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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have cloned and sequenced a cDNA for the rabbit gastric proton-potassium pump (H+/K(+)-ATPase) alpha-subunit. The deduced peptide contains 1035 amino acids (Mr 114,201) and shows 97% sequence identity with the respective rat and hog proteins. A monoclonal antibody 146-14 has been shown previously to react with the extracytoplasmic side of the catalytic H+/K(+)-ATPase subunit and here we show that the epitope is in the region between amino acids 855 and 902 (the numbering of the H+/K(+)-ATPase catalytic subunit throughout the paper refers to the rabbit sequence). The localization of this epitope in conjunction with previously observed trypsin cleavage sites in the C-terminal one third of the enzyme and the hydrophobicity plot of the deduced peptide sequence are evidence for a structural model for the alpha-subunit of the H+/K(+)-ATPase which contains at least ten membrane spanning segments, similar to that deduced for the Ca(2+)-ATPase of sarcoplasmic reticulum.
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PMID:cDNA cloning and membrane topology of the rabbit gastric H+/K(+)-ATPase alpha-subunit. 131 71

cDNAs for mutant alpha-subunits of Torpedo californica (Na,K)ATPase variously truncated at the N-terminal end were constructed and transcribed in vitro. Each of the mRNAs thus synthesized was co-injected into Xenopus oocytes together with mRNA for wild-type beta-subunit. Truncation of the alpha-subunit at trypsin accessible site T2(removal of the N-terminal 36 residues, alpha delta K37) led to a decrease in ouabain-sensitive ATPase activity and ouabain-binding capacity, leaving the amount of immunoprecipitable alpha-subunit unchanged. The Km values for Na+ and K+ of alpha delta K37 were about 10mM and 2mM, respectively, and fall in the same range for the wild-type ATPase. Truncation of the alpha-subunit leaving lysine-54(alpha delta K54) or alanine-79(alpha delta A79) resulted in the loss of the ATPase activity as well as a substantial decrease in the amount of immunoprecipitable alpha-subunit. Since the beta-subunit assembles with and thereby stabilizes the alpha-subunit, which is otherwise degraded rapidly, these results suggest that the segment of the alpha-subunit between lysine-37 and lysine-54 is involved in the assembly with the beta-subunit leading to the formation of the stable and active alpha beta complex.
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PMID:Expression and assembly of Torpedo californica (Na,K)ATPase alpha-subunit truncated at N-terminal end in Xenopus oocytes. 131 53

The purpose of this study was to purify and identify the proteinase-like substance previously recognized as responsible for the Na+/K(+)-ATPase stimulating property of plasma from insulin-dependent diabetic subjects. Anion-exchange chromatography followed by two-step heparin affinity chromatography resulted in a fraction highly enriched in both potent Na+/K(+)-ATPase stimulating activity and potent proteolytic activity. Approx. 400 micrograms of purified protein was isolated from 62 mg of starting plasma proteins. When analyzed on sodium dodecyl sulfate gels the active fraction consisted mainly of one polypeptide band with an apparent molecular mass of 66 kDa under either reducing or nonreducing conditions. The proteinase-like properties of the purified fraction were further revealed by its ability to clot plasma, split fibrinogen with production of fibrinopeptide A and induce shape change in human platelets and irreversible platelet aggregation in the presence of the stable analogue of endoperoxides U46619. Its additional capacity to affect platelet phosphoinositol metabolism was shown by the stimulation of protein kinase C-dependent phosphorylation of 47 kDa platelet membrane protein. In designing an identification protocol for the purified fraction, it was postulated that plasma proteinases are probably bound to their inhibitors, to form a stable covalently linked complex. The possibility that a proteinase-proteinase inhibitor complex was purified instead of single proteinase(s) was investigated. Neither trypsin nor neutrophil elastase were present in the active fraction whereas, among the possible plasma proteinase inhibitors tested, immunoreactivity was observed only in the presence of alpha 1-antitrypsin (alpha 1 AT) antiserum. Double immunodiffusion showed that control human alpha 1 AT and the plasma-purified fraction shared common antigens. Furthermore, both isoelectric focusing and amino acid composition analysis showed that the two substances were similar. The results obtained indicate that alpha 1 AT is apparently the only active component of the purified fraction from the plasma of insulin-dependent diabetics, thus suggesting that an altered form of the inhibitor is responsible for the broad range of proteinase-like effects elicited by the plasma-purified fraction.
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PMID:Purification of proteinase-like and Na+/K(+)-ATPase stimulating substance from plasma of insulin-dependent diabetics and its identification as alpha 1-antitrypsin. 131 11

