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)

Dithiobis (succinimidyl propionate) has been used to cross-link sarcoplasmic reticulum microsome proteins. Although the 100,000 dalton calcium stimulated ATPase and the 60,000 dalton calcium-binding protein calsequestrin were readily cross-linked to form homopolymers, no heteropolymer formation between these two proteins were detected. The 90,000 dalton protein A1 which is always observed in our preparations appeared to preferrentially form dimers on cross-linking. When calsequestrin was solubilized using 0.1 mg deoxycholate/mg protein, this protein was not cross-linked even at dithiobis(succinimidyl propionate) concentrations ten times those used to cross-link this protein in the intact membrane. In a similar manner the deoxycholate-solubilized ATPase (0.5 mg deoxycholate/mg protein) was not cross-linked by dithiobis (succinimidyl propionate). These results suggest that the state of aggregation of the sarcoplasmic reticulum proteins may be modified when solubilized in detergents such as deoxycholate. When the 100,000 dalton ATPase polypeptide was cleaved with trypsin to two fragments with molecular weights of approximately 55,000, these could be readily cross-linked. The fragments were capable of forming polymers with either other 55,000 dalton fragments or with the 100,000 dalton ATPase. The 29,000 and 22,000 dalton fragments, produced by further tryptic cleavage of the 55,000 dalton fragments, were not cross-linked at dithiobis (succinimidyl propionate) concentrations which readily cross-linked the 55,000 dalton fragments. Thus tryptic cleavage of the ATPase to fragments smaller than 55,000 dalton altered associations made by the ATPase in the membrane.
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PMID:The effects of deoxycholate and trypsin on the cross-linking of rabbit skeletal muscle sarcoplasmic reticulum proteins. 15 Feb 90

The amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.
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PMID:Calcium-binding protein of bovine intestine. The complete amino acid sequence. 117 41

The tryptic hydrolysis of bivine intestinal calcium-binding protein in the presence and absence of excess calcium has been investigated. Calcium-binding activity and immunological reactivity of the protein were not significantly affected in the presence of 1.0 mM CaCl2 following 24 h incubation at 38 degrees C with trypsin at ratios of 1:9 of enzyme to calcium-binding protein. Some modification of the protein did occur under these conditions, however, since analysis by analytical acrylamide gel electrophoresis indicated the formation of a more rapidly-migrating species from the slower-moving original protein band. Omission of added calcium from the incubation medium resulted in rapid and essentially complete destruction of calcium-binding activity and immunological reactivity, and the formation of peptides of low molecular weight. This provides evidence that the conformation of the calcium-binding protein in the presence of calcium differs from that in its absence.
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PMID:The effect of calcium on the tryptic digestion of bovine intestinal calcium-binding protein. 123 20

We previously described a novel molecular chaperone (designated p88) that participates in the assembly of murine class I histocompatibility molecules (Degen, E., and Williams, D. B. (1991) J. Cell Biol. 112, 1099-1115). Our findings suggest that p88 may either promote proper assembly of class I molecules or retain them, probably within the endoplasmic reticulum (ER), until assembly of the ternary complex of heavy chain, beta 2-microglobulin, and peptide ligand is complete. In this report, we compare p88 to calnexin, a calcium-binding 90-kDa phosphoprotein of the ER membrane (Wada, I., Rindress, D., Cameron, P. H., Ou, W.-J., Doherty, J.-J., II, Louvard, D., Bell, A.W., Dignard, D., Thomas, D. Y., and Bergeron, J. J. M. (1991) J. Biol. Chem. 266, 19599-19610). We show that p88 and calnexin share antigenic epitopes defined by a polyclonal anti-calnexin antiserum. Furthermore, both proteins were immunoprecipitated in association with an intracellularly retained variant of the class I H-2Kb molecule. Since p88 and calnexin were also indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, were resistant to digestion with endoglycosidase H, and exhibited virtually identical patterns of peptide fragments following digestion with either V8 protease or trypsin, we conclude that p88 and calnexin represent the same protein. The identification of the p88 chaperone as a phosphorylated, calcium-binding protein of the ER membrane suggests possible means whereby its interaction with class I molecules may be regulated.
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PMID:The p88 molecular chaperone is identical to the endoplasmic reticulum membrane protein, calnexin. 135 Feb 81

