J.J.H.H.M. De Pont

Adenylate cyclase in the rat pancreas properties and stimulation by hormones

Abstract 1. 1. In view of evidence for a role of cyclic AMP in the secretin-stimulated water and electrolyte secretion and the pancreozymin-stimulated enzyme secretion by the pancreas, the occurrence of adenylate cyclase in the pancreas has been investigated. 2. 2. The presence of the enzyme in a particulate fraction of rat pancreas has been demonstrated. Basal activity is very low under standard conditions (less than 3 pmoles/min per mg protein), but 10 mM NaF stimulates the enzyme to 15–40 pmoles/min per mg protein. 3. 3. Properties of the enzyme have been determined in the presence of 10 mM NaF. The apparent K m for ATP, derived from a Lineweaver-Burk plot is 0.3 mM. At ATP concentrations above 1.5 mM there is substrate inhibition. The optimal pH is 7.4. The enzyme requires Mg 2+ for its activity, concentrations of 5 mM and higher giving maximal activity. Ca 2+ in concentrations above 0.1 mM inhibits the enzyme. 4. 4. The enzyme is stimulated by a synthetic secretin preparation (half maximally activating concentration 1.5·10 −8 M) as well as by a purified pancreozymin preparation (half maximally activating concentration 1.5·10 −6 M). The latter activation is not due to a contamination of the pancreozymin preparation by secretin. The secretin-stimulated enzyme has the same optimal pH of 7.4 and shows the same Ca 2+ inhibition as the NaF-stimulated enzyme. 5. 5. The enzyme is not stimulated by adrenalin and its analogues, by acetylcholine and its analogues, and by the hormones glucagon and gastrin (synthetic). 6. 6. Specific enzyme activity (per mg protein) in enzyme preparations obtained from rats with free access to food and stimulated in vivo by secretin and pancreozymin is twice as high as that in enzyme preparations from starved animals. 7. 7. The physiological significance of these findings is discussed.

Synergistic effect of A23187 and a phorbol ester on amylase secretion from rabbit pancreatic acini

The combination of the ionophore A23187 and the phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) stimulates amylase secretion from rabbit pancreatic acini up to a level equal to, or slightly higher than when carbachol is used as stimulant. Each of the two compounds alone gives only a minor stimulation. This synergistic effect of A23187 and TPA supports a role of protein kinase C in pancreatic enzyme secretion.

Role of negatively charged phospholipids in highly purified (Na+ + K+)-ATPase from rabbit kidney outer medulla. Studies on (Na+ + K+)-activated ATPase, XXXIX

1. The requirement for specific polar head groups of phospholipids for activity of purified (Na+ + K+)ATPase from rabbit kidney outer medulla has been investigated. 2. Comparison of content and composition of phospholipids in microsomes and the purified enzyme indicates that purification leads to an increase in the phospholipid/protein ratio and in phosphatidylserine content. 3. The purified preparation contains 267 molecules phospholipid per molecule (Na+ + K+)-ATPase, viz. 95 phosphatidylcholine, 74 phosphatidylethanolamine, 48 spingomyelin, 35 phosphatidylserine and 15 phosphatidylinositol. 4. Complete conversion of phosphatidylserine into phosphatidylethanolamine by the enzyme phosphatidylserine decarboxylase has no effect on the (Na+ + K+)-ATPase activity of the purified preparation. 5. Complete hydrolysis of phosphatidylinositol by a phospholipase C from Staphylococcus aureus, which is specific for this phospholipid, has no effect on the (Na+ + K+)-ATPase activity. 6. Hydrolysis of 95% of the phosphatidylcholine and 60--70% of the spingomyelin and phosphatidylethanolamine by another phospholipase C (Clostridium welchii) lowers the (Na+ + K+)-ATPase activity by about 20%. 7. Combination of the phospholipid-converting enzymes has the same effect as can be calculated from the effects of the enzymes separately. Only complete conversion of both phosphatidylserine and phosphatidylinositol results in a loss of 44% of the (NA+ + K+)-ATPase activity and 36% of the potassium 4-nitrophenylphosphatase activity. 8. These experiments indicate that there is no absolute requirement for one of the polar head groups, although in the absence of negative charges the activity is lower than in their presence.

Studies on (Na+ +K+) activated ATPase. XLI. Effects of N-ethylmaleimide on overall and partial reactions.

1. Preincubation with N-ethylmaleimide inhibits the overall activity of highly purified (Na+ +K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations of rabbit kidney outer medulla. 2. This inhibition is decreased by addition of ATP or 4-nitrophenylphosphate under non-phosphorylating conditions, and also by addition of ADP or adenylylimidodiphosphate. 3. N-ethylmaleimide treatment leads to inhibition of K+-stimulated 4-nitrophenylphosphatase activity, Na+-stimulated ATPase activity, and phosphorylation by ATP as well as by inorganic phosphate. These inhibitions strictly parallel that of the overal (Na+ +K+)-ATPase reaction. 4. N-ethylmaleimide lowers the number of sites which are phosphorylated by inorganic phosphate, without affecting the dissociation constant of the enzyme-phosphate complex. 5. N-ethylmaleimide does not affect the relative stimulation by ATP of the K+-stimulated 4-nitrophenylphosphatase activity. 6. These effects of N-ethylmaleimide can be explained as a complete loss of active enzyme, either by reaction of N-ethylmaleimide inside the active center, or by alterations in the quaternary structure through reactions outside the active center.

