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)

Control of the rate of cardiac cell division by oxygen occurs most probably by altering the redox state of a control substance, e.g. NAD(+)right harpoon over left harpoonNADH. NAD(+) (and not NADH) forms poly(ADP-ribose), an inhibitor of DNA synthesis, in a reaction catalysed by poly(ADP-ribose) polymerase. Lower partial pressure of oxygen, which increases the rate of division, would shift NAD(+)-->NADH, decrease poly(ADP-ribose) synthesis, and increase DNA synthesis. Chick-embryo heart cells grown in culture in 20% O(2) (in which they divide more slowly than in 5% O(2)) did exhibit greater poly(ADP-ribose) polymerase activity (+83%, P<0.001) than when grown in 5% O(2). Reaction product was identified as poly(ADP-ribose) by its insensitivity to deoxyribonuclease, ribonuclease, NAD glycohydrolase, Pronase, trypsin and micrococcal nuclease, and by its complete digestion with snake-venom phosphodiesterase to phosphoribosyl-AMP and AMP. Isolation of these digestion products by Dowex 1 (formate form) column chromatography and paper chromatography allowed calculation of average poly(ADP-ribose) chain length, which was 15-26% greater in 20% than in 5% O(2). Thus in 20% O(2) the increase in poly(ADP-ribose) formation results from chain elongation. Formation of new chains also occurs, probably to an even greater degree than chain elongation. Additionally, poly(ADP-ribose) polymerase has very different K(m) and V(max.) values and pH optima in 20% and 5% O(2). These data suggest that poly(ADP-ribose) metabolism participates in the regulation of heart-cell division by O(2), probably by several different mechanisms.
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PMID:Poly(adenosine dephosphate ribose) metabolism and regulation of myocardial cell growth by oxygen. 2 65

Dialyzable Lawrence-type transfer factor was prepared from the spleen cells of CF1 mice inoculated with Coccidioides immitis- and Candida albicans-killed vaccines and with live Mycobacterium tuberculosis vaccine (BCG). These preparations were shown to transfer antigen-specific cell-mediated immunity to naive mice, as measured by the delayed skin test and footpad-swelling methods. Reactivity could be demonstrated when the test antigens were given 24 h after the transfer factor, but not when they were given simultaneously. Coccidioides-specific transfer factor was shown to be sensitive to Pronase and resistant to trypsin and ribonuclease. A preparation of BCG transfer factor was sensitive to snake venom phosphodiesterase.
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PMID:Transfer of delayed hypersensitivity in mice to microbial antigens with dialyzable transfer factor. 6 30

ColE1 DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein-denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open-circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or [14C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36-kDa protein which, we believe, is covalently bound to ColE1 DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethane-treated-trypsin and the DNA-peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE-Sephadex chromatography. The only 5'-dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. O-Phosphoserine was found in acid hydrolysates of the DNA-peptide(s). We suggest that in the relaxation event the 36-kDa protein becomes covalently linked to ColE1 DNA via a phosphodiester bond between dC and the serine residue.
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PMID:A protein covalently bound to ColE1 DNA. 304 96

The mechanism of poly ADPR synthesis and the transfer of poly ADPR to histone H1 molecule by electrophoretically homogenous calf thymus poly ADPR polymerase containing DNA was examined. 1) An acid insoluble radioactive complex (I) was obtained after incubation of purified enzyme with [3H] NAD. The stability of (I) was examined by SDS-polyacrylamide gel electrophoresis. The complex (I) was stable against acid, SDS, urea, DNase and RNase, but labile against pronase, trypsin, alkali and snake venom phosphodiesterase treatment. The molecular weight of (I) was about 130 000 daltons estimated by SDS-gel electrophoresis. The radioactive products of successive alkali, venom phosphodiesterase and Pronase hydrolysis of (I) were PR-AMP and AMP. The mean chain length of poly ADPR of (I) was 20--30. These results suggest that the complex (I) is poly ADP-ribosylated poly ADPR polymerase. 2) Besides (I), a second radioactive peak (II) was observed when acid insoluble products obtained from an incubation mixture containing purified poly ADPR polymerase, [3H] NAD and purified histone H1 were analyzed on SDS-polyacrylamide gel electrophoresis. The molecular weight of (II) was estimated to be about 23 000 daltons. The complex (II) is eluted like histone H1 on CM-cellulose columns and hydrolyzed by alkali, trypsin and snake venom phosphodiesterase but not by DNase, or RNase. The comples (II) was extracted selectively by 5 per cent perchloric acid or 5 per cent trichloroacetic acid from mixture of (I) and (II). The mean chain length of poly ADPR of complex (II) and 5--20; these results suggest that the complex (II) is poly ADP-ribosylated histone H1. 3) Results 1) and 2) indicate that purified DNA containing, thus DNA independent, poly ADPR polymerase catalyzes two different reactions, the ADPR transfer onto the enzyme itself and onto histone H1 and the elongation of ADPR chains. Dimeric forms of ADP-ribosylated histone H1 was not observed. Free poly ADPR was observed only when very small quantities of enzyme were used for incubation.
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PMID:Adenosine diphosphate ribosylation of histone H1 by purified calf thymus polyadenosine diphosphate ribose polymerase. 624 65

