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)

In advanced osteoarthritis, all of the cartilaginous components are lost from the joint surface. Although mechanisms exist for proteoglycan degradation, there is not known to be any system for removal of the collagen. This study suggests that the loss of the collagen components may be a function of articular cartilage collagenase. The enzyme in normal human cartilage is bound to an inhibitor and appears to be present in very small amounts. Attempts to demonstrate collagenase activity in ground human articular cartilage or in its lysosomal fraction were unsuccessful. 7-Day cartilage tissue cultures also failed to demonstrate the presence of the enzyme; but the same culture fluid, incubated with trypsin, showed significant degradation of collagen, suggesting that trypsin destroyed the inhibitor. 7-Day culture fluids were then chromatographed on a heparin-charged Sepharose 4B affinity column that had been activated with cyanogen bromide. This removed the inhibitor, and the chromatographed fluid from osteoarthritic cartilage released 42% of the incorporated counts of the collagen substrate, whereas normal cartilage released 10.1% and a trypsin control, 6.4%. Electrophoresis of the degradation products of the enzyme-collagen complex incubated at 37 degrees C revealed breakdown was complete to small dialyzable fragments, while at 25 degrees C larger fragments were split off.
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PMID:Collagenase and collagenase inhibitors in osteoarthritic and normal cartilage. 18 66

A noncovalent complex of the apoprotein (1-104) and cyanogen bromide heme fragment containing residues 1 to 65, (1-65) H, has been prepared from horse heart cytochrome c. Conditions under which the redundant portions of the ferrous complex can be removed by limited trypsin digestion have been devised. The complementing fragments have been isolated from the derived complexes and four apofragments and one heme fragment have been identified in the amino acid sequence of cytochrome c. They are (39-104), (40-104), (54-104), (56-104), and (1-53)H. The formation of an ordered ferric complex composed of one heme fragment and one apofragment for the cases (1-53)H (39-104), (1-53)H-(40-104), (1-53)H-(54-104), and (1-53)H-(56-104) has been demonstrated by the quenching of the tryptophan 59 fluorescence and the regain of biological activity in a cytochrome b2 assay. The apparent dissociation constant has been estimated as less than 3 X 10(-7) M in all the aforementioned cases. Thus, the region (between residues 38 and 57) of the amino acid sequence permissible for cleavage without disruption of the ordered structure indicated by the present in vitro experiments corresponds to that (between residues 38 and 57) evolutionally deleted in the three-dimensional structure of Pseudomonas aeruginosa cytochrome c551 discovered by Dickerson et al. (Dickerson, R.E., Timkovich, R., and Almassy, R.J. (1976) J. Mol. Biol. 100, 473-491).
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PMID:Formation of a biologically active, ordered complex from two overlapping fragments of cytochrome c. 19 Feb 31

Bovine liver and mammary UDP-galactose-4-epimerases were investigated with respect to various inhibitors and inactivators. Uridine nucleotides and NADH are potent inhibitors with Ki values in the low micromolar range. The NAD+/NADH ratio may be an important physiological control mechanism for it affects markedly the activity of the enzyme with 50% inhibition occurring at a ratio of 20:1. In the presence of uridine nucleotides binding of NADH to the epimerases is enhanced. Consequently, the effect of changes in the NAD+/NADH ratio in vivo would not be immediately apparent as uridine nucleotides would slow down the displacement of NADH by NAD+. Neither uridine nor galactose 1-phosphate inhibits the purified enzymes as previously reported with the impure liver enzyme. Uridine nucleotides provide almost total protection against the apparent first order inactivation of the epimerases by trypsin and allow determination of dissociation constants. NAD+ partially protects against trypsin inactivation. Inactivation with various sulfhydryl reagents is complex and the results indicate that at least three sulfhydryl groups may be modified before total inactivation occurs. Partial inactivation occurs upon modification of the epimerases with 2-hydroxy-5-nitrogenzyl bromide. Some protection against this modification is provided by the combination of NAD+ and UDP.
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PMID:Inhibition and inactivation of bovine mammary and liver UDP-galactose-4-epimerases. 19 53

