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)

The biosynthetic origin of the 10,000 molecular weight neurophysins, carriers of the peptide hormones oxytocin and vasopressin, has been studied by cell-free synthesis, Poly(A)-RNA was isolated from bovine hypothalamus and translated in a wheat germ system containing (35)S- or (3)H-labeled amino acids. A number of unique [(35)S]cysteine- but few [(35)S]-methionine-labeled proteins were coded by hypothalamic mRNA. A single, major, isotopically labeled protein (molecular weight 23,000-25,000) was immunoprecipitated from these translation mixtures by addition of purified antibodies against bovine neurophysin II and subsequent addition of Cowan I strain of Staphylococcus aureus. Specificity of the immunoprecipitation was demonstrated by competition with unlabeled authentic neurophysins and the absence of competition with structurally unrelated ovalbumin. Furthermore, neither nonimmune serum nor purified antibodies against ribonuclease immunoprecipitated the protein. The [(35)S]cysteine-labeled protein that was specifically immunoprecipitated was oxidized with performic acid and digested with trypsin in the presence of unlabeled, authentic bovine neurophysin II. Peptide mapping revealed that most of the major [(35)S]cysteine-labeled peptides (of the translation product) were identical to major cysteine-containing peptides of authentic neurophysin. The data show that hypothalamic mRNA directs the translation of several unique cysteine-rich proteins in an in vitro cell-free system. Furthermore, one of these proteins, which has a higher molecular weight than authentic neurophysin, is recognized by purified antibodies to bovine neurophysin II and has cysteine-containing tryptic peptides in common with those of authentic neurophysin. The data suggest that this protein is the primary translation product, pre-pro-neurophysin.
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PMID:Immunological and chemical identification of a neurophysin-containing protein coded by messenger RNA from bovine hypothalamus. 29 Oct 40

Poly(A)-containing mRNA isolated from the islets of Langerhans obtained from two species of fish, angler fish (Lophius americanus) and sea raven (Hemitripterus americanus), stimulated protein synthesis 16-fold in a wheat germ cell-free system. Characterization of the translation products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed a major polypeptide weighing 11,500 daltons that was specifically precipitated by an antibody against angler fish insulin. Partial sequence analysis of the amino terminal revealed that this polypeptide is preproinsulin, in which the amino terminus of proinsulin is preceded by either 23 (angler fish) or 25 (sea raven) amino acid residues. Translation of fish islet mRNA in a wheat germ cell-free system in the presence of dog pancreas microsomal membranes led to the correct cleavage of the nascent preproinsulin, resulting in the synthesis of authentic fish proinsulin, as verified by partial sequence analysis. Moreover, the synthesized fish proinsulin was segregated, presumably into the luminal space of the dog pancreas microsomal vesicles, because it was found to be resistant to proteolysis by added trypsin and chymotrypsin. Our data thus suggest that the mechanisms and information for the transfer of secretory proteins across the microsomal membrane are highly conserved during evolution.
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PMID:Cell-free synthesis of fish preproinsulin, and processing by heterologous mammalian microsomal membranes. 32 65

Alterations in the structure of the DNA-binding protein specified by gene 32 of bacteriophage T4 have been detected using partial trypsin digestion as a conformational probe. Limited tryptic hydrolysis of the gene 32 protein removes a fragment ("B" region), of 21 amino acids from the NH2 terminus and a 6,200-dalton fragment ("A" region) from the COOH terminus. Poly(dT), poly(dC), and single-stranded DNA increase the rate of tryptic hydrolysis of the "A" region but decrease the rate of tryptic hydrolysis of the "B" region. Oligonucleotides, which are too short to permit cooperative binding of the gene 32 protein, do not alter the rate of tryptic hydrolysis of either the "A" or "B" regions. A model which accounts for these findings requires that the "B" region be involved in gene 32 protein:gene 32 protein interactions when the gene 32 protein: DNA complex is formed. As a consequence of the gene 32 protein:DNA interaction, the "A" region should be able to participate more effectively in vivo and in vitro with other proteins involved in T4 DNA metabolism.
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PMID:Structural changes in the T4 gene 32 protein induced by DNA polynucleotides. 63 79