Adenosine 3′,5′-monophosphate phosphodiesterase assay in tissue homogenates

Abstract 1. 1. A fast, reliable and sensitive method for the assay of 3′,5′-AMP phosphodiesterase (adenosine 3′,5′-monophosphate phosphohydrolase, EC 3.1.4.c) is described, which is also applicable to homogenates and crude enzyme preparations containing enzymes catalyzing the breakdown of the reaction product 5′-AMP. The method has been worked out for rat pancreatic homogenate. 2. 2. The method is based on existing methods in which 3′,5′-[3H]AMP is used as substrate and in which the resulting 5′-AMP is dephosphorylated in a second step by means of added 5′-nucleotidase. 3. 3. It is shown that already during the first incubation step a mixture of radioactive products is formed: 5′-AMP, 5′-IMP, 3′,5′-IMP, adenosine, inosine, hypoxanthine and adenine. During the second incubation step 5′-AMP and 5′-IMP are converted into the corresponding nucleosides. 4. 4. The reaction mixture after the second incubation step is applied to a column of anionic exchange resin. All products of the phosphodiesterase reaction can be eluted with 0.1 M NaHCO3, while the unchanged 3′,5′-AMP and the product of the side reaction 3′,5′-IMP, are retained. After liquid scintillation counting of the eluate, the 3′,5′-AMP phosphodiesterase activity can be calculated.

Is there a plasma membrane-located anion-sensitive ATPase?

A study of the intracellular localization of HCO-3-stimulated, SCN--inhibited magnesium-dependent ATPase was performed in gill tissue of the rainbow trout (Salmo irideus), rabbit kidney and rabbit gastric mucosa. Tissue homogenates were subjected to centrifugal fractionation, and the microsomal (60 min 100 000 X g) and light mitochondrial (20 min 20 000 X g) fractions were further fractionated by density gradient centrifugation. Subfractions were characterized by marker enzyme assays and electron microscopic observation. In trout gill indications for an exclusively mitochondrial localization were found. In kidney no definite conclusions could be drawn. In rabbit gastric mucosa initially an apparently non-mitochondrial HCO-3-stimulated ATPase, in addition to a mitochondrial one, was found and its characteristics were studied. Further studies showed that this ATPase also appears to be of mitochondrial origin and probably represents mitochondrial inner membranes. Possible explanations for earlier conflicting reports concerning the localization of this enzyme in gastric mucosa and other tissues are discussed.

Role of negatively charged residues in the fifth and sixth transmembrane domains of the catalytic subunit of gastric H+,K+-ATPase

The role of six negatively charged residues located in or around the fifth and sixth transmembrane domain of the catalytic subunit of gastric H+,K+-ATPase, which are conserved in P-type ATPases, was investigated by site-directed mutagenesis of each of these residues. The acid residues were converted into their corresponding acid amides. Sf9 cells were used as the expression system using a baculovirus with coding sequences for the α- and β-subunits of H+,K+-ATPase behind two different promoters. Both subunits of all mutants were expressed like the wild type enzyme in intracellular membranes of Sf9 cells as indicated by Western blotting experiments, an enzyme-linked immunosorbent assay, and confocal laser scan microscopy studies. The mutants D824N, E834Q, E837Q, and D839N showed no 3-(cyanomethyl)-2-methyl-8(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATP dependent phosphorylation capacity. Mutants E795Q and E820Q formed a phosphorylated intermediate, which, like the wild type enzyme, was hydroxylamine-sensitive, indicating that an acylphosphate was formed. Formation of the phosphorylated intermediate from the E795Q mutant was similarly inhibited by K+ (I50 = 0.4 mM) and SCH 28080 (I50 = 10 nM) as the wild type enzyme, when the membranes were preincubated with these ligands before phosphorylation. The dephosphorylation reaction was K+-sensitive, whereas ADP had hardly any effect. Formation of the phosphorylated intermediate of mutant E820Q was much less sensitive toward K+ (I50 = 4.5 mM) and SCH 28080 (I50 = 1.7 μM) than the wild type enzyme. The dephosphorylation reaction of this intermediate was not stimulated by either K+ or ADP. In contrast to the wild type enzyme and mutant E795Q, mutant E820Q did not show any K+-stimulated ATPase activity. These findings indicate that residue Glu820 might be involved in K+ binding and transition to the E2 form of gastric H+,K+-ATPase.

Studies on (Na+ + K+)-activated ATPase. XLVII. Chemical composition, molecular weight and molar ratio of the subunits of the enzyme from rabbit kidney outer medulla.

