M) on the excess of the substrate (Cahn et al, 1962, Dawson et al, 1964) It was also stated that L D H in the whole organisms (Altman & Robin, 1969, Tdgner & Mlchejda, 1972) or in particular tissues (Dawson et al, 1964, Rablnowltz et al. 1967, Hellung-Larsen & Andersen, 1969, Crlbbs & Kllne, 1971) cultivated in aerobic or anaerobic con&t~ons shows adaptative properties to changlng environment requirements It can appear as the increase of synthesis of one from the subunlts (H or M), or as conformatlonal changes of enzyme molecule, in both cases it can be reflected in L D H kinetic alteration An important role in the regulation of metabolic processes m crustaceans play neurohormones produced in the eyestalk X-organ (Hlghnam & Hill, 1969) It was stated that an eyestalk removal induces the moulting process in crustaceans and the changes in respiratory (Frost et al, 1951, Bliss, 1953, McWhlnme & Klrchenberg, 1962, McWhlnnle & Corkill, 1964, Obuchowlcz, 1966, Sdverthorn, 1973) as well as in carbohydrate metabolism (Scheer, 1959, McWhlnnle & Corklll, 1964, Parvathy, 1972) The observed metabolic changes resemble those, which occur in the mltml phase of moulting process Because L D H is one of the enzymes taking part directly m the carbohydrate metabohsm and indirectly in the respiratory metabolism regulation, studies concerning an

MATERIALS AND METHODS

Materials Specimens of Orconectes hmosus Raf (Cambarus affims Say) caught In September in the environs of Poznan were kept In running water tank at 8-12°C They were regularly fed with meat and starved during 2 days before each experiment The specimens put to the eyestalk removal were kept in similar conditions For studies there were used the crayfishes being in the "ratermoult" phase LDH of hepatopancreas and abdominal muscle of crayfishes without eyestalks were investigated after 3 hr (only LDH of hepatopancreas), 2, 6 and 27 days since the moment of eyestalk removal Puromycm dissolved In crayfish physiological solution was injected (50 #g/1 g of wet wt) through articulating membrane of left crayfish chelae After 15 rain their eyestalks were removed Reagents used in this mvestlgahon were provided by the following firms nicotlnamide adenine dlnucleotide (NAD), nicotinamlde adenine dlnucleotlde (reduced) (NADH), pyruvlc acid (PA), DE-lactic acid (DL-LA), bovine serum albumin (BSA)--Slgma, trlshydroxymethylamlnomethan (Tns) --LOBA Chemic Wien, phenazlne methasulphate (PMS), starch--BDH, nitro-blue-tetrazohum (NBT)--Gurr, 2merkaptoethanol--Koch Light Lab, agar (Special Agar Noble)--Dffco, puromycln--SERVA Other chemicals were made m Poland T,ssue preparation From muscle excised out of three first abdominal segments as well as from hepatopancreas a 20% homogenate in Isolating solution was prepared The composition of this solution was as follows phosphate buffer, l0 mM, pH 7 4, EDTA (ethylenedlamlnetetraacetate sodmm salt) 1 mM, 2-merkaptoethanol 1 mM, NAD 7 5 mM The homogenates were centrifuged m a freezing lll

112

HANNA TILGNER AND ANDRZEJ LESICKI

centrifugal for 30 mln at 17,600 g The pellet was removed and the clear supernatants of both preparations were investigated as the crude lactate dehydrogenase extracts Preparatlon and storage of extracts were carried out at 0°C Measuring of enzymatic acttvtty Lactate dehydrogenase activity of both tissues was measured spectrophotometncally (Carl Zelss Jena VSU-1) in the direction of NADH oxidation and NAD reduction, at 340 nm and 23 _+ 0 5~C For the measurements glass-cuvettes of 1 cm light-path were used The 1 5 ml of mcubatlon soluUon contained Tns HC1 buffer 100 mM pH 7 4 (or 9 0), NADH 0 08 mM (or NAD 1 5 mM), KCN 1 mM (only In the direction of NADH oxidaUon) and variable quantity of PA (or DL-LA) The enzymatic reaction was started by addition 1(~30#1 of the preparation and the enzymatic activity was marked in mlcromoles of NADH oxidized (or NAD reduced) by 1 ml of preparation in 1 min The determination of protein was carried out with the modified method of Lowry et al (1951) using BSA as a standard Electrophoreuc separations were made with the methods described by Tllgner & Mlchejda (1969) (electrophoresls on agar gel) as well as those described by Fine & Costello (1963) (electrophoresls on starch gel)

