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 Lactate dehydrogenase (LDH, E C 1 1 1 27) occurs usually m the form of five lsozymes which are te- tramers resulting from the combination of two poly- peptlde subumts of heart (H) and muscle (M) types (Cahn et al, 1962, Markert, 1963) Proportions of LDH lsozymes occurring in particular tissues depend upon the degree of their oxygenation, what is con- nected 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 LDH in the whole organisms (Altman & Robin, 1969, Tdgner & Mlchejda, 1972) or in particular tissues (Dawson et al, 1964, Rablnowltz et al. 1967, Hel- lung-Larsen & Andersen, 1969, Crlbbs & Kllne, 1971) cultivated in aerobic or anaerobic con&t~ons shows adaptative properties to changlng environment re- quirements It can appear as the increase of synthesis of one from the subunlts (H or M), or as conforma- tlonal changes of enzyme molecule, in both cases it can be reflected in LDH kinetic alteration An important role in the regulation of metabolic processes m crustaceans play neurohormones pro- duced 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, Mc- Whlnme & Klrchenberg, 1962, McWhlnnle & Cor- kill, 1964, Obuchowlcz, 1966, Sdverthorn, 1973) as well as in carbohydrate metabolism (Scheer, 1959, McWhlnnle & Corklll, 1964, Parvathy, 1972) The ob- served metabolic changes resemble those, which occur in the mltml phase of moulting process Because LDH is one of the enzymes taking part directly m the carbohydrate metabohsm and indirectly in the respir- atory metabolism regulation, studies concerning an influence of neurohormones synthetlsed in eyestalks on the lactate dehydrogenase kinetics m hepatopan- creas and abdominal muscle of Orconectes hmosus Raf crayfish were taken up MATERIALS AND METHODS Materials Specimens of Orconectes hmosus Raf (Cam- barus 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 "rater- moult" phase LDH of hepatopancreas and abdominal muscle of cray- fishes without eyestalks were investigated after 3 hr (only LDH of hepatopancreas), 2, 6 and 27 days since the mo- ment 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), pyr- uvlc acid (PA), DE-lactic acid (DL-LA), bovine serum albu- min (BSA)--Slgma, trlshydroxymethylamlnomethan (Tns) --LOBA Chemic Wien, phenazlne methasulphate (PMS), starch--BDH, nitro-blue-tetrazohum (NBT)--Gurr, 2- merkaptoethanol--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