Comp. Biochem. Physiol. Vol. 88B, No. 3, pp. 983-987, 1987 0305-0491/87 $3.00+ 0.00 Printed in Great Britain © 1987 Pergamon Journals Ltd GLYCOGEN PHOSPHORYLASE IN LOBSTER HEPATOPANCREAS: ACTION OF PROTEASES ON THE ENZYME JAUME ROSELL, ISABEL V. BAANANTE* a n d FELIPE FERNANDEZ Biologla General, Facultad de Biologla, Universidad de Barcelona, Avda. Diagonal, 645. 08028 Barcelona, Spain *Dept. Bioquimiea, Facultad de Farmacia, Universidad de Barcelona, Avda. Diagonal, 643. 08028 Barcelona, Spain (Received 30 January 1987) Abatraet--1. In lobster hepatopancreas, extra~:elluiar protreases cause the inactivation of glycogen phosphorylase. 2. The proteolysis of glycogen phosphorylase purified from rabbit muscle by these proteases has been shown by SDS-polyacrylamide gel electrophoresis. 3. A cell isolation technique has allowed us to remove proteases of extraceilular digestion and to measure glycogen phosphorylase activity in lobster hepatopancreas. 4. The glycogen phosphorylase activity seems to be mainly associated with R cells while it could not be detected in B cells. INTRODUCTION Crustacean hepatopancreas has two main functions: (a) secretion of digestive enzymes for extracellular food hydrolysis and (b) absorption and accumulation of nutrient reserves (Van Weel, 1974; Barker and Gibson, 1977). It is well known that these functions are carried out by different cell types. F (fibriUar) and B (blister) cells are linked in the processes of synthesis and secretion of enzymes like lipases, proteases and amylases that are involved in the extracellular digestion of food. Otherwise, the R cells (resorptive) are responsible for nutrient absorption, storage and metabolization of glycogen and lipids (Gibson and Barker, 1979; Loizzi, 1971). Several authors found difficulties in determining the activity of different enzymes from crustacean hepatopancreas (Wang and Scheer, 1963; Wieser and Lackner, 1982; Lackner, 1985). These difficulties seem to be associated with the functional character- istics of the bepatopancreas and specifically with the enzymatic inactivation caused by hepatopancreatic proteases. Usually, protease inhibitors have been used in order to avoid this inactivation (Lackner, 1985). In our assays, the use of different kinds of protease inhibitors failed to prevent glycogen phosphorylase inactivation in crude hepatopancreas extracts from the spiny lobster Panulirus regius. Cell isolation techniques have allowed us to mea- sure phosphorylase activity in extracts obtained from the cells or in extracts made with one of the cell types isolated from the hepatopancreas. To our knowledge this is the first time that glycogen phosphorylase has been measured in lobster hepatopancreas. MATERIALS AND METHODS Experimental animals Live spiny lobsters Panuliru~ regius (Zadquiey, 1968) were obtained through a commercial supplier. Lobsters were kept in aerated filtered open circuit sea-water tanks long enough to recover from stress caused during transportation. Three weeks before each assay, the animals were transferred to individual closed circuit sea-water tanks. They were fed mussels and clams. Males and females were used in the assays. The animals' weights were within a range of 250-400 g and all the lobsters were in intermoult. Animals were killed by cutting with a guillotine between cepha- lothorax and abdomen. Muscles were immediately dis- sected, frozen with liquid nitrogen and stored at -25°C. Part of the hepatopancreas was frozen in the same way and the remaining tissue was immediately used for cell isolation. Hepatopancreatic cell isolation The method for cell isolation was based on that of Ahearn et al. (1983) modified as follows. Fifty millimolar HEPES was added to all saline solutions used for pH 7.5 mainte- nance. This pH is the optimum for collagenase action (Seglen, 1976). The hepatopancreas, washed with divalent eation-free saline, was minced in small pieces and incubated in the same saline solution for 30 rain (1 ml saline/1 g tissue). After this time, collagenase previously dissolved in control lobster saline, was added to the incubation medium for 50 rain. After collagenase action, cells were recovered by centrifugation at 300 rpm (room temperature) for 6 rain in a Heraeus 7.-320 centrifuge. The ceils were resuspended in 10 ml of control lobster saline and centrifuged at 200 rpm for 5 rain. This wash process was repeated five times to complete elimination of extracellular proteases. To measure glycogen phosphorylase activity in the whole hepatopancreatic cells, the cells obtained after the last wash were homogenized in buffer solution A. To isolate different cell types, the pellet of cells obtained was dissolved in 3 ml of control lobster saline and applied to a Percoll solution 983