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 zeta potential of washed Tice substrain BCG organisms was measured over a range of ionic strengths from I = 0.005 to 0.1 M. No change in the isoelectric point of 3.4-3.7 was evident. Proteolytic enzymes (trypsin/chymotrypsin, pepsin, papain and pronase) and fluorodinitrobenzene abolished the cationic charge, suggesting that this is substantially due to amino groups associated with protein. Neither hot HCI nor cold trichloroacetic acid affected the charge, indicating that ionic groups are not associated with extractable polysaccharides. Methanolysis, treatment with HF and carbodiimide, and cationic detergent (cetyltrimethylammonium bromide) binding indicated that the negative charge was provided by carboxylic acids, phosphoesters and strong acidic groups, possibly sulphates. Standardless quantitative X-ray microanalysis revealed the presence of phosphorus and sulphur on the surface of actively growing BCG colonies.
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PMID:Origins of BCG surface charge: effect of ionic strength and chemical modifications on zeta potential of Mycobacterium bovis BCG, Tice substrain, cells. 140 39

The scheme for the isolation and purification of low-molecular cell-wall protein without type specificity, including the extraction of the cell walls of group A streptococci, type M 29, with 1% solution of Triton X-100, the separation of the extract by ion-exchange chromatography in DEAE-trisacryl M with the subsequent two-stage gel filtration in superfine Sephadex G-50, is described. The isolated protein had a molecular weight of 4,000 daltons and contained no admixtures of group-specific polysaccharide A, phosphorus, nucleic acids and Fc receptors and interacted with antisera to group A streptococcal cells of heterologous type M in the enzyme immunoassay (EIA). Purified protein was characterized by a high content of glycine. The antigenic determinants of immobilized protein, recognized by antibodies in EIA, were sensitive to the action of trypsin and resistant to the action of pepsin, papain, pronase E and sodium periodate.
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PMID:[The low-molecular protein of the cell wall in streptococci group A devoid of serological type specificity]. 171 20

Rat heart myocardial membranes exposed to the free radical generating system, Fe2+/ascorbate, undergo lipid peroxidation as evidenced by the accumulation of thiobarbituric acid-reactive substances, loss of polyunsaturated fatty acids from phospholipids, and formation of conjugated dienes and fluorescent substances. In addition, the treated membranes exhibit a dramatic decrease in extractable phospholipids. This decrease is even more pronounced in individual phospholipid classes isolated by high-performance liquid chromatography. The decrease in lipid phosphorus under oxidant stress is accompanied by an increase in the phosphorus content of the aqueous phase after Folch extraction and by an even greater increase of phosphorus in the protein residue. In addition, increased amounts of saturated and monounsaturated fatty acyl groups are found in the protein residue of Fe2+/ascorbate-treated membranes. Extraction of the oxidant-treated membranes with acidic solvents does not enhance the recovery of phospholipids and neither does treatment with detergents, trypsin, and chymotrypsin prior to lipid extraction. However, treatment with the bacterial protease, Pronase, markedly enhances the recovery of phospholipids from the peroxidized membranes. These results indicate that membrane phospholipids undergoing free radical-induced peroxidation may form lipid-protein adducts, which renders them inextractable with lipid solvents.
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PMID:Peroxidative modification of phospholipids in myocardial membranes. 235 24

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

Two different nuclear magnetic resonance experiments were conducted to elucidate the properties of the Ca(II) binding locus on serine proteases in solution. Trypsin, alpha-chymotrypsin, and subtilisin were inactivated with diisopropyl fluorophosphate, and the distance of the phosphorus from Gd(III) in place of Ca(II) was determined from the lanthanide-induced relaxation on the 31P resonance. The distances found (between 20 and 21 A) were in excellent agreement with those reported in the X-ray crystallographic structures of trypsin and subtilisin, demonstrating that the method has wide applicability to systems for which no X-ray structure is available. Subsequently, the 113Cd spectra [in place of Ca(II)] were examined in the presence of the native enzymes. At ambient temperatures only a single 113Cd resonance could be observed, presumably representing the weighted average of the variously weakly bound ions and the free ion. At 280 K for trypsin and chymotrypsin, and at 268 K for subtilisin there was observed a resonance at ca. 65-70 ppm higher field than the previous averaged resonance that could be attributed to tightly bound Cd. The chemical shift of the resonance was consistent with its assignment to an octahedral environment around Cd with oxygen ligands.
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PMID:Multinuclear magnetic resonance studies on the calcium (II) binding site in trypsin, chymotrypsin, and subtilisin. 269 2