Rabbit lung flavin-containing monooxygenase (FMO, EC 1.14.13.8) was denatured, reduced, carboxymethylated, digested with endoproteinase Glu-C or trypsin, and subjected to mass spectrometric analysis. The amino acid sequences of selected peptides were determined by tandem mass spectrometry. Over 90% of rabbit lung FMO was mapped by liquid secondary ion mass spectrometry (LSIMS). The FMO N-terminal amino acid was found to be N-acetylated, and the N-terminal 23 amino acid peptide contained an FAD binding domain consisting of Gly-X-Gly-X-X-Gly. Another peptide was found to contain a NADP+ binding domain consisting of Gly-X-Gly-X-X-Ala. The mapped and/or sequenced peptides were found to be completely consistent with the peptide sequence deduced from the cDNA data and the previously published gas-phase sequencing data. Further mass spectrometry and protein analytical work unambiguously showed that rabbit lung FMO existed in tight association with a calcium-binding protein, calreticulin. Over 68% of rabbit lung calreticulin was mapped by LSIMS. Tandem mass spectrometric and gas-phase sequencing studies provided direct evidence for the identification of the N-terminal and other rabbit lung calreticulin-derived peptide sequences that were identical to other previously reported calreticulins. The complexation of calreticulin to rabbit lung FMO could account for some of the unusual physical properties of this FMO enzyme form.
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PMID:Evidence for complex formation between rabbit lung flavin-containing monooxygenase and calreticulin. 191 80

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] has been found to exert its effects in a manner entirely analogous to that of other steroid hormones and is known to induce the synthesis of a calcium-binding protein (CaBP). The effects of 1,25-(OH)2D3 and dietary alteration on genomic expression in rat kidney were studied by in vitro translation of poly(A+)-containing RNA and by immunoprecipitation. Poly(A+)RNA from rat kidneys was translated in a rabbit reticulocyte lysate system in the presence of [35S]methionine, and the renal CaBP mRNA translation product was identified and quantitated by specific immunoprecipitation. Total translation products and specific immunoprecipitable products were visualized on sodium dodecyl sulfate-gels, followed by fluorography. After the addition of affinity purified rat renal CaBP antiserum to the 35S-labeled translation products, only one protein band, electrophoretically indistinguishable from that of purified renal CaBP (mol wt, 28,000), was observed. When 10 micrograms purified renal CaBP were added to the translation product before addition of the antiserum, immunoprecipitation of the 35S-labeled 28,000 mol wt protein was not observed. A comparison of the peptides produced after limited digestion with trypsin of 125I-labeled CaBP and [3H]tyrosine-labeled translation product indicated a good coincidence of peaks from purified 125I-labeled CaBP and the immunoprecipitated translation product, suggesting that the immunoprecipitated translation product is indeed vitamin D-dependent renal CaBP. When 100 ng 1,25-(OH)2D3 were injected for 7 days to 8-week-old vitamin D-deficient rats, there was a 4-fold increase in CaBP mRNA levels in the kidney (quantitated by densitometry of immunoprecipitates analyzed on sodium dodecyl sulfate-polyacrylamide gels). This increase in mRNA was accompanied by a corresponding increase in the concentration of renal CaBP, as measured by RIA, thus establishing a bridge between CaBP and the putative transcriptional effect of 1,25-(OH)2D3 in rat kidney. Similarly, both the concentration of renal CaBP and renal CaBP mRNA levels increased 4-fold in rats fed low phosphorus diets, increased 2-fold in rats fed low calcium diets, and decreased 67% in rats fed low sodium diets, providing evidence that the nutritional induction or decrease in renal CaBP is accompanied by a corresponding alteration in the concentration of its specific translatable mRNA. These results are consistent with a transcriptional control mechanism for the induction of renal CaBP.
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PMID:Cell-free translational analysis of messenger ribonucleic acid coding for vitamin D-dependent rat renal calcium-binding protein. 241 31