The amino acid sequence of a 51-residue tryptic peptide of citraconylated [1-14C]carboxamidomethyl-labeled Escherichia coli GMP synthetase was determined by sequenator analyses of the intact peptide and fragments obtained by cleavage of the peptide with cyanogen bromide, trypsin, and Staphylcoccus aureus strain V8 protease. The cysteine residue of this peptide fragment is essential for glutamine-dependent GMP synthesis activity and is implicated in formation of a hypothetical covalent glutamyl-enzyme intermediate. There is essentially cysteine-containing regions of two other glutamine amidotransferases, Pseudomonas putida anthranilate synthetase Component II and chicken liver formylglycinamide ribonucleotide amidotransferase. There is, however, a cluster of amino acids with "antipathy" for helix formation and a "nonessential" cysteine of anthranilate synthetase Component II.
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PMID:Amino acid sequence of a peptide containing an essential cysteine residue of Escherichia coli GMP synthetase. 21 34

Acetate kinase (ATP:phosphotransferase E.C.2.7.2.1) has been purified to a high state of purity from Veillonella alcalescens. The native enzyme had a molecular weight of 88,000, as determined by Sephadex G-150 gel filtration. The molecular weight of the monomeric enzyme, estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42,000. The enzyme was determined to be a homodimer from the amino acid composition and the results of trypsin digestion and cyanogen bromide cleavage. Two moles of phosphate were incorporated into the dimer upon incubation of the enzyme with ATP and acetate. These results support the conclusion that each subunit of the dimeric enzyme consists of a single active catalytic center. Succinate enhanced the rate of ATP-ADP phosphoryl group exchange 20-fold and the binding of ATP 10-fold. These results are considered in light of data from previous reports (Pelroy, R. A., and Whiteley, H. R. (1971) J. Bacteriol. 105, 259-267; Bowman, C. M., Valdez, R. O., and Nishimura, J. S. (1976) J. Biol. Chem 251, 3117-3121).
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PMID:Acetate kinase from Veillonella alcalescens. Purification and physical properties. 21 74

Deuterium exchange at the C(2)-H position of the two histidine residues of native soybean trypsin inhibitor (Kunitz) in 2-H2O was followed by 1-H nuclear magnetic resonance (NMR) spectroscopy. The two histidine residues of soybean trypsin inhibitor exchange at significantly different rates at pH* 5.00, 40 degrees. Half-times observed were: peak H1, t1/2=61 plus or minus 2 days; peak H2, T1/2=24 plus or minus 2 days. Differentially deuterated soybean trypsin inhibitor was cleaved by cyanogen bromide into two fragments each containing one histidine residue. The deuterium content of the histidine residue of each separated fragment was analyzed by 1H NMR spectroscopy. Hisidine-71 in fragment 1-114 showed approximately twice the deuterium content of His-157 in fragment 115-181. These results lead to the assignment of 1H NMR peak H1 to His-157 and peak H2 to His-71. These assignments were extended to the histidine peaks of trypsin-modified soybean trypsin inhibitor by converting the differentially deuterated virgin soybean trypsin inhibitor to the modified form. The correlation of histidine peaks in virgin amd modified soybean trypsin inhibitors was the same as proposed earlier on the basis of pK arguments. The results demonstrate that His-71 is the residue whose pK value is raised from 5.27 to 5.91 on trypsin modification of soybean trypsin inhibitor [Markley, J. L., (1973), Biochemistry 12, 2245].
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PMID:Assignment of the histidine proton magnetic resonance peaks of soybean trypsin inhibitor (Kunitz) by a differertial deuterium exchange technique. 23 87

Neurospora glutamate dehydrogenase (NADP-specific) is rapidly inactivated upon reaction with tetranitromethane. This inactivation is completely prevented by the presence of coenzyme (NADP) or nicotinamide mononucleotide (NMN) but not by substrate. NADH, or 2'-monophosphoadenosine-5'-diphosphoribose. Amino acid analysis indicates that the primary effect of modification is nitration of a single residue of tyrosine per polypeptide chain. We have identified the reactive tyrosine by isolation of a single, uniquely labeled peptide after hydrolysis with trypsin followed by cleavage with cyanogen bromide. The modified residue proved to be tyrosine-168 in the linear sequence. This residue is not present in the part of the sequence that had been previously implicated as involved in the binding of the adenylate portion of the coenzyme. Both NMN and 2-monophosphoadenosine-5'-diphosphoribose act as competitive inhibitors of NADP in the oxidation of glutamate with Ki values of 4.65 x 10(-4) M and 4.30 x 10(-4) M, respectively. Thus, the specific protection afforded by NADP and NMN, but not by 2'-monophosphoadenosine-5'-diphosphoribose, indicates that tyrosine-168 is involved in binding the nicotinamide portion of the coenzyme.
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PMID:Nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase of Neurospora. III. Inactivation by nitration of a tyrosine residue involved in coenzyme binding. 23 46