RESULTS AND DISCUSSION

Investigation of specific L D H activity in both crayfish tissues have demonstrated that the activity of this enzyme in abdommal muscle ]s almost ten-fold higher than that of hepatopancreas Specific L D H activity m both tissues and ItS proportion after eyestalk removal were stable (Table 1) Electrophoretlc separation of hepatopancreas and abdominal muscle preparations of O hmosus on agar and starch gels have revealed the presence of one anode L D H fracUon having an identical electrophoretlc mobility Even the electrophoretlc separation of abdominal muscle L D H in O hmosus made by Urban (1969) on polyacrylamade gel showed a presence of only one lsozyme In our investigations neither changes in electrophoretlc moblhty nor the appearance of a supplementary L D H band have been observed after eyestalk removal The effect of PA as a substrate on the L D H actlvlty in crayfish hepatopancreas and abdominal muscle is shown in Figs 1 and 2 Maximal L D H activity in both tissues was reached at 5 m M PA concentration The L D H activity was inhibited insignificantly (10-25%) by higher PA concentrations (20mM) In order to illustrate more adequately the mhlbmon of enzymatic actlwty by higher substrate concentrations the ratio L H, i e relation of L D H actlvlty measured at low (5 mM) to the activity at high (20mM) PA concentrations, was calculated For L D H of "muscle" type. not mhlb]ted by higher substrate concentrations. Table 1 Spec]fic LDH actlv]ty (EU/mg protein) in hepatopancreas and abdominal muscle of the O hmosus crayfish Hepa~op~ucreas

Abdominal muscle

Ioo

~. o° ~

so

a:

I 5

I I0

PA

O~utrol

0,145 + 0,023

1,23 +- 0,15

0,132 4" 0,01~

1,07 +- 0,20

Average values +- S.D. are given. Ac~ivlty was m e a s u r e d with

5 mI~ YA as a eubstrate.~

20

(raM)

Effect of eyestalk removal on the LDH activity of hepatopancreas with PA as a substrate -e = control (3). after eyestalk removal

Fig 1 crayfish

@ [] [] = 3hr (4), + - - + = 2 days (4), × - - × = 6 days (3), © © = 27 days (1) Numbers in brackets designate quantity of experiments

this ratio should approximately be equal to 1 The ratio L H for hepatopancreas L D H was equal to 1 3 and for muscle L D H 1 1 (Table 2) Michaehs constants K,.0pAJ for hepatopancreas and abdominal muscle of control crayfishes equalled 065 and 0 67 m M respectively (Table 2) In the presence of urea (2 M) only 24% of the lnmal L D H activity in hepatopancreas was stated (Fig 3) Slmdar activity inhibition of L D H from rabbit skeletal muscle was obtained in the same conditions by Wlthycombe et al (1965) The above data in&cite the similarity between the L D H kinetics of crayfish hepatopancreas and abdominal muscle and this of "muscle" type in vertebrates given by Cahn et al (1962) To such a similarity of L D H kinetics of abdominal muscle pointed

I00

"6 0

O • 50 U-, 0 n.-

i I S

I I0

PA Eyestalkless

I 15

I 15

20

(raM)

Fig 2 Effect of eyestalk removal on the LDH activity of crayfish abdominal muscle with PA as a substrate = control (4), after eyestalk removal × - - × = 2 days (3), + + = 6 days (3), C) (3 = 27 days (1) Numbers in brackets designate quantity of experiments

113

Kinetic changes of lactate dehydrogenase Table 2 Mlchaehs costants KronA)and L H ratio for hepatopancreas and abdominal muscle LDH of O l,mosus crayfish Hepatopancreas

KW,r ~ / ~ '

