Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Isolated rat pancreatic islets were pulse-labeled for 5 min with [3H]leucine then chased for 25 min, during which time endogenously labeled [3H]proinsulin becomes predominantly compartmented in immature secretory granules. The islets were then homogenized in isotonic sucrose (pH 7.4) and a beta-granule preparation obtained by differential centrifugation and discontinuous sucrose gradient ultracentrifugation. This preparation was enriched 8-fold in beta-granules. Aside from contamination with mitochondria and a limited number of lysosomes, the beta-granule preparation was essentially free of any other organelles involved in proinsulin synthesis and packaging (i.e. microsomal elements and, more particularly, Golgi complex). Conversion of endogenously labeled [3H]proinsulin was followed in this beta-granule fraction for up to 2 h at 37 degrees C in a buffer (pH 7.3) that mimicked the cationic constituents of B-cell cytosol, during which time 92% of the beta-granules remained intact. Proinsulin conversion was analyzed by high performance liquid chromatography. The rate of proinsulin conversion to insulin was stimulated by 2.2 +/- 0.1-fold (n = 6) (at a 60-min incubation) in the presence of ATP (2 mM) and an ATP regenerating system compared to beta-granule preparations incubated without ATP. This ATP stimulation was abolished in the presence of beta-granule proton pump ATPase inhibitors (tributyltin, 2.5 microM, or 1,3-dicyclohexylcarbodiimide, 50 microM). Inhibitors of mitochondrial proton pump ATPases (sodium azide, 20 mM, or oligomycin, 10 micrograms/ml) had no effect on the ATP stimulation of proinsulin conversion. When granules were incubated in a more acidic buffer (pH 5.5), proinsulin conversion was increased relative to that at pH 7.3. At pH 5.5, ATP no longer stimulated conversion, and tributyltin and 1,3-dicyclohexylcarbodiimide had no effect. Disrupted granules only converted proinsulin to a limited extent, and neither ATP nor the inhibitors affected conversion. It is therefore suggested that ATP stimulation of proinsulin conversion in isolated, intact, beta-granules is secondary to intragranular acidification by an ATP-dependent proton pump (reflecting the low pH optimum for proinsulin conversion), rather than ATP dependence of converting activity per se.
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PMID:Stimulation by ATP of proinsulin to insulin conversion in isolated rat pancreatic islet secretory granules. Association with the ATP-dependent proton pump. 244 Aug 73

The direct addition of insulin to highly purified nuclear envelopes prepared from the livers of diabetic rats resulted in a decrease in the incorporation of 32P into trichloroacetic acid-precipitable proteins. Autoradiography of 32P-labeled envelopes, solubilized in sodium dodecyl sulfate and subjected to electrophoresis, revealed that insulin decreased the phosphorylation of all major protein bands. Insulin produced detectable effects at concentrations between 0.1 and 1 pM, maximal effects at 10 pM, and progressively diminished effects at higher concentrations. Two insulin analogs, desdipeptide proinsulin and desoctapeptide insulin, had approximately 10% and 1%, respectively, the activity of native insulin. When nuclear envelopes were first phosphorylated with [gamma-32P]ATP and insulin was then added with an excess of unlabeled ATP, dephosphorylation was enhanced, suggesting that insulin was regulating nuclear envelope phosphatase activity. The direct addition of insulin to isolated rat liver nuclei in the presence of ATP stimulated the release of previously 14C-labeled trichloroacetic acid-precipitable mRNA-like material, and the direct addition of insulin to nuclear envelopes stimulated the activity of nucleoside triphosphatase, the enzyme that participates in mRNA nucleocytoplasmic transport. Moreover, the dose-response curves for these functions mirrored insulin's inhibition of nuclear envelope phosphorylation. These data suggest, therefore, a mechanism whereby insulin directly inhibits the phosphorylation of the nuclear envelope, leading in turn to the regulation of mRNA metabolism.
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PMID:Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism. 629 83

