Stimulated Phosphatase Activity Research Articles

Sarcolemmal enzymes and Na+ -Ca2+ exchange in hypoxic, ischemic, and reperfused rat hearts.

Isolated spontaneously beating rat hearts were perfused in the Langendorff mode and divided into four groups, i.e., control (aerobic), hypoxic (95% N2-5% CO2), ischemic, and ischemic reperfused. After a total of 90 min of perfusion, the sarcolemma was isolated and enzymatically characterized. Ouabain-sensitive Na+-K+-ATPase was inhibited in all three experimental groups, whereas K+-stimulated phosphatase activity was decreased only in the ischemic and reperfused groups compared with control. 5'-Nucleotidase activity was inhibited (P less than 0.05) only in the ischemic group. Mg2+-ATPase activity was not different from control. Passive Ca2+ uptake and Ca2+ efflux were not significantly altered by any of the interventions. Na+-Ca2+ exchange rate, but not capacity, was decreased (by 32-42%) in the ischemic group but this was partially reversed on reperfusion. These results suggest that changes secondary to lack of flow rather than O2 play a major role in the etiology of ischemic damage to the membrane and that a 15-min period of reperfusion after 60 min of ischemia does not exacerbate this damage.

Interaction of Ca 2+ with (Na + + K +)-ATPase: Properties of the Ca 2+-stimulated phosphatase activity

Ca 2+ inhibited the Mg 2+-dependent and K +-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na + + K +)-ATPase. In the absence of K +, however, a Mg 2+-dependent and Ca 2+-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K +-stimulated phosphatase. The Ca 2+-stimulated phosphatase, like the K +-stimulated activity, was inhibited by either ouabain or Na + or ATP. Ouabain sensitivity was decreased with increase in Ca 2+, but the K 0.5 values of the inhibitory effects of Na + and ATP were independent of Ca 2+ concentration. Optimal pH was 7.0 for Ca 2+-stimulated activity, and 7.8–8.2 for the K +-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca 2+-stimulated phosphatase is catalyzed by (Na + + K +)-ATPase; (b) there is a site of Ca 2+ action different from the site at which Ca 2+ inhibits in competition with Mg 2+; and (c) Ca 2+ stimulation can not be explained easily by the action of Ca 2+ at either the Na + site or the K + site.

Catalytic site of calmodulin-dependent protein phosphatase from bovine brain resides in subunit A.

The calmodulin-dependent protein phosphatase from bovine brain is composed of two subunits: subunit A, Mr 60,000, and subunit B, Mr 16,500. Using in vitro immunization techniques, we have produced a monoclonal antibody specific for the phosphatase. The antibody was immobilized to Sepharose 4B to prepare an immunoabsorbent column, which was used to purify the enzyme. Phosphatase isolated from the column showed a polypeptide with Mr 60,000, equivalent to subunit A, which showed calmodulin-dependent phosphatase activity. Subunit B was not obtained from the column. Limited trypsin digestion stimulated phosphatase activity, yielding polypeptides of Mr 59,000, 43,000, and 16,000; the phosphatase activity after trypsin digestion was calmodulin independent. Chromatography of trypsin-treated phosphatase on an immunoaffinity column yielded two proteins, Mr 59,000 and 43,000, that were catalytically active and calmodulin independent. In a separate experiment, the two subunits of the phosphatase were separated by gel filtration in 6 M urea. Subunit A isolated from the filtration column showed little or no activity in the presence of Ca2+ and calmodulin, but it showed calmodulin-dependent phosphatase activity in the presence of 0.8 mM Mn2+. Subunit B was catalytically inactive. Collectively, these results indicate that subunit A and its proteolytic fragment contain the catalytic site and the antigenic determinant for the monoclonal antibody.

A role of renal K +-stimulated phosphatase activity on elevation of urinary sodium excretion in fluoride-treated rats

Fluoride administered to nonfasted rats caused an elevation of urinary sodium excretion but not an increased urinary potassium excretion as previously observed by one of us in fasted rats. This elevated urinary sodium excretion was associated with decreased renal K +-stimulated phosphatase activity. The decreased phosphatase activity was reversed by treatment with aldosterone, but not by treatment with prednisolone.