Abd oainal m~ole

T.,H

Km/r.t//roW/

T.,H

0,65 + 0,02

1,5

0,67 + 0,02

1t,1

0,63

1,5

0,65

1,1

5 hours

0,56 -+ 0,02

1,6

-

3 hours + puromycin

0,55

1,6

-

2 dayP

0,44 + 0,02

1,5

0,65 + 0,006

2 da~,'l + p u r t ~ l n

0,44

1,5

0,65

1,1

6 days

0 , 4 5 - 0,02

1,7

0,61 + 0,01

1,5

0,55

1,7

0,56

1,8

0,65

1,5

0,69

1,1

Control

+ puro~yein Eyes+,.alkl s s s

27 d a y s 2 days after

chelae removal

Average values + S.E. are given /S.E.

was c l a c u l a t e d

-

1,1

i f t h e number o f e x p e r i m e n t s

e x c e e d e d 3,/,

also U r b a n (1969) in O hmosus crayfish as well as Kaloustlan & Kaplan (1969) in Homarus amencanus lobster These authors have obtamed lower KmO'A) values The cause of this difference is probably the fact that they worked with purified L D H preparatlons L D H of both Ussues demonstrated a little affinity to DL-LA as a substrate (Fig 4) Accordmg to Kaloustian's & Kaplan's (1969) suggestions it may be supposed that the use of acetyl-pyrldin analogue of N A D could increase this affinity LDH of both investigated tissues of O hmosus crayfish was specific for L(+ )-LA L D H kinetic change for PA as a substrate in crayfish hepatopancreas (Fig 1) and abdominal muscle (Fig 2) was stated after eyestalk removal After the lesion a progresswe Inhibition of enzymatic actiwty at 20 m M PA concentration, so the increase of L H ratio and a diminution of K,.a,AI values, in comparison with results obtained in crayfishes from control group, was observed (Table 2) Moreover, In the hepatopancreas after 6 and 27 days since eyestalk removal a displacement of L D H actw]ty maximum in the d]i

rectxon of lower PA concentrations (1 e from 5 to 4 m M ) was stated, whereas such a change m the muscle was not observed These results suggest that hepatopancreas LDH reacts more rapidly and to a higher degree upon the changes at the neurohormone level in crayfishes' hemolymph than the muscle L D H This is also confirmed by stu&es carried out w]th LA as a substrate (Table 3) In both cases a diminution of Km~AI values after eyestalk removal is observed, but it is marked more strongly in hepatopancreas

A traumatic st]mulus (removal of chelae) did not cause any changes in L D H kinetics (Table 2) It confirms that the changes observed m L D H kinetics of both investigated tissues result from the removal of neurohormone source

?i

°J°f°

o

clO o e,,

I00 O'sN

~OSW @

\

E O

0

N I I

°~. [ 2

I ~ a _ _ :a 4

Urea (M)

Fig 3 Effect of urea on the LDH activity of crayfish hepatopancreas (3) The act]vlty was measured In a reaction of NADH ox~datlon m presence of 5 mM PA

I I00

I

I 300

I

I 5O0

DL- LA (mM)

Fig 4 Dependence of LDH activity of crayfish hepatopancreas ~ and abdominal muscle C ) ~ C) on DL-LA concentration

114

HANNA TILGNER AND ANDRZEJ LESICKI

Table 3 Mlchaehs constants Km(LA) for hepatopancreas and abdominal muscle LDH of the O hmosus crayfish

Acknowledgement--The authors are obliged to Prof Dr L Obuchowlcz (the head of Ammal Physiology Department) for the revision and discussion of this paper