Strains of Streptococcus mutans synthesized bacteriocins in agar plates, but synthesis of detectable bacteriocins in liquid media took place only under certain culture conditions. The composition of the medium proved to be crucial. Trypticase Soy Broth with 4% Yeast Extract meeting the requirements. The effect of the Yeast Extract is obscure, for some strains also formed detectable bacteriocins in a special Trypticase medium without this agent. It was noted that the broth should be filter-sterilized rather than autoclaved and only a few days old. Attempts at liberating cell-bound bacteriocins from washed cells were unsuccessful, even when they were treated with ultrasound, EDTA, or various chemicals followed by ultrasound. On the basis of size and sensitivity to heat the bacteriocins could be divided into two groups, while their resistance to ether and chloroform and to trypsin did not follow this pattern. Dependence on plasmids could not be demonstrated by attempts at curing with acridine orange or ethidium bromide; and the involvement of phages was unlikely, since the inhibition was not transmissible and phage-like structures were not observed in the electron microscope.
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PMID:Synthesis of bacteriocins in liquid cultures of Streptococcus mutans. 26 10

Although the tripeptides Glu-O-Phosphoserine-Tyr and Glu-O-Phosphoserine-Leu have been identified in embryonic bovine enamel proteins, 1, 2 the issue of whether both sequences occur in each of the phosphopeptides, or whether certain sequences occur in specific peptides only, has recently been resolved by isolating homogeneous samples of E33 and E44. All three of the Ser residues of both peptides are phosphorylated. All three in E3 are in the sequence Glu-O-Phosphoserine-Leu, and all three in E4 are in the sequence Glu-O-Phosphoserine-Tyr. It was not possible to sequence either of the polypeptide chains directly by automatic peptide sequencing. However, a partial sequence of E4 was constructed from data derived from peptides isolated after cyanogen bromide, trypsin and chymotrypsin digestions. The presence of Glu, Tyr and Leu adjacent to and near the O-Phosphoserine [Ser(L)] residues and the 2 degrees, 3 degrees and higher ordered structures of the enamel phosphopeptides may be important in calcium binding and mineralization.
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PMID:Phosphopeptides of enamel matrix. 28 20

A myeloma IgD immunoglobulin (designated WAH) that was present in high concentration in plasma ( approximately 3.5 g/dl) was purified in >90% yield by a two-step procedure of ammonium sulfate precipitation plus AcA 34 gel filtration. Although the plasma had been stored for 2 years without the addition of a proteolytic inhibitor, no "spontaneous" degradation was apparent and the isolated IgD remained structurally intact. However, the purified IgD showed extreme susceptibility in vitro to various proteolytic enzymes; e.g., Fab(delta) (M(r) approximately 47,000) and Fc(delta) (M(r) approximately 80,000) fragments were generated quantitatively after only 10 min of incubation with papain in the absence of cysteine. By combining limited enzymatic digestion, reductive cleavage, and cyanogen bromide fragmentation, several series of well defined fragments corresponding to the different regions and domains of the IgD molecule were generated. These fragments are useful for physical, chemical, and immunological studies, as well as for the sequence determination of the IgD delta chain. A model of the IgD molecule was derived from such studies and from overlapping of the series of fragments. The possible existence of an extra constant domain in the delta chain appears unlikely in view of our finding of an extended hinge region of about 50 residues which can be cleaved off the amino terminus of the papain Fc(delta) by brief treatment with trypsin. In addition to a distinct stretch of carbohydrate attachment sites, the delta-chain hinge region contains a segment unusually rich in electrical charge. This charged segment is responsible for the lability of IgD to spontaneous degradation and may be related to its biological role as a B lymphocyte receptor.
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PMID:Structural studies of human IgD: isolation by a two-step purification procedure and characterization by chemical and enzymatic fragmentation. 29 45


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