Reaction of Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) with phosphonoacetaldehyde or acetaldehyde in the presence of NaBH4 resulted in complete loss of enzymatic activity. Treatment of phosphonatase with NaBH4 in the absence of substrate or product had no effect on catalysis. Inactivation of phosphonatase with [3H]NaBH4 and phosphonoacetaldehyde, NaBH4 and [14C]acetaldehyde, or NaBH4 and [2-3H]phosphonoacetaldehyde produced in each instance radiolabeled enzyme. The nature of the covalent modification was investigated by digesting the radiolabeled enzyme preparations with trypsin and by separating the tryptic peptides with HPLC. Analysis of the peptide fractions revealed that incorporation of the 3H- or 14C-radiolabel into the protein was reasonably selective for an amino acid residue found in a peptide fragment observed in each of the three trypsin digests. Sequence analysis of the 3H-labeled peptide fragment isolated from the digest of the [2-3H]phosphonoacetaldehyde/NaBH4-treated enzyme identified N epsilon-ethyllysine as the radiolabeled amino acid. The ability of the phosphonatase competitive inhibitor (Ki = 230 +/- 20 microM) acetonylphosphonate to protect the enzyme from phosphonoacetaldehyde/NaBH4-induced inactivation suggested that the reactive lysine residue is located in the enzyme active site. Comparison of the relative effectiveness of phosphonoacetaldehyde and acetaldehyde as phosphonatase inactivators showed that the N-ethyllysine imine that is reduced by the NaBH4 is derived from the corresponding N-(phosphonoethyl) imine. On the basis of these findings, a catalytic mechanism for for phosphonatase is proposed in which phosphonoacetaldehyde is activated for P-C bond cleavage by formation of a Schiff base with an active-site lysine. Accordingly, an N-ethyllsysine enamine rather than the high-energy acetaldehyde enolate anion is displaced from the phosphorus.
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PMID:Investigation of the Bacillus cereus phosphonoacetaldehyde hydrolase. Evidence for a Schiff base mechanism and sequence analysis of an active-site peptide containing the catalytic lysine residue. 313 6

A wall-surface protein antigen, designated 32K antigen, was extracted from whole cells of Clostridium difficile strain ATCC 11011 with phosphate buffered saline and purified by ion-exchange chromatography, gel filtration, and chromatofocusing. The 32K antigen preparation was determined to be highly homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition of the antigen was characteristic in the predominance of the acidic amino acids, the very low contents of methionine and histidine, and the lack of cysteine. A monomeric molecular weight of the 32K antigen was estimated to be 32,000 by SDS-PAGE and 30,200 by sedimentation equilibrium. The antigen exhibited two isoelectric forms (IP, 4.12 and 3.96). Neither carbohydrate nor phosphorus was detectable in the antigen. The antigen was relatively resistant to trypsin but sensitive to pepsin. Immunoblot analysis of the wall proteins isolated from other strains of C. difficile probed with monospecific antiserum against the antigen from ATCC 11011 showed that the antigenicity of 32K wall protein was common among some of the strains containing 32K wall proteins.
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PMID:Purification and immunochemical properties of a wall protein antigen from Clostridium difficile ATCC 11011. 332 Jun 89