r~/~, /~4 Hepa~opancreas

Control

176

Abdonlnal muscle

185

Eyeatalklaaa 2 daya

163

182

6 days

136

175

The rejection of puromycln to crayfishes did not cause the inhibition of changes in the L D H kinetics (Km~PA) as well as the ratio L H) in hepatopancreas a n d a b d o m m a l muscle observed after eyestalk removal (Table 2) The reports of Frost et al (1951), Bliss (1953), McW h l n m e & Klrchenberg (1962), M c W h l n m e & Corkill (1964) as well as Sllverthorn (1973) a b o u t a n Increase of oxygen c o n s u m p t i o n after eyestalk removal as well as K a p l a n ' s group hypothesis (Cahn et al, 1962, Dawson et al, 1964) connecting the "heart" type of L D H with the oxygen metabolism, suggest a n a d a p t a t i o n of Investigated tissues L D H to the changed respiratory metabolism a p p e a r m g after tl~s operation. It is confirmed by a displacement of kinetics of "muscle" type L D H in crayfish's hepatopancreas a n d a b d o m i n a l muscle in the direction of a characteristic kinetics for the lsozyme of "heart" type L D H of vertebrates If the kinetic change of this enzyme results from a d a p t a t i o n to the c h a n g m g respiratory metabohsm, it may be that a different degree of L D H kinetic changes m hepatopancreas a n d abdominal muscle can reflect differences in the oxygen assimilation by these two tissues Results of electrophoretlc a n d kinetic studies carn e d out with the use of puromycln suggest that the changes of L D H kmetlcs b o t h crayfish tissues effect from conformatlonal alterations of this enzyme a n d are not related to its synthesis In crustaceans after eyestalk removal the level of steroid moulting-hormone increases (Hlghnam & Hill, 1969) Steroid hormones can selectively regulate synthesis of the L D H subunlts (Goodfrtend & Kaplan, 1964, Spellman & Fottrell, 1972) as well as cause conformatlonal changes of some enzymes (Klmberg & Yielding, 1962) Therefore it is suggested that the m o u l t i n g - h o r m o n e can be a n allosterlc effector regulatmg the L D H properties in crustaceans Beside the steroid hormones this enzyme can be influenced allostertcally by metabohtes of citric acid cycle In this way the LDH-5 lsozyme (of "muscle" type) from rabbit muscle (Fritz, 1965) is regulated F r o m the studtes of other authors (Bliss, 1953, M c W h m n t e & Klrchenberg, 1962, M c W h l n n i e & Corklll, 1964, Zerbe et al, 1970) it appears that the activity of this cycle increases after eyestalk removal Consequently, the observed L D H kinetic change despite whether It is caused by allosteric effect resultlng from increased metabolic level of citric acid cycle, or by the level of moulting-hormone, IS a result of L D H a d a p t a t i o n to the changing tissue m e t a b o h s m after eyestalk removal