Plasma membrane has been prepared from pea seedlings in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Calmodulin has been detected in these plasma membrane preparations using calcium overlay techniques, immunoblots, quantitation with antibodies raised against spinach calmodulin, phosphodiesterase activation, mobility shift, and heat stability. EGTA-stable calmodulin represents 0.5-1% of the total plasma membrane protein, and it is the only detectable calcium-binding protein in plasma membrane isolated under these conditions. The anti-spinach calmodulin reacts only with the N-terminal region of spinach calmodulin representing residues 1-106. The positioning of EGTA-stable calmodulin in the plasma membrane has been probed with trypsin and anti-spinach calmodulin. The data suggest that the calmodulin N-terminal region representing residues 1-106 projects from the membrane and could be available for binding other proteins. Calcium-dependent calmodulin binding to the plasma membrane has also been detected. Calcium-dependent calmodulin-binding proteins have been characterized using calmodulin overlay methods. The exposure of calmodulin-binding domains of most of these proteins from the plasma membrane is further suggested by their reaction with azidoiodinated calmodulin.
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PMID:The location of calmodulin in the pea plasma membrane. 249 18

Calbindin D-28k is a calcium-binding protein which is not expressed by dorsal root ganglion cells cultured from 6-day-old (E6) chick embryos. When soluble muscle extracts from embryos at E11, E18 or chickens 2 weeks after hatching were added immediately after seeding, dorsal root ganglia cells grown at E6 displayed neuronal subpopulations expressing calbindin immunoreactivity with time; the effect of muscle extract on the percentage of calbindin-immunoreactive dorsal root ganglia cells followed a dose-response curve. When muscle extract was added to cultures after a 3 day delay, the percentage of calbindin-expressing neurons was unchanged. The effect produced by muscle extract and, to a lesser degree, skin extract on the appearance of calbindin-positive neurons was not reproduced by brain or liver extracts while all four exerted a trophic action on cultured neurons. Hence it is assumed that muscle extract contains a factor which produces an inductive effect on the initiation of calbindin-expression by uncommitted subpopulations of sensory neurons rather than a trophic influence on the selective survival of covertly committed neuronal subpopulations. The fact that muscle extract promoted calbindin expression by dorsal root ganglia cells in neuron-enriched as well as in mixed dorsal root ganglion cell cultures indicates that the factor would act directly on sensory neurons rather than indirectly through mediation of non-neuronal cells. Since the active muscular factor was non-dialysable, heat-inactivated, trypsin-sensitive and retained by molecular filters with a cut-off of 30 K, this factor is probably a protein.
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PMID:Inducing effect of skeletal muscle extracts on the appearance of calbindin-immunoreactive dorsal root ganglion cells in culture. 276 94

The phosphorylation of intact calmodulin and of fragments obtained by trypsin digestion was studied, using a protein kinase partially purified from bovine brain. Brain extracts were made in the presence of the detergent CHAPS (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). The protein kinase catalyzed the incorporation of nearly 1 mol of 32P from [gamma-32P]ATP into calmodulin fragment 1-106. Incorporation was exclusively into serine 101. With fragment 78-148, the extent of phosphorylation was somewhat less and 32P appeared mainly in threonine residues. Fragment 1-90 was also a fairly good substrate, but the phosphorylation of intact calmodulin never exceeded 0.01 mol per mol. Little or no phosphorylation was seen with parvalbumin, the brain Ca2+-binding protein (CBP-18) and intestinal calcium-binding protein. The protein kinase had no requirement for cAMP or phospholipids. High levels of Mg2+ (60-70 mM) stimulated phosphorylation of the fragments 20-fold. Millimolar concentrations of Ca2+ were inhibitory. It is suggested that the calmodulin fragments were in a conformation more favorable for phosphorylation than intact soluble calmodulin.
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PMID:The phosphorylation of calmodulin and calmodulin fragments by kinase fractions from bovine brain. 284 73

Tubulin is a calcium-binding protein. Two different modes of interaction of calcium with tubulin have been described: a high affinity interaction to one or two binding sites and lower affinity interactions to several other binding sites. In the present study, we have used limited proteolysis of tubulin with trypsin, chymotrypsin, and subtilisin to localize the high affinity calcium-binding sites. Our results indicate that two sites are located in the carboxyl-terminal region of both tubulin subunits, and that tubulin deprived of its carboxyl-terminal region is able to polymerize in the presence of 0.5 mM calcium.
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PMID:Localization of the high affinity calcium-binding site on tubulin molecule. 370 Apr 28


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