The Streptococcus mutans group b antigen of strain FA1 has been defined as to chemical composition and immunological specificity. The antigen in cold trichloroacetic acid extracts was fractionated on diethylaminoethyl-Sephadex A-25 at pH 8.5. Two forms were isolated: a polysaccharide and a mucoprotein. The two polymers reacted as a single substance in agar gel diffusion against specific adsorbed FA1 rabbit antisera but were separated by gel immunoelectrophoresis. No reaction with any other S. mutans or streptococcal group sera occurred. Galactose composed about one-third and galactosamine about 3% of the total weight of each polymer. Rhamnose was a major component of the polysaccharide (47%) but was present only in traces in the mucoprotein. The protein content of the latter was about 40%. No significant quantities of glycerol, phosphorus, or muramic acid were present in either case. Pepsin and trypsin had no effect on the serological specificity of the mucoprotein. d-Galactose and d-galactosamine were strong inhibitors (70%) of the precipitin reaction, whereas d-glucose, d-glucosamine, and N-acetyl-d-glucosamine inhibited between 25 and 35%. The results indicate that the antigen is a major antigenic component of the cell wall and that the specificity of the antigen resides in binding sites which contain both d-galactose and d-galactosamine. Agglutination of whole cells by specific group b antiserum indicates the antibody receptor sites of the polysaccharide antigen are at the surface of the streptococcal cell. The mucoprotein, but not the polysaccharide, was released from the cell by lysozyme. Lysis did not occur. The immunological specificity and other characteristics of the antigen establishes it as the identifying antigen of S. mutans group b.
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PMID:Structure and immunological specificity of the Streptococcus mutans group b cell wall antigen. 412 3

Australia antigen [Au(1)], a particle associated with viral hepatitis, was isolated from the plasma of a patient with chronic anicteric hepatitis and leukemia who had received radioactive phosphorus. We have found that the immunoreactivity and appearance of Au(1) in the electron microscope were not altered by treatment with enzymes including trypsin, pronase, lipase, phospholipase C, ribonuclease, deoxyribonuclease, amylase, and neuraminidase. In contrast, other serum constituents were degraded by these enzymes. Therefore, treatment of the patient's plasma with many enzymes was exploited as an initial step for the isolation of Au(1). Subsequently, Au(1) was purified from the enzyme-treated (32)P-labeled plasma by gel filtration through Sephadex G-200 and centrifugation through sucrose and in cesium chloride gradients. There were no detectable human serum components in the purest fractions, as tested by immunoelectrophoresis and immunodiffusion. The density of the purified Au(1) was 1.21 in CsCl. The particle measured about 200 A in diameter, was predominantly spherical in shape and appeared to be composed of subunits. Nucleic acids were not detected by spectrophotometric, radiochemical, and chemical analyses. Immunoreactivity of purified Au(1) was destroyed by heating for 1 hr at 85 degrees C but was stable at 56 degrees C. Treatment with Carnoy's solution (3 parts ethanol:1 part glacial acetic acid) followed by pronase disrupted the particles as seen with the electron microscope. These findings, combined with other published information on Australia antigen and viral hepatitis, suggest that the bulk of Australia antigen in the blood of this patient is an incomplete virus or virus capsid.
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PMID:Australia antigen (a hepatitis-associated antigen): purification and physical properties. 424 40

Cell walls isolated from two strains of Blastomyces dermatitidis were examined. Whereas strain Ga-1 was practically avirulent for mice, strain KL-1 produced death by 21 days in 50% of the mice inoculated. Analyses of the trypsin-treated cell walls of the two strains revealed a higher chitin and protein content in strain KL-1, whereas a higher polysaccharide content was observed in the cell walls of strain Ga-1. Extraction of the walls with 1 n NaOH revealed a threefold difference in the amount of alkali-soluble cell wall material present. The alkali-soluble material could be further fractionated into a water-soluble and a water-insoluble fraction. Previous reports have indicated that the water-insoluble fraction of B. dermatitidis consists of an alpha-linked glucan; however, we report that in addition a phospholipid moiety is covalently bound to the polysaccharide. Furthermore, on the basis of organic phosphorus content, considerably more phospholipid is associated with the alpha-linked glucan of the more virulent KL-1 strain. These results suggest that this cell wall constituent might be one of the factors related to the virulence of this fungus.
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PMID:Cell wall composition of two strains of Blastomyces dermatitidis exhibiting differences in virulence for mice. 463 84


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