Rat peritoneal macrophages contain a chymotrypsin-like protease and its specific inhibitor, both being associated with chromatin of the cells. The inhibitor was separated from the protease by gel filtration through a Sephadex G-75 column, further treated with trypsin, DNase and RNase, and then purified successively on Sephadex G-75, Sephadex G-25, and dihydroxyboryl Bio-Gel P-60 columns. The purified inhibitor had a molecular weight in the range from 2,000 to 3,500 and an absorption maximum at 260 nm at pH 7.0. When the inhibitor was digested by snake venom phosphodiesterase, the inhibitory potency was lost, yielding 5'-AMP and 2'-(5'-phosphoribosyl)-5'-AMP as the digestion products which were identified by high pressure liquid chromatography. The inhibitory potency was neutralized specifically by anti-poly(ADP-ribose) antiserum. The profile of inhibition by the isolated inhibitor was nearly identical with that produced by authentic poly(ADP-ribose). It was therefore concluded that the inhibitor isolated was identical with poly(ADP-ribose), whose chain length ranged from 4 to 7 ADP-ribosyl units. This is the first demonstration that a intracellular protease inhibitor can be endogenous poly(ADP-ribose).
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PMID:Identification of a protease inhibitor from rat peritoneal macrophages as poly(ADP-ribose). 624 11

Polyacrylamide gel electrophoresis of cell-free extracts of Escherichia coli that had been grown in a medium containing 32Pi disclosed the presence of several 32P-labeled proteins. Comparison of the electrophoretic patterns obtained in the presence of carrier unlabeled purified E. coli glutamine synthetase before and after treatment with trypsin, subtilisin, or snake venom phosphodiesterase showed that most of the 32P was present in the adenylyl moieties of adenylylated glutamine synthetase. Low molecular weight 32P-labeled degradation products of glutamine synthetase were also observed in extracts prepared by treatment of cells with lysozyme but not in extracts prepared by sonic oscillation. The degradation of glutamine synthetase in lysozyme-prepared extracts is likely due to an intrinsic proteolytic activity of egg white lysozyme. Proteolysis probably occurs at the esterase site of lysozyme described by Piszkiewicz and Bruice [Piszkiewicz, D. & Bruice, T.C. (1968) Biochemistry 7, 3037-3047]. Selective carboxymethylation of lysozyme histidine-15 leads to simultaneous loss of esterase and protease activities but only to partial loss of lytic activity. In view of these findings, caution is needed in the interpretation of results obtained with extracts of cells prepared by lysozyme treatment, especially when such extracts are used to investigate the properties of proteolytic enzymes.
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PMID:A proteolytic artifact associated with the lysis of bacteria by egg white lysozyme. 634 Jan 15

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.
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PMID:Mass spectral characterization of a protein-nucleic acid photocrosslink. 1063 98

Phage varphiX174 A(*) protein cleaves single-stranded DNA and then binds to the 5'-phosphorylated terminus of the cleaved DNA fragment, forming a covalent protein-DNA complex. The bound A(*) protein can religate the termini to form covalently closed single-stranded circles. To determine the nature of the covalent linkage and the amino acid involved, we used A(*) protein to cleave DNA synthesized in vivo with [alpha-(32)P]dATP to form the A(*)-single-stranded DNA complex. The complex was then digested with DNase I and the (32)P-labeled A(*) protein was isolated by electrophoresis on polyacrylamide gels. The isolated complex was digested with either trypsin or Pronase. Incubation of the tryptic digest with snake venom phosphodiesterase gave (32)P-labeled products that migrated on electrophoresis on cellulose plates to the cathode, indicating covalent linkage of (32)P-labeled dAMP residues to a tryptic peptide. High concentrations of snake venom phosphodiesterase released all of the (32)P label as free dAMP. Formic acid/diphenylamine depurination (Burton reaction) of the [alpha-(32)P]dATP-labeled peptide-oligonucleotide complexes caused a transfer of the labeled phosphate from dAMP to the peptide. The phosphorylated peptides were isolated on cellulose plates and shown to be sensitive to bacterial alkaline phosphatase, indicating that a phosphodiester bond linked the peptides to the dAMP. The phosphorylated product of the Pronase digest was identified as free phosphotyrosine by its mobility in three different chromatography systems. Likewise, acid hydrolysis (5.6 M HCl, 110 degrees C, 2 hr) of the phosphorylated tryptic peptides revealed linkage of the phosphate to a tyrosine. Thus, A(*) protein cleaves single-stranded DNA and binds covalently to the 5'-phosphorylated terminus via a tyrosyl-dAMP phosphodiester bond.
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PMID:Cleavage of single-stranded DNA by the varphiX174 A protein: The A-single-stranded DNA covalent linkage. 1659 85

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