Oligomerization of newly synthesized alpha- and beta-subunits is a prerequisite for the structural and functional maturation of Na,K-ATPase. In this study, we have tested the competence of presynthesized alpha- and beta-subunits to assemble into functional enzyme complexes. Antisense oligonucleotides complementary to alpha-mRNA were used to inhibit alpha-subunit synthesis in Xenopus oocytes leaving a presynthesized trypsin-sensitive alpha-subunit pool. beta-Subunits expressed in these oocytes from injected cRNA assembled with the preexisting alpha-subunits, rendered them trypsin-resistant, and permitted the expression of more ouabain binding sites at the plasma membrane. Similarly, presynthesized beta 1- or beta 3-subunits produced in Xenopus oocytes by injection of beta-cRNA and later of specific antisense oligonucleotides were stabilized and transported out of the endoplasmic reticulum when alpha-cRNA was injected into oocytes. These data indicate that alpha- and beta-subunits can insert into endoplasmic reticulum membranes independent of each other in an assembly-competent form and retain their ability for oligomerization after synthesis.
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PMID:Beta 1- and beta 3-subunits can associate with presynthesized alpha-subunits of Xenopus oocyte Na,K-ATPase. 132 7

Na,K-ATPase from rectal glands of Squalus acanthias has been subjected to proteolysis with trypsin. The E1- and E2-forms of the enzyme can be distinguished from the inactivation patterns at low trypsin concentrations, as previously seen with kidney enzyme. Extensive degradation by trypsin in the presence of 5 mM Rb+ yields membrane fragments with a 19 kDa peptide as the major proteolytic fragment of the alpha-subunit. The sequence of the N-terminal 40 residues of this peptide is almost identical to that of a similar proteolytic fragment isolated by Capasso et al. (Capasso, J.M., Hoving, S., Tal, D.M., Goldshleger, R. and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1150-1158) using kidney Na,K-ATPase. Rb+ occlusion can be fully retained under these circumstances, supporting the findings with kidney enzyme that only minor parts of the alpha-subunit are required to form a functional occlusion-site.
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PMID:Occlusion of Rb+ after extensive tryptic digestion of shark rectal gland Na,K-ATPase. 132 76

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

Using extracted human deciduous teeth undergoing physiologic root resorption, this author studied the ultrastructural and cytochemical features of odontoclasts. The scanning electron microscopic observation of trypsin-treated dentin and cementum surfaces of resorption lacunae showed the exposure of collagen fibrils and prominent loss of the peritubular matrices around the dentinal tubules. In the resorption lacunae formed in enamel, there was dissolution of either the rod or the interrod regions. The odontoclasts developed extensive ruffled borders apposed to these resorbing matrices and had round phagosomes containing tannic acid-stainable fine amorphous inclusions, which were identical to those in the extracellular canals of the ruffled borders. The odontoclasts did not phagocytose the collagen fibrils. The odontoclasts showed the enzymatic activities of the acid trimetaphosphatase and acid p-nitrophenyl phosphatase (p-NPPase) in the Golgi-lysosome system, the ruffled border region, and along the resorbing dentin surfaces. The p-NPPase activity was inhibited by sodium tartrate. Also, the odontoclasts showed H(+)-K(+)-ATPase activity in the cytoplasm along the plasma membranes including those of ruffled border and the limiting membranes of the lysosomes. These results suggest that: 1) the odontoclasts are associated with resorption of non-collagenous organic matrices and/or extracellularly-degraded collagenous fragments rather than the incorporation of intact collagen fibrils; 2) the odontoclasts release the hydrolytic enzymes onto the lacunal surfaces and/or the lysosomes for the extra/intracellular degradation of the organic matrices; and 3) they also have H(+)-K(+)-ATPase for extracellular demineralization of the inorganic crystals.
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PMID:Ultrastructural and cytochemical study of the odontoclasts in physiologic root resorption of human deciduous teeth. 132 51