The hypothesis that the COOH-terminal heptapeptide mediates the aldosterone-stimulating activity of angiotensin II was evaluated by comparing the relative effects on aldosterone production of angiotensin II and the heptapeptide to angiotensin analogues that are poorly metabolized to the heptapeptide and to a nonapeptide, des-Asp-1-angiotensin I, that is directly converted to the heptapeptide. In in vivo studies utilizing the adrenocorticotropic hormone-suppressed bilaterally nephrectomized dog, angiotensin II and the heptapeptide produced statistically significant increases in both aldosterone and cortisol secretory rates (P less than 0.001 for both relations). Sar-1-angiotensin II stimulated the production of both steroids to the same extent, but had much longer duration of action. [Poly(oAc)Seryl]angiotensin II was a weak agonist, having only about 30% of the steroidogenic potency of either angiotensin II or the heptapeptide. In equimolar concentrations des-Asp-1-angiotensin I had about one-half of the aldosterone-stimulating activity of the hepatpeptide. In in vitro studies, employing the trypsin-dispersed cat adrenal zona glomerulosa cells, the steroidogenic potency of angiotensin II and the heptapeptide was identical to maximal aldosterone production of 10(-8) M peptide concentration. By contrast, the response to Sar-1-angiotensin II was approximately a 10-fold increase in relative potency, while the response to [poly(oAc)Seryl]angiotensin II demonstrated a 10-fold decrease. These findings suggest that, although the heptapeptide may play an important role in the regulation of aldosterone production, the possibility remains open that angiotensin II could stimulate aldosterone biosynthesis without prior conversion to the heptapeptide.
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PMID:The role of angiotensins in aldosterone production. 126 9

Microcapsules made from alginate-poly(L-lysine)-alginate membranes have been studied as vehicles for cell culture in a number of laboratories. We have examined their permeability, robustness and ultrastructure in detail. Permeability to globular proteins could be controlled by using poly-lysine of different mean MW in their construction. However, this parameter also affected the degree to which microencapsulated living cells leaked out of the capsules during and after preparation. Poly-lysine of low MW produced a relatively permeable and robust membrane whereas a high MW produced capsules with the reverse characteristics. A MW of 22,000 appears to be optimal in forming robust capsules which are relatively impermeable to high MW species such as immunoglobulins. The structure of the semipermeable membrane was investigated by electron microscopy and found to be complex but entirely consistent with the data on protein permeability and cell leakage. Microcapsules were not disrupted by gentle treatment with trypsin or chelating agents but dissolved with the addition of heparin, sodium dodecyl sulphate or sodium hydroxide. Empty microcapsules implanted into the peritoneal cavity of rats elicited a host cellular reaction but remained intact for at least three months.
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PMID:Semi-permeable microcapsules for cell culture: ultra-structural characterization. 194 37

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

With a view toward the determination of nucleic acid binding domains and sites on nucleic acid helix-destabilizing (single strand-specific) proteins (HDPs), we have studied the interactions of the copolymer polynucleotide photoaffinity label, poly(adenylic, 8-azidoadenylic acid), (poly(A,8-N3A] with the T4 bacteriophage HDP, 32 protein. Poly(A,8-N3A) quenched the intrinsic tryptophan fluorescence of 32 protein in a manner similar to that observed with other polynucleotides, and the effect could be reversed by addition of sufficient NaCl. The binding affinity and site size of this noncovalent interaction of poly(A,8-N3A) with 32 protein are similar to the values obtained for poly(A) and this protein. When [3H]poly(A,8-N3A)/32 protein mixtures were irradiated at 254 nm, fluorescence quenching was not reversed by NaCl, suggesting that the label was covalently bound to the protein. Mixtures of photolabel and protein subjected to short periods of irradiation (generally 1 min, 2000 erg mm-2) formed high molecular weight complexes, which when electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels were radioactive and stained with Coomassie Blue R. Under the same conditions, [3H]poly(A) failed to label 32 protein. The radioactivity of [3H]poly(A,8-N3A)-labeled complexes subjected to micrococcal nuclease after irradiation was seen to migrate just behind the free 32 protein monomer on SDS-polyacrylamide gels, indicating that portions of the photolabel not in direct contact with protein were accessible to this enzyme. By several criteria, we conclude that 32 protein was photolabeled specifically at its single-stranded nucleic acid binding site. Single-stranded nucleic acids with affinities for protein greater than that of poly(A,8-N3A) effectively inhibited photolabeling. The [NaCl] dependence of photolabeling monitored on SDS gels paralleled the NaCl reversal of (noncovalent) poly(A,8-N3A)-32 protein binding. Photolabeling reached a plateau after 1-2 min. The formation of high molecular weight complexes with increasing [poly(A,8-N3A)] paralleled the disappearance of free protein on SDS gels, and reached a saturation level of about 75% labeling. Several chromatographic procedures appear to be useful for the separation of the photolabeled complexes from free protein and photolabel. Limited trypsin hydrolysis of photolabeled 32 protein indicated that all the label was within the central ("III") portion of the protein. This approach should have general applicability to the identification of nucleic acid binding sites on helix-destabilizing proteins.
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PMID:Photoaffinity labeling of T4 bacteriophage 32 protein. 243 10