L-Glutamine at a near-physiological concentration (1.0mM) was rapidly taken up and metabolized in rat pancreatic islets. The rate of glutamine deamidation much exceeded that of glutamate conversion into 2-oxoglutarate, the latter conversion being mediated mainly by transamination reactions. The production of 14CO2 from L-[U-14C]glutamine, which reflected the generation of ATP through the metabolism of exogenous glutamine, appeared to be regulated by the redox state of nicotinamide nucleotides and the ATP content of the islet cells. The influence of environmental factors on glutamine oxidation was examined in order to identify ATP-requiring processes. Glutamine oxidation was decreased in the absence of extracellular Ca2+, under conditions aiming at inhibition of the (Na+ + NA+)-dependent ATPase and, provided that glucose was present in the incubation medium, by cycloheximide. These findings were interpreted to suggest that the handling of Ca2+ by the islet cells, the active transport of univalent cations and the biosynthesis of proinsulin represent three major ATP-consuming processes in this fuel-sensor organ.
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PMID:The stimulus-secretion coupling of glucose-induced insulin release. Environmental influences on L-glutamine oxidation in pancreatic islets. 704 29

The NS-1 gene of the parvovirus minute virus of mice encodes a multifunctional protein essential for viral DNA replication and gene expression. In addition to possessing DNA helicase and ATPase activities, NS-1 forms a covalent linkage with the 5' ends of viral DNA and is a strong candidate for the site-specific nicking-closing enzyme postulated to be involved in the resolution of concatemers and terminal hairpin structures that arise during parvoviral DNA replication. Since the covalent linkage between NS-1 and the 5' terminus of MVM DNA resists alkali and mild acid treatment, a tyrosine phosphodiester is likely to be involved. To map domains responsible for this activity, mutations converting tyrosine to phenylalanine were introduced into the NS-1 gene using oligonucleotide-directed mutagenesis and their effect on the DNA replication and transcriptional activation functions of NS-1 was examined in transient in vivo transfection assays. Replacement of Tyr-188, Tyr-197, Tyr-210, Tyr-310, Tyr-422, or Tyr-550 with phenylalanine greatly reduced the ability of NS-1 to complement the replication of the target genome ins 20B in COS-7 cells. However, a Ser-545 to Thr-545 substitution in the Phe-550 mutant restored DNA replication activity. Replacement of 5 other tyrosines in NS-1 with phenylalanine either enhanced (Phe-6), had a moderate inhibitory effect (Phe-209) or had no effect (Phe-47, Phe 227 and Phe-543) on its DNA replication activity. Two of the 11 phenylalanine substitution mutations, Phe-188 and Phe-197, also greatly reduced the ability of NS-1 to transactivate the p38 promoter and displayed a dominant negative phenotype with respect to transactivation. Since the remaining tyrosines in MVM NS-1, Tyr-152, Tyr-252, Tyr-374, and Tyr-595, are not conserved among the NS-1 proteins encoded by porcine and feline parvoviruses, they are presumed to be nonessential for the normal functioning of NS-1. The results point to a role for either Tyr-188, Tyr-197, Tyr-210, Tyr-310, or Tyr-422 in forming a covalent linkage with viral DNA and further suggest a regulatory role for several tyrosines in other DNA replication and transcriptional activation functions of NS-1.
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PMID:Mutational analysis of conserved tyrosines in the NS-1 protein of the parvovirus minute virus of mice. 850 71

The relationship between capacitative Ca2+ influx and activation of Ca2+-dependent Cl- channels was monitored in intact Xenopus oocytes following stimulation of 5-hydroxytryptamine (5-HT) receptors, through the activity of Ca2+-dependent Cl- channels using the double-electrode voltage-clamp technique. Under voltage-clamp conditions, 5-HT evoked a rapid transient inward current followed by a slowly developing secondary inward current. The secondary current reflected depletion-activated Ca2+ entry. Hyperpolarising pulses evoked sustained Ca2+-dependent Cl- currents when applied during the transient inward current, but evoked hump-like currents which inactivated rapidly when applied during the secondary inward current. Hump currents arose from Ca2+ entering through the depletion-activated pathway. The hump currents inactivated with hyperpolarising pulses at < 5-s intervals, and recovered monoexponentially with a time constant of around 8 s. Currents in response to hyperpolarising pulses during the transient current did not inactivate, suggesting that inactivation was associated with Ca2+ entry. When ca2+ release evoked by inositol 1,4,5-triphosphate [ins(1,4,5)p3] was prevented by heparin injection, hyperpolarising pulses during ca2+ ionophore application also generated hump currents that were dependent on external ca2+, inactivated and recovered from inactivation with a similar time course as the humps following 5-ht treatment. Pretreatment with the Ca2+ adenosine 5'-triphosphatase (Ca2+ATPase) inhibitor thapsigargin reduced the rate of rise of the hump current, increased the time-to-peak of the current and slowed the rate of decay. Pharmacological interventions to disrupt the cytoskeleton reduced the amplitude of the hump current. It is suggested that, following hyperpolarisation in the presence of Ca2+ entry, the ensuing Ca2+ influx interacts with Cl- channels in a way that might reflect both Ca2+ inhibition of Ca2+ entry and clustering of Cl- channels in the plasma membrane.
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PMID:Interaction between capacitative Ca2+ influx and Ca2+-dependent Cl- currents in Xenopus oocytes. 859 48