REFERENCES ALTMAN M & ROBIN E D (1969) Survival during prolonged anaeroblOSlS as a function of an unusual adaptation lnvolvmg lactate dehydrogenase subumts Comp Btochem PhysIol 30, 1179-1187 BLISS D E (1953) Endocrine control of metabolism m the land crab Gecarclnu9 laterahs--I Differences in the respiratory metabolism of sxnus-glandless and eyestalkless crabs Bwl Bull mar htol Lab Woods Hole 104. 275-296 CAHN R D, KAPLAN N O, LEVINI L & ZWILLIN(, E (1962) Nature and development of lactic dehydrogenase Science, N Y 136, 962-969 CRIBBS R & KLINF E S (1971) The effect of environment on lactate dehydrogenase lsozymes of cultured somatic cells J Cell Ph)~lol 78, 59-64 DAWSON D M GOODVRII~DT L & KAPLAN N O (19641 Lactic dehydrogenases functions of the two type Scwnee N Y , 143, 929-933 FINE J H & COSTELLO L A (1963) The use of starch gel electrophoresls in dehydrogenases studies In Methods m Enzymology (Edited by COLOWlCK S P & KAPLAN N O), Vol 6, pp 958 972 Academic Press, New York FRITZ P J (1965) Rabbit muscle lactate dehydrogenase 5 A regulatory properties Science, N Y 150, 364-366 FROST R, SALOUM R & KLEINHOLZ L H (1951) Effect of sinus gland and of evestalk removal on rate of ox',gen consumption m Astacus 4,at Re¢ i11, 572 GOODFRIEND T L & KAPLAN N O (1964) Effects of hormone administration on lactic dehydrogenase J btol Chem 239, 13(~135 HELLUNG-LARSEN P & ANDFRSEN V (1969) Kinetics of oxygen-induced changes in lactate dehydrogenases lsoenzymes of human lymphocytes in culture E~pl Cell Res 54, 201-204 HIGHNAM K C & HILL L (1969) Endocrine metabolism in Crustacea In The Comparative Endocrmolog) o! the Invertebrates pp 169 212, Arnold, London KALOUSTIAN H D & KAPLAN N O (1969) Lactate dchydrogenase of lobster (Hormarus amertcanus) tall muscle--II Kinetics and regulatory properties J hlol Chem 244, 2902-2910 KIMBERG D V & YIELDING K L (1962) Pyruvate klnase structural and functional changes Induced by dlethylstllbestrol and certain steroid hormones J h~ol Chem 237, 3233-3239 LOWRY O H, ROSEBROUGHN J, FARR A L & RANDALL R J (1951) Protein measurements with the Fohn phenol reagent J htol Chem 193, 265 275 MARKERT C L (1963) Lactate dehydrogenase lsoenzymes dissociation and recombination of subumts Scwn~e rV y 140. 1329-1330 M(WHINNIE M A & CORKILL A J {1964) The hexosemonophosphate pathway and its vanahon In the intermolt cycle m crayfish Comp Blochem Phystol 12, 81 93 MCWHINNIE M A & KIRCHENBERG R J (1962)Crayfish hepatopancreas metabolism and the mtermolt cycle Comp Blochem Physlol 6, 117-128 OBucnowlcz L (1966) Hormonal regulation of metabolism in the hepatopancreas mltochondrla of crayfish Astacus leptodaculus FEBS Third Meeting, Abstracts M-97. p 173, Warsaw PARVATHY K (1972) Endocrine regulation of carbohydrate metabolism during the moult cycle in c r u s t a c e a n ~ I Effect of eyestalk removal m Ocvpoda platvtarsts Mat Blol Berlin 14, 58452

Kinetic changes of lactate dehydrogenase RABINOWITZ J, LUBRANO T , WILHITE B A & DIETZ A A (1967) Lactic dehydrogenase of cultured lymphocytes response to environmental conditions Expl Cell Res 48. 675-678 SCHH R B T (1959) The hormonal control of metabolism m crustaceans--IX Carbohydrate metabohsm m the transition from mtermoult to premoult in Carcmtdes maenas Btol Bull mar btol Lab, Woods Hole 116, 175 183 SILVERTHOr~N S U (1973)Resplrahon in .eyestalkless Uca (Crustacea Decapoda) acclimated to two temperatures Comp Btochem Phystol 45A, 417-420 SV~LLMAN M & FOTTRELL P F (1972) Inhlbmon of human lactate dehydrogenase lsoenzymes by oestradlol-17fl m rltro FEBS Lett 21, 186-188 TILGNFR H & MI(HFJDA J W (1969) Studies on malate dehydrogenase isoenzymes--II Quantitative evaluation of agar gel electrophoretlc pattern Bulletin de la Socwte des Amls de Sczences et de Lettres de Poznafi D9, 157-169

115

TILGNER H & MICHEJDA J W (1972) Kinetic changes of cytozol and mltochondnal dehydrogenases (LDH and MDH) in amoeba reduced by hypoxlc culture conditions 10th Meeting of Pohsh Biochemical Society, Abstracts F-45, p 245, Poznafi URBAN J (1969) Lactat-Dehydrogenase des Flusskrebses Orconectes hmosus Isoherung und Charaktenmerung Hoppe-Seyler's Z phystol Chem 350, 721-733 WITHYCOMBE W A, PLUMMER D T & WILKINSON J H (1965) Organ specificity and lactate dehydrogenase activity Dlfferentml inhibition by urea and related compounds Bwchem J 94, 384-389 ZFRBE T . KLEPKF A K & OBUCHOWlCZ L (1970) Hormonal regulahon of metabohsm m hepatopancreas mltochondrm of crayfish Astacus leptodactylus--I The influence of eyestalks removal on the oxtdo-reduchon enzyme achvlhes Bulletin de la Socwte des Amls de Scwnces et de Lettres de Po:nah D l l , 45-51