Inhibition of dog brain synaptosomal Na +-K + ATPase and K +-stimulated phosphatase activities by long chain n-alkyl-amine and -piperidine, and N″-alkylnicotinamide derivatives

The effects of piperidine, primary amine, and nicotinamide aliphatic derivatives on dog brain snyaptosomal Na +-K + ATPase were investigated. These derivatives inhibited the enzyme activity in a manner dependent on alkyl chain length. Kinetic studies revealed that inhibition of Na +-K + ATPase activity by long chain alkyl derivatives (C 12-C 18) was biphasic and non-competitive with respect to the inhibitor and substrate (ATP) concentrations respectively. These long alkyl derivatives caused changes in the Hill coefficient that suggest the occurrence of a possible conformational change in the enzyme molecule. Dual-inhibitor experiments showed that both saturated and unsaturated pentadecylpiperidine (C 15-pip) derivatives inhibited Na +-K + ATPase by the same mechanism. Oleylamine (C 18:1-NH 2), and N-dodecylnicotinamide (C 12-NA +Cl −) derivatives apparently inhibited the enzyme activity by a mechanism different from that of piperidine derivatives. Low concentrations of C 15:1-pip, C 18:1-NH 2, and C 12-NA +Cl − inhibited dog brain Na +-K + ATPase activity only, but at higher inhibitor concentrations K +-stimulated phosphatase activity was inhibited as much as Na +-K + ATPase. It is concluded that long chain n-alkyl derivatives of piperidines, amines, and nicotinamide have a cationic detergent-like action on Na +-K + ATPase, possibly by the disruption of protein-phospholipid interactions. The mechanism by which these compounds inhibit the overall Na +-K + ATPase may involve two different inhibitory sites: one, a high affinity inhibitory site, binding to which inhibits the Na +-stimulated phosphorylation reaction, and the other, a low affinity inhibitory site, binding to which inhibits the K +-stimulated dephosphorylation reaction. These possible mechanisms may provide an explanation for the observed biphasic inhibition kinetics.

Somatostatin and vitamin D3 metabolites in rat calvarium: in vitro evidence for physiological interaction.

The hypotheses that bone is a target organ for somatostatin and that vitamin D3 metabolites interact with this peptide on bone were tested in vitro, using calvaria from newborn rats with the following results. 1) [125I]iV-Tyr1 -somatostatin bound specifically to intact calvaria tissue and cells (0.1–7.5% at the 30th–50th min of incubation) and to the crude cytosol fraction (23.6 ± 4.8% of the total radioactivity incubated with cytosol). Three binding sites with different affinities could be detected; the Kd of one of them was 1.17 ± 0.17 × 10-10 M, with a binding site capacity of 0.34 ± 0.05 pmol/mg protein. 2) Low doses of somatostatin (6 × 10-9 to 3 × 10-7 M) affected the cAMP content of calvaria incubated in a protein-free medium for 5–60 min. The somatostatin-induced change was calcium dependent; there was an increase in cAMP with 0 or 1 mM calcium and a decrease with 3 mM calcium. 3) Somatostatin and vitamin D3 metabolites influenced the effects of one another on cAMP content and phosphatase activities. In the presence of 2.4 × 10-9 M 1,25-dihydroxyvitamin D3, the somatostatin affected cAMP levels at a concentration (6 × 10-11 M) that was 100 times lower than that necessary in the absence of 1,25-dihydroxyvitamin D3. The cAMP response to somatostatin was also influenced by 24,25-dihydroxyvitamin D3 (2.4 × 10-9 M), but in a different manner, with a decrease rather than an increase using 1 mM calcium. The stimulation of phosphatase activities by both vitamin D3 metabolites was decreased by somatostatin. These results support the hypotheses of a physiological role for somatostatin on bone and an interaction of this peptide with vitamin D3 metabolites.

The K +- and HCO 3−-stimulated phosphatase activities in renal microsomes of rats

The K +-stimulated phosphatase activity of microsomes from rat kidney was not inhibited by l-phenylalanine, but the HCO 3 −-stimulated phosphatase activity was markedly inhibited by l-phenylalanine. Valinomycin enhanced the HCO 3 −-stimulated phosphatase activity, but did not enhance the K +-stimulated phosphatase activity. Ouabain did not inhibit the HCO 3 −-stimulated phosphatase activity, but inhibited the K +-stimulated phosphatase activity. The renal K +-stimulated phosphatase activity was suppressed to 40% of the control values by adrenalectomy, but the renal HCO 3 −-stimulated phosphatase activity was little suppressed by adrenalectomy. The renal K +-stimulated phosphatase activity in intact and adrenalectomized rats was found to be significantly elevated, in a manner similar to the elevation of the renal (Na + + K +)-ATPase activity by aldosterone treatment ( P < 0.02).