This paper summarises results and conclusions from experiments with renal Na/K-ATPase, utilising proteolytic digestion to define minimal peptide structures involved in cation occlusion and chemical modification with dicyclohexylcarbodiimide (DCCD) to investigate the role of carboxyl groups and location of K (Rb) and/or Na binding residues. Extensive digestion with trypsin or non-selective proteases in the presence of Na or Rb and absence of divalent cations reveals an essential C-terminal 19Kd fragment of the alpha chain (N-terminal asn 830) and indicates that occlusion sites of Na or K ions must reside within transmembrane segments. The bulk of the beta chain is not involved. Kinetics of inactivation of Rb or Na occlusion and covalent labelling with DCCD indicate that each of two Rb(K) or Na sites contains a carboxyl group. The third Na site may contain only neutral ligating groups. One carboxyl group is located on the 19Kd fragment and the other on tryptic fragment of about 9Kd. When cyanogen bromide was used to digest labelled alpha chain, glu 953 was found to be labelled in a Rb-protectable fashion. In tryptic "19Kd-membranes", fragments containing all putative transmembrane segments of the alpha chain have been identified (i.e. 19, 10.9, 8.7 and 8.0 Kda respectively). The cation occlusion "cage" is apparently composed of ligating groups from different trans-membrane segments, including segments of the 19Kd fragment. Construction of models is hampered by the fact that the number of the transmembrane segments is still uncertain, particularly in the crucial C-terminal domain. Alternative ways of arranging the tryptic fragments across the membrane are discussed.
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PMID:Identification of the cation binding domain of Na/K-ATPase. 133 62

The sodium pump or Na,K-ATPase, maintains the Na+ and K+ gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss of ATPase activity were prevented by co-incubation with ADP (acting as an ATP analog) or KCl. Only the alpha-subunit was labeled by SITS. The alpha-subunit from the inhibited enzyme was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino acid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated that SITS modifies Lys-501 (X) on the alpha-subunit of Na,K-ATPase.
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PMID:Inactivation of the Na,K-ATPase by modification of Lys-501 with 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS). 133 19

The regulation of acid secretion was clarified by the development of H2-receptor antagonists in the 1970s. It appears that gastrin and acetylcholine exert their effects on acid secretion mainly by stimulation of histamine release from the enterochromaffin-like (ECL) cell of the fundic gastric mucosa. The isolated ECL cell of rat gastric mucosa responds to gastrin/cholecystokinin (CCK), acetylcholine, and epinephrine with histamine release and to somatostatin and R-alpha-methyl histamine by inhibition of histamine release. Histamine and acetylcholine stimulate the parietal cell by elevation of cAMP or [Ca]i by activation of H2 or M3 receptors, respectively. These independent pathways converge to activate the gastric acid pump, the H+,K+ ATPase. Activation is a function of the association of the ATPase with a potassium chloride transport pathway that occurs in the membrane of the secretory canaliculus of the parietal cell. Hence the secretory canaliculus is the site of acid secretion, the acid being pumped into the lumen of the canaliculus. The pump is composed of two subunits, a large catalytic and a smaller glycosylated protein. This final step of acid secretion has become the target of drugs also designed to inhibit acid secretion. The target domain of the benzimidazole class of acid pump inhibitors is the extracytoplasmic domain of the pump that is secreting acid, and the target amino acids are the cysteines present in this domain. The secondary structure of the pump can be analyzed by determining trypsin-sensitive bonds in intact, cytoplasmic-side-out vesicles of the ATPase, and it has been shown that the alpha subunit has at least eight membrane-spanning segments. Omeprazole, the first acid pump inhibitor, forms a disulfide bond with cysteines in the extracytoplasmic loop between the fifth and sixth membrane-spanning segment and to a cysteine in the extracytoplasmic loop between the seventh and eight segments, preventing phosphorylation of the pump by ATP. As a result of the effective and long-lasting inhibition of acid secretion by the acid pump inhibitor, superior clinical results have been found in all forms of acid-related disease.
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PMID:Acid secretion and the H,K ATPase of stomach. 134 Oct 65


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