This study was performed in order to test the hypothesis that the connecting peptide of proinsulin, C-peptide, might in itself possess biological activity. Renal tubular Na+, K(+)-ATPase, which is a well-established target for many peptide hormones, was chosen as a model. Rat C-peptide (I) was found to stimulate Na+, K(+)-ATPase activity in single, proximal convoluted tubules dissected from rat kidneys. C-peptide increased the Na+ affinity of the enzyme and all subsequent studies were performed at non-saturating Na+ concentrations. C-peptide stimulation of Na+, K(+)-ATPase activity occurred in a concentration-dependent manner in the dose range 10(-8)-10(-6) mol/l. The presence of neuropeptide Y, 5 x 10(-9) mol/l, enhanced this effect and stimulation of Na+, K(+)-ATPase activity then occurred in the C-peptide dose range 10(-11)-10(-8) mol/l. C-peptide stimulation of Na+, K(+)-ATPase activity was abolished in tubules pretreated with pertussis toxin. It was also abolished in the presence of FK 506, a specific inhibitor of the Ca2(+)-calmodulin-dependent protein phosphatase 2B. These results indicate that C-peptide stimulates Na+, K(+)-ATPase activity, probably by activating a receptor coupled to a pertussis toxin-sensitive G-protein with subsequent activation of Ca2(+)-dependent intracellular signalling pathways.
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PMID:C-peptide stimulates rat renal tubular Na+, K(+)-ATPase activity in synergism with neuropeptide Y. 863 72

Recent findings suggest that proinsulin C-peptide improves renal and nerve function as well as microcirculation in patients with insulin-dependent diabetes possibly by stimulating Na-K+-ATPase activity. Furthermore, in vitro studies on proximal rat renal tubule cells show that the effect of C-peptide on Na+, K+-ATPase activity is potentiated in the presence of the vasoconstrictor peptide neuropeptide Y. The aim of the present study was to examine whether the effects of neuropeptide Y on resting forearm blood flow in insulin-dependent patients is altered in the presence of C-peptide. Forearm blood flow was measured by a plethysmographic method in eight insulin-dependent patients and six healthy control subjects. Neuropeptide Y (20, 200 and 2000 pmol min(-1)) was infused into the brachial artery before and during an i.v. infusion of C-peptide (5 pmol kg(-1) min(-1)). Basal blood flow was 36.7 +/- 2.2 mL min(-1) L(-1) tissue. It decreased in a dose dependent manner by 11 +/- 2, 18 +/- 3 and 25 +/- 3%, respectively, during infusion of neuropeptide Y. Administration of C-peptide increased basal blood flow by 25 +/- 6%, to 46.3 +/- 3.5 mL min(-1) L(-1) tissue (P < 0.01) and forearm glucose uptake by 76 +/- 34% (P < 0.05). Infusion of the three doses of neuropeptide Y during administration of C-peptide decreased forearm blood flow by 14 +/- 4, 22 +/- 3 and 42 +/- 4%. There was a significant difference (43%, P < 0.001) between the reduction in blood flow evoked by the high dose (2000 pmol min(-1)) of neuropeptide Y before and during C-peptide infusion. Similar differences were also obtained when data were calculated as changes in vascular resistance. C-peptide did not affect resting forearm blood flow or the response to neuropeptide Y in healthy controls. In conclusion, the present data demonstrate that C-peptide increases resting forearm blood flow and augments the vasoconstrictor effects of neuropeptide Y in insulin-dependent patients.
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PMID:C-peptide potentiates the vasoconstrictor effect of neuropeptide Y in insulin-dependent diabetic patients. 1007 95