Effects of vanadate on Na +,K +-ATPase and on the force of contraction in guinea-pig hearts

Vanadate has been shown to be a potent inhibitor of isolated Na +,K +-ATPase. Since the inhibition of this enzyme system has been implicated in a mechanism for the positive inotropic action of cardiac glycosides, the cardiac actions of vanadate were examined in connection with its action on Na +,K +-ATPase. Vanadate inhibited isolated Na +,K +-ATPase obtained from various tissues. The differences in the vanadate sensitivity due to enzyme source were relatively small. K +-stimulated phosphatase activity was more sensitive than Na +,K +-stimulated ATP hydrolysis. The compounds was more potent than phosphate in supporting [ 3H] oubain binding in the presence of Mg 2+, indicating a higher affinity of the enzyme for vanadate. It, however, failed to inhibit oubain sensitive 86Rb uptake in electrically stimulated atrial muscle of guinea-pig hearts in concentrations which would inhibit isolated Na +,K +-ATPase. These latter concentrations of vanadate also failed to produce positive inotropic effects in electrically stimulated left atrial preparations of guinea-pig hearts. Higher concentrations produced marked negative inotropic effects associated with a shortening of the action potential duration. These results indicate that vanadate is a potent inhibitor of isolated Na +,K +-ATPase, but cannot inhibit the enzyme in intact myocardial cells or produce positive inotropic effects when applied extracellularly. Inhibitory sites on the enzyme are probably located at the internal surface of the cell membrane which are normally inaccessible to vanadate in intact tissue.

Cation-dependent phosphatase activites in a rat pancreatic islet plasma membrane fraction prepared by one-step gradient centrifugation.

A plasma membrane-enriched fraction was prepared from homogenized rat pancreatic islets by a one-step sucrose gradient centrifugation. Using 125I-wheat germ agglutinin as a plasma membrane probe, a fraction was obtained at a sucrose density of about 1.10 that was enriched in 5'-nucleotidase, Mg2+-ATPase and alkaline phosphatase. The fraction contained little, if any, monoamino oxidase activity, insulin or DNA. Hydrolysis of 3-0-methyl-fluoresceinphosphate was stimulated by K+ (10mM) at a pH optimum of pH 8.2. Hydrolysis of ATP-gamma-32P in the presence of MgCl2 was of high specific activity and was optimum at pH 7.0 and 8.2. K+ did not affect ATP-hydrolysis. At pH 8.2, a small fraction of the total Mg2+-ATPase activity was inhibited by ouabain in the presence of Na+ and K+. Since K+-stimulated phosphatase activity does not correlate with Mg2+-ATPase, the two assay systems define separate enzymatic processes.

Differential pattern of developmental changes of Na+ - stimulated and nonstimulated alkaline phosphatose activities in a microsomal fraction of rat submandibular gland.

Differential pattern of developmental changes of Na+ - stimulated and nonstimulated alkaline phosphatose activities in a microsomal fraction of rat submandibular gland.

Vitamin A deficiency stimulated phosphatase activity in epiphyseal chick cartilage.

This paper reports studies of the epiphyseal cartilage in normal and vitamin A deficient chicks. The organic composition and the phosphatase activity in the resting cartilage, ossifying cartilage and new bone were measured. The ossifying cartilage and new bone had a higher content of inorganic material, phosphate and collagen than the resting cartilage. Vitamin A deficiency caused increase in the phospholipid content of all three tissues. The resting cartilage from vitamin A deficient tissue had, after homogenisation and centrifugation, a supernatant with an activity of alkaline phosphatase and glycerophosphatase higher than that in control samples. It is considered that effects of vitamin A deficiency on enzymes are related to defects of the lysosomal membrane with release of phosphatases, and that normal mineralisation also involves phosphatases activity.