Tryptase was previously shown to undergo rapid inactivation under physiological conditions unless stabilized by the presence of heparin. The current study shows that increasing the concentration of free tryptase enhances the preservation of enzymic activity, consistent with dissociation of the tetramer, rather than autodegradation, as the mechanism of inactivation. Heparin glycosaminoglycan fragments of Mr greater than 5700 are necessary for complete stabilization of tryptase activity. This stabilizing effect depends upon negative charge density rather than carbohydrate composition. Thus, keratan sulphate or hyaluronic acid were no better than physiological buffer alone; chondroitin monosulphates and heparan sulphate each prolonged the t1/2 about 20-fold over buffer alone; chondroitin sulphate E prolonged the t1/2 69-fold; and dextran sulphate and heparin provided complete stabilization of tryptase activity for 120 min. Poly-D-glutamic acid prolonged the t1/2 55-fold. In each case the loss of tryptase activity followed apparent first-order kinetics. Increasing the NaCl concentration from 0.01 M to 1.0 M increased the stability of free tryptase. In contrast, increasing the NaCl concentration in the presence of stabilizing polysaccharides decreased the stability of tryptase until dissociation of tryptase from each polysaccharide presumably occurred; thereafter tryptase stability increased as did that of free tryptase. The effect of salt concentration on heparin-stabilized tryptase activity (as opposed to stability) was also evaluated. The mast cell proteoglycans heparin and chondroitin sulphate E, by virtue of containing the naturally occurring glycosaminoglycans of highest negative charge density, may play a major role in the regulation of mast cell tryptase activity in vivo.
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PMID:Regulation of human mast cell tryptase. Effects of enzyme concentration, ionic strength and the structure and negative charge density of polysaccharides. 244 72

Polycations such as poly-L-lysine powerfully stimulated cultured endothelial cells from pig aorta to release prostacyclin and cytoplasmic purines in a dose (charge)-dependent and molecular weight (size) dependent manner. Neutral or anionic polymers were inactive. Qualitatively similar findings were made in vivo where poly-L-lysine induced charge and size-dependent local edema formation after intradermal injection in the rabbit. Pretreatment of cultured endothelium with heparin or trypsin (but not neuraminidase) effectively reduced the response of the cells to subsequent exposure to poly-L-lysine suggesting an interaction of polycations with integral membrane proteins. Edema responses to poly-L-lysine were reduced in the presence of indomethacin suggesting that generation of an endogenous vasodilator prostaglandin, perhaps endothelial cell-derived, was an important component of the response. Poly-L-lysine-induced edema formation was not dependent on endogenous histamine release but was reduced by locally administered trasylol while soybean trypsin inhibitor failed to inhibit the response. Our results indicate that polycations such as poly-L-lysine can induce responses of vascular endothelium in vitro and in vivo and that the effects are not only charge-related but are also dependent on the size of the polycation. We suggest that naturally occurring polycations such as those derived from leukocytes and platelets may play an important role in various pathologic processes and that this may be closely related to their size.
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PMID:Endothelial functional responses and increased vascular permeability induced by polycations. 245 1

Aggregation and autophosphorylation of the insulin receptor-protein kinase, from cultured 3T3-L1 adipocytes, were studied in the presence of cationic polyamino acids. Poly-L-lysine and poly-L-arginine produced the following effects with the purified receptor: first, the autophosphorylation rate was increased by polycations. Half-maximal stimulation was proportional to polymer length. The rate enhancement was greater at lower ATP concentrations. Second, near-endpoint (equilibrium) autophosphorylation was greater in the presence of the polycations. Polycations inhibited the reverse reaction: ADP + phosphoreceptor yielding ATP + aporeceptor. Third, the [32P]phosphopeptides generated by trypsin digestion of the 32P-beta-subunit, showed that no new autophosphorylation sites resulted from the presence of polycations. Fourth, the polycations, but not insulin, promoted receptor aggregation, and phosphoreceptor aggregated more readily than aporeceptor. Insulin receptor enriched through the wheat germ agglutinin eluate step was compared with purified receptor. Higher concentrations of poly-L-arginine were required to stimulate autophosphorylation and to promote aggregation. Finally, several polycation-dependent substrates present in the wheat germ agglutinin eluate co-aggregated with the insulin receptor. Polycation-stimulated receptor autophosphorylation is linked to a lower KM,app for ATP, but substrate phosphorylation may require the aggregation.
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PMID:Insulin receptor aggregation and autophosphorylation in the presence of cationic polyamino acids. 259 62


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