1. (1) The subunits of a purified preparation of (Na+ + K+)-ATPase from rabbit kidney outer medulla have been completely separated by gel filtration in sodium dodecyl sulfate. During the gel filtration procedure 3–4% of the phospholipids present in the purified enzyme preparation remain bound to the separated subunits even after repeated gel filtration. 2. (2) The composition of the bound phospholipids does not greatly differ from that in the original enzyme preparation, which indicates that there is no specific binding of phospholipids to the subunits. The isolated β-subunit contains twice as much phospholipid per mg protein as the α-subunit. 3. (3) The α-subunit is more hydrophobic (43%) in its amino acid composition than the β-subunit (38%). The α-subunit contains more alanine and histidine and much less tyrosine and lysine than the β-subunit; the other amino acids show little difference. The amino acid composition of each subunit closely resembles that of the corresponding subunit of the enzyme from dog and lamb kidney, duck salt gland, shark rectal gland and electric eel electroplax. 4. (4) Both subunits are glycoproteins, although the β-subunit contains 5-times more carbohydrate per g protein than the α-subunit. Both subunits contain glucosamine, galactose, mannose, sialic acid and also glucose, which appears to be covalently bound. Galactosamine, in small amounts, is only detected in the β-subunit.

Dose-dependent recruitment of pancreatic acinar cells during receptor-mediated calcium mobilization.

Digital-imaging microscopy of Fura-2-loaded rabbit pancreatic acinar cells was used to simultaneously monitor the cholecystokinin-octapeptide (CCK8)-induced changes in free cytosolic Ca2+ concentration, [Ca2+]i, in large numbers of individual acinar cells. CCK8 typically evoked a switchlike increase in [Ca2+]i which was preceded by a concentration-dependent latency. The threshold concentration for the CCK8-induced rise in [Ca2+]i differed greatly among individual acinar cells, resulting in the dose-dependent recruitment of acinar cells in terms of CCK8-induced Ca2+ mobilization. The EC50 value for CCK8-induced cell-recruitment was estimated to be 15 pM. The hormone was equally potent in stimulating amylase secretion from acinar cells in suspension. At a CCK8 concentration of 100 pM, virtually all cells responded to the hormone with an increase in [Ca2+]i and the number of responding cells remained unchanged upon further increase of the CCK8 concentration. The dose-response curve for cell-recruitment coincides with that of the apparent [Ca2+]i increase in a suspension of acinar cells. This suggests that the most likely interpretation of the latter dose-response curve is not a generalized increase in [Ca2+]i but an increase in the number of responding cells. The initial rise in [Ca2+]i, which was transient by nature, was followed by repetitive [Ca2+]i transients of long duration. The dose-response curve for the effect of CCK8 on the percentage of acinar cells displaying these distinct [Ca2+]i oscillations was biphasic. A maximum of 99% of the cells showing oscillatory behaviour was reached at 100 pM CCK8, beyond which concentration the number of oscillating cells dose-dependently decreased again. The latter decrease was paralleled by a dose-dependent increase of the percentage responding but non-oscillating cells, indicating that beyond 100 pM CCK8 an increasing number of acinar cells became desensitized towards hormonal induction of oscillatory changes in [Ca2+]i. CCK8 was approximately 100-fold more potent in reducing the percentage of oscillating cells than in inhibiting amylase secretion. Oscillating acinar cells responded to a stepwise increase of the medium CCK8 concentration with a rapid change in amplitude and frequency of the oscillations. Thus, with increasing CCK8 concentration the frequency gradually increased, whereas the amplitude only slightly increased at first, reached a maximum, and decreased thereafter. In some cells full extinction was reached. Again, large differences in dose-dependency were observed among individual acinar cells. The observations presented demonstrate the existence of a marked functional heterogeneity among pancreatic acinar cells in terms of CCK8-induced Ca2+ mobilization.

Studies on (K+ + H+)-ATPase. I. Essential arginine residue in its substrate binding center.

1. A membrane vesicle fraction containing a high (K+ + H+)-ATPase activity was isolated from porcine gastric mucosa. The enzyme has a pH optimum of 7.0 and is stimulated by T1+, K+, Rb+ and NH4+ with KA values of 0.13, 2.7, 7.6 and 26 mM, respectively, at this pH. 2. Incubation of the isolated membrane fraction with butanedione leads to inactivation of the (K+ + H+)-ATPase activity. The pH-dependence of the (K+ + H+)-ATPase activity. The pH-dependence of the inactivation and the reversibility of the reaction, observed after removal of excess butanedione and borate, indicate that modification of arginine is involved. 3. The inactivation of (K+ + H+)-ATPase activity by butanedione is time-dependent and follows second-order kinetics. From the dependence of the inactivation rate on the reagent concentration it appears that a single arginine residue is involved in the inactivation of the (K+ + H+)-ATPase activity. 4. ATP, deoxy-ATP, ADP and adenylyl imidodiphosphate (AMPPNP), but not CTP, GTP and ITP which are poor substrates, protect the enzyme against butanedione inactivation, suggesting that the essential arginine residue is located in the ATP binding centre. 5. In the presence of Mg2+ the butanedione inactivation is increased, and the protection by ATP, deoxy-ATP and ADP (but not that by AMPPNP) is less pronounced. This suggests that Mg2+ induces a conformational change in the enzyme, exposing the arginine group and coinciding with phosphorylation and subsequent release of ADP from its binding site.