Isolation of plasma membranes from Ehrlich ascites tumor cells Influence of amino acids on (Na + + K +)-ATPase and K +-stimulated phosphatase

A method was developed for isolating plasma membranes from Ehrlich ascites tumor cells. The plasma membranes appeared as highly irregular shrunken sacs or ghosts. Enzymatic characterization of the plasma membranes showed them to be high in (Na + + K +-ATPase activity and K +-stimulated phosphatase activity. A detailed study showed that both of these latter enzymic functions were stimulated by various amino acids. Such stimulation occurred in the 1–15 mM range of amino acids and was most effective for aromatic species, e.g. phenylalanine and histidine. The amino acid stimulation, which appeared to show little or no stereospecificity, was eliminated by a one carbon separation of NH 2 and COOH groups. Since the metal chelating agent EDTA was also effective in mimicking the stimulation by amino acids, and since a mild washing procedure did not render membranes insensitive to subsequent amino acid or EDTA stimulation, it is proposed that the operation of the (Na + + K +)-ATPase (and K +-stimulated phosphatase) is to some extent controlled by a tightly bound metal. The possible physiological function of an amino acid-regulated transport ATPase is discussed.

The zinc-stimulated acid and neutral p-nitrophenyl phosphatase activities of chick liver and duodenum

1. 1.|Some properties of the soluble Zn 2+-stimulated p-nitrophenyl phosphatases active at acid and neutral pH have been studied with (NH 4) 2SO 4-precipitated preparations from the soluble cytoplasm fraction of homogenates from chick liver, duodenum and metanephros. Analogous Zn 2+-stimulated phosphatase activity was not found in the particulate fraction from these tissues or in any fraction from homogenates of chick brain or heart muscle. 2. 2.|The pH optima of these soluble, Zn 2+-stimulated enzymes were at pH 4.9, 6.2 and 6.7, respectively, for duodenum, liver and metanephros. The enzymes responsible for the low level of soluble activity without added Zn 2+ or with added Mg 2+ showed maximal activity at pH 5.4–5.8 in all three tissues. 3. 3.|The soluble Zn 2+-stimulated p-nitrophenyl phosphatase enzymes of liver and duodenum were similar in most other properties examined. With preparations from both tissues, a marked (10- to 20-fold) stimulation of p-nitrophenyl phosphatase activity was elicited only by Zn 2+, but Mg 2+, Mn 2+ and Co 2+ caused some stimulation. The optimal concentration of Zn 2+ varied over the range 3–10 mM, depending on pH and type of buffer used. A marked and specific stimulatory effect of Zn 2+ was seen only for hydrolysis of p-nitrophenyl phosphate, of the many phosphate compounds tested. Hydrolysis of ATP by these preparations was moderately stimulated by both Mg 2+ and Zn 2+. Indirect evidence suggests that the Zn 2+-stimulated hydrolysis of p-nitrophenyl phosphate was not due to the inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1), fructose-1,6-diphosphatase (fructose-1,6-diphosphate 1-phosphohydrolase, EC 3.1.3.11), or ATPase (ATP phosphohydrolase, EC 3.6.1.3) present in these preparations.

DIFFERENCE IN ACTIVITY BETWEEN 2,4-DICHLORO-PHENOXYACETIC ACID AND OTHER AUXINS, AND ITS SIGNIFICANCE IN HERBICIDAL ACTION

Many reports are available in the literature on the effects of 2,4-D on numerous physiological and biochemical processes. Among these are increased cell proliferation (5), increased respiration (20), increased carbohydrate depletion (17), decreased respiration of roots (27), decreased uptake of potassium by roots (18), decreased accumulation by the root system of KNO3 and KC1 (13), inhibition of lipase activity (9), inhibition of ascorbic acid oxidase (27), stimulation of phosphatase activity (15), stimulation of beta-amylase activity (22, 28), inhibition of alphaand beta-amylase activity (14), etc. Useful though these observations are for the understanding of auxin action, they do not tell us if these physiological changes are the specific reasons 2,4-D has such a high herbicidal activity. It would rather seem that these phenomena are not specific for 2,4-D but are caused as well by other auxins such as indoleacetic acid and naphthaleneacetic acid (for terminology see 22). Thus, an increased respiration, a stimulated proliferation, and a considerable loss of carbohydrate are caused in the Jerusalem artichoke tuber by indoleacetic acid (8). Increased phosphatase activity is found in Avena roots after treatment with indoleacetic acid and other auxins (4). Inhibition of amylase activity by indoleacetic acid and by a variety of other auxins has been demonstrated (26). Indoleacetic acid, though capable of affecting many reactions in a fashion similar to 2,4-D is definitely not an efficient herbicide. The clue to the solution of why 2,4-D displays such a high herbicidal activity appears to lie in a different direction. Rather than only asking: What physiological changes does 2,4-D bring about? it would appear to be more profitable to ask first: In what fundamental aspect does 2,4-D differ from the other auxins? The present paper will largely deal with this question.