Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.
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PMID:Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. Kinetic properties of the basal and trypsin-stimulated activities. 12 30

The possibility that platelet activation may also involve membrane (Na-K)ATPase was investigated by testing the effects of both proteinases on platelet shape change and aggregation in the absence and presence of the specific (Na-K)ATPase inhibitor ouabain. Ouabain (8 to 80 microM) completely antagonized trypsin-induced platelet shape change and aggregation when it was preincubated with platelet suspension before the addition of trypsin. Unlike trypsin, thrombin-induced platelet activation was significantly enhanced by ouabain. It was also observed that on partially purified beef heart (Na-K)ATPase preparation, thrombin significantly enhanced the enzyme inhibition caused by submaximal inhibitory concentrations of ouabain. Soybean trypsin inhibitor (4 micrograms/ml) employed as the agent capable to counteract proteinase effects on the (Na-K)ATPase, was shown both to prevent and antagonize the platelet activation induced by trypsin (0.3 to 1.5 micrograms/ml), but it failed to modify the responses evoked by thrombin. It is concluded that membrane (Na-K)ATPase is involved differently in platelet activation by trypsin and thrombin probably because receptor sites to which either proteinase on the platelet surface binds, are distinct. Direct enzyme involvement is indeed apparent only in trypsin-induced platelet activation.
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PMID:Differential involvement of membrane (Na-K)ATPase in thrombin- and trypsin-mediated platelet activation. 132 84

Catecholamines and insulin have been reported to hyperpolarize skeletal muscle fibers via stimulation of the electrogenic Na-K pump (Flatman and Clausen, 1979, Nature, 281:580-581). Therefore, the electrogenic Na-K pump current was investigated in cultured colcemid-treated rat skeletal myoballs using whole-cell voltage clamp. Skeletal muscles were taken from newborn rat hindlegs, trypsin digested, and cultured. By day 7, all myoblast cells fused into myotubes. After treatment with the microtubule disrupter colcemid (10(-7) M) for 2 days, some of the myotubes became transformed into spherical myoballs, having an average diameter of 41.2 +/- 1.5 microns (n = 21). The resting membrane potential averaged -56.8 +/- 1.7 mV (n = 40). Ouabain (1 mM) quickly depolarized the myoballs to -51.1 +/- 1.1 mV (n = 27), showing the existence of an electrogenic Na-K pump in the skeletal myoball preparation. The values for the specific membrane resistance and capacitance were 5.5 +/- 1.0 K omega-cm2 (n = 21) and 3.7 +/- 0.3 microF/cm2 (n = 21), respectively. The pump current averaged 0.28 +/- 0.03 pA/pF (n = 10), with the membrane potential at -60 mV and 10 mM intrapipette Na+. The Na-K pump contribution to resting membrane potential was calculated to be 5.7 mV, matching the ouabain-induced rapid depolarization. When the Na-K pump was stimulated with 50 mM intrapipette Na+, the pump current was about doubled (0.52 +/- 0.08 pA/pF; n = 10). Isoproterenol (1 microM) and 8-Br-cAMP (1 mM) also significantly increased pump current by 50% (0.42 +/- 0.04 pA/pF; n = 9) and 64% (0.46 +/- 0.09 pA/pF; n = 7), respectively. In contrast, although insulin and phorbol ester also increased pump current, this increase was not statistically significant. The ineffectiveness of insulin and phorbol ester may be due to colcemid interfering with Na-K pump translocation from internal vesicles to the sarcolemma.
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PMID:Electrogenic Na-K pump current in rat skeletal myoballs. 813 86

Amino acids N886-A911 of the rat Na+-K+-ATPase alpha3-subunit were replaced by the corresponding region (Q905-V930) of the rat gastric H+-K+-ATPase alpha-subunit. The chimera (NGH26) was expressed in yeast with the rat Na+-K+ -ATPase beta1-subunit (rbeta1), the rat H+-K+-ATPase beta-subunit (HKbeta), the chimeric beta-subunit NHbeta1 (containing the carboxy-terminal ectodomain of HKbeta), or the chimeric beta-subunit HNbeta1 (containing the carboxy-terminal ectodomain of rbeta1). Increased resistance to trypsin digestion indicated that NGH26 preferentially assembled with HKbeta and NHbeta1 rather than with rbeta1 or HNbeta1. Ouabain binding also indicated that more functional complexes were assembled when NGH26 was expressed with HKbeta or NHbeta1. These results suggest that the sequence Q905-V930 interacts with the HKbeta-subunit on the extracellular side of the cell membrane. The NGH26 + HKbeta complex is less stable than alpha3 + HKbeta when heated and also has a lower binding affinity for ouabain [dissociation constant (Kd) = 63 nM] compared with alpha3 + rbeta1 or alpha3 + HKbeta (K(d) = 5-10 nM). In contrast, the NGH26+NHbeta1 complex is thermally as stable as alpha3 + rbeta1 complexes, and its ouabain binding affinity (K(d) = 10 nM) is the same as the wild type. These results indicate that the amino acids Q905-V930 of the rat gastric H+-K+-ATPase alpha-subunit preferentially associate with the extracellular domain of H+-K+-ATPase beta-subunit to form functional pump complexes and that the cytoplasmic and/or transmembrane region of the beta-subunit influences the stability of the alpha beta complexes.
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PMID:Na+-K+-ATPase alpha-subunit containing Q905-V930 of gastric H+-K+-ATPase alpha preferentially assembles with H+-K+-ATPase beta. 912 28