Hypoglycemia with a low serum immunoreactive insulin (IRI) level and serum immunoreactive C-peptide (IRC) level was found in a 74-yr-old female. Although a fasting test induced hypoglycemia, the responses of IRI and IRC during the fasting test, and the results of a glucose tolerance test, glucagon test, and secretin test did not indicate the presence of an insulinoma. However, the serum proinsulin level before the fasting test was 130.5 pmol/L (N: 3.0-10.0 pmol/L), and this high level was maintained throughout the test. Soon after surgical enucleation of the tumor, the patient's blood glucose levels increased. Postoperatively, the hypoglycemic status resolved, and the serum proinsulin levels returned to normal (2.8 pmol/L). Histopathological studies revealed a typical insulinoma. Immunohistochemical studies by the recently developed method for vacuolar-type H+ (V-ATPase), which is responsible for acidification of the intracellular compartments in eukaryotic cells, showed that normal islets stained positive, but not the tumor. This finding indicates that the insulin-secretory granules in the insulinoma cells existed in a microenvironment in which V-ATPase activity had been lost. This suggests that the reduced activity of V-ATPase on the endomembrane of the insulin-secretory granules in insulinomas may result in loss of the acidic microenvironment and impaired conversion of proinsulin by converting enzymes.
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PMID:Insulinoma with hyperproinsulinemia during hypoglycemia and loss of expression of vacuolar-type H(+)-ATPase (V-ATPase) in the tumor tissue. 1021 16

Menkes disease is an X-linked recessive disorder of the copper membrane transport system caused by mutations to the Menkes (MNK) gene. We identified three novel mutations of the MNK gene in three unrelated Japanese patients with classical Menkes disease by analyzing reverse-transcriptase polymerase chain reaction products and genomic DNA of the MNK gene. Firstly, an insertional mutation was found, 1173 ins A, which led to a premature termination and resulted in a very immature Menkes protein. Secondly, we found a point mutation, T2763G, resulting in a leucine-to-arginine conversion, which we predicted would cause a change in the secondary structure of the Menkes protein. Finally, we identified a splicing mutation, 2317 + 5G > C, which resulted in the skipping of both exons 8 and 9 or exon 9 only, and led to a truncation of the protein. Each of these mutations is hypothesized to destroy copper-ATPase-mediated copper transport. We propose that each of these mutations in the MNK gene plays a causative role in the disease.
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PMID:Identification of three novel mutations in the MNK gene in three unrelated Japanese patients with classical Menkes disease. 1031 89

This study was conducted to evaluate the influence of proinsulin C-peptide on erythrocyte Na(+),K(+)-ATPase and endothelial nitric oxide synthase activities in patients with type I diabetes. In a randomized double-blind study design, ten patients with type I diabetes received intravenous infusions of either human C-peptide or physiological saline on two different occasions. C-peptide was infused at a rate of 3 pmol.min(-1).kg(-1) for 60 min, and thereafter at 10 pmol.min(-1).kg(-1) for 60 min. At baseline and after 60 and 120 min, laser Doppler flow (LDF) was measured following acetylcholine iontophoresis or mild thermal stimulation (44 degrees C), and venous blood samples were collected to determine plasma cGMP levels and erythrocyte membrane Na(+),K(+)-ATPase activity. The LDF response to acetylcholine increased during C-peptide infusion and decreased during saline infusion [18.6+/-19.2 and -13.2+/-9.4 arbitrary units respectively; mean+/-S.E.M.; P<0.05). No significant change in LDF was observed after thermal stimulation. The baseline plasma concentration of cGMP was 5.5+/-0.6 nmol.l(-1); this rose to 6.8+/-0.9 nmol.l(-1) during C-peptide infusion (P<0.05). Erythrocyte Na(+),K(+)-ATPase activity increased from 140+/-29 nmol of P(i).h(-1).mg(-1) in the basal state to 287+/-5 nmol of P(i). h(-1).mg(-1) during C-peptide infusion (P<0.01). There was a significant linear relationship between plasma C-peptide levels and erythrocyte Na(+),K(+)-ATPase activity during the C-peptide infusion (r=0.46, P<0.01). No significant changes in plasma cGMP levels or Na(+),K(+)-ATPase activity were observed during saline infusion. This study demonstrates an effect of human proinsulin C-peptide on microvascular function, which might be mediated by an increase in NO production and an activation of the erythrocyte Na(+),K(+)-ATPase. These mechanisms are compatible with the previous observed microvascular effects of C-peptide in patients with type I diabetes.
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PMID:Effects of proinsulin C-peptide on nitric oxide, microvascular blood flow and erythrocyte Na+,K+-ATPase activity in diabetes mellitus type I. 1067 86


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