Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The H-D exchange of the black-eyed pea trypsin and chymotrypsin inhibitor (BTCI) in D2O was studied by an ultraviolet spectroscopic method recently proposed (J. J. Englander, D. B. Calhoun, and S. W. Englander, (1979) Analytical Biochemistry, 92, 517-524). Isotopic exchange data are presented as plots of X (the fraction of unexchanged peptide hydrogen atoms at time t) versus log(kot), where ko is the pH dependent rate constant for peptide groups exposed to the solvent. In the range of pD 2.25-6.9, at 20 degrees C, BTCI shows a continuous exchange curve which indicates that the exchange mechanism is of the EX2 type and no detectable conformational changes occur in the protein. Deviations from this exchange curve are found at pD 7.3 and 8.0. About 60% of the peptide hydrogens of BTCI are exchanged for delta Go less than or equal to 2 kcal/mol, and 90% for delta Go less than 6 kcal/mole. For reduced and carboxymethylated BTCI, exchange data suggest a much more open conformation in comparison with the unmodified protein. However, some residual structure appears to be maintained, after scission of the disulfide bonds. The exchange data indicate that, as a consequence of the formation of the beta-trypsin-BTCI complex, part of the peptide groups of the enzyme and/or inhibitor become less accessible to the isotopic exchange.
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PMID:Hydrogen-deuterium exchange in the black-eyed pea trypsin and chymotrypsin inhibitor and its complex with beta-trypsin. 721 61

The problem of determining small but significant amounts of carbohydrates, in purified proteins, has been studied using the membrane protein, cytochrome b5. A newly developed method that involves direct gas chromatography-mass spectrometry of sugars obtained by hydrolysis of proteins purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) allows the identification and determination of small amounts of carbohydrates (e.g., 20 micrograms of glycoprotein containing a minimum of 0.1% monosaccharide), even in the presence of relatively high amounts of impurities. Application of this method to cytochrome b5 fragments obtained by tryptic digestion from rat liver microsomes and purified by combined gel filtration and ion exchange chromatography, followed by SDS PAGE, has consistently yielded values below 0.07 mol of the individual sugars and aminosugars per mole cytochrome b5. It is concluded that cytochrome b5, at least its trypsin-released major amino-terminal fragment, is not constitutively glycosylated.
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PMID:Absence of sugars in electrophoretically purified cytochrome b5 demonstrated by combined gas chromatography-mass spectrometry. 725 67

1. Treatment of low K goat red cells with trypsin stimulated the Na-K pump more than twofold. Dose dependence and time course experiments indicated a half-maximal stimulation at 1.6 mg trypsin/ml. (37 degrees C, 3 hr), and a maximum effect after 5 hr (10 mg/ml). 2. Trypsin had only a small and variable effect on the ouabain-insensitive component of K influx. 3. The Na-K pump activity of high K goat red cells was not affected by trypsinization. 4. When intracellular K was varied by the PCMBS technique, it was found that the trypsin stimulation was greatest (2-5-fold) in cells with the highest K (40 m-mole/1. cells) and lowest (1.1-fold) in cells with low K (<1 m-mole/1. cells). 5. The trypsin effect was reversed by nystatin treatment or hypotonic lysis. 6. Trypsin did not increase the number of ouabain-binding sites. 7. It is concluded that trypsinization modifies the L antigen in low K goat red cells to decrease the apparent internal affinity for K of the Na-K pump in these cells.
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PMID:Stimulation of the sodium-potassium pump by trypsin in low potassium type erythrocytes of goats. 741 31

Proteinase inhibitors adsorbed from human serum on DEAE Sephadex A 25 0.25 or 0.3 mol/l NaCl were purified by affinity chromatography on a trypsin-Sepharose 4B column, by gel filtration, and by DEAE cellulose chromatography. Small amounts of TCI-I (desorbed from the ion-exchange between 0.25-0.3 mol/l NaCl), and TCI-II (desorbed at NaCl concentration above 0.3 mole/l) with high specific activity were obtained. The low recovery of inhibitory activity (below 9% was due to a molecular transformation of these inhibitors after contact with the immobilized trypsin. The low-molecular weight derivatives were formed that lost their ability to adsorbe on ion-exchanger at 0.25 or 0.3 mole/l salt concentration.
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PMID:Further studies on proteinase inhibitors separated from human serum by deae sephadex chromatography. 744 41

Treatment of relaxed skinned rabbit psoas muscle fibers with 0.1 mM N-phenylmaleimide (NPM) for 1 h locks all of the crossbridges in a weakly-binding state resembling that of the myosin.ATP crossbridge. Under these conditions, NPM reacts mainly with myosin heavy chain (Barnett et al. (1992) Biophys. J. 61, 358-367). Here the specific sites for that reaction are explored. Small bundles of rabbit psoas muscle fibers were treated with Triton X-100 to make the fiber sarcolemmas permeable. The bundles were treated with 0.1 mM [14C]NPM for 1 h, and homogenized for SDS-PAGE. 43 +/- 2.2% of the muscle fiber protein ran in the myosin heavy chain band, the same as for untreated fibers. An alkylating stoichiometry of 2.2 +/- 0.33 moles NPM per mole myosin heavy chain was determined. Exhaustive trypsin digestion followed by two-dimensional thin-layer chromatography and reverse-phase HPLC revealed two major sites on myosin heavy chain for NPM binding. The sites contained about the same amount of linked NPM, suggesting that the reaction stoichiometry of each site under the conditions studied is approx. 1 mol NPM/mol myosin heavy chain. Comparison of the labeled tryptic peptides with NPM-reacted synthetic SH1 and SH2 tryptic peptides and analysis of the treated fiber bundles' ATPase activity suggested that the sites for NPM reaction on myosin heavy chain when it locks crossbridges in a weakly-binding state are Cys-697 (SH2) and Cys-707 (SH1).
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PMID:The site and stoichiometry of the N-phenylmaleimide reaction with myosin when weakly-binding crossbridges are formed in skinned rabbit psoas fibers. 749 34

CD59 is an 18-kDa glycoprotein widely expressed on human cells. An important structural feature of CD59 is its attachment to the cell surface via a glycosyl-phosphatidylinositol (GPI) anchor. CD59, like many GPI-anchored proteins, has been found in urine, serum, and other body fluids. The structures of the GPI anchor and the asparagine-linked sugar chain of a soluble form of CD59 in urine, U-CD59, were determined. Purified U-CD59 released 1 mol of inositol per mole of protein by nitrous acid deamination, which cleaved between glucosamine and inositol present commonly in the GPI anchor. This indicates that a GPI anchor, which ended with inositol, is linked at the carboxy terminus of U-CD59. The peptide containing an asparagine-linked sugar chain and the peptide containing a glycan portion of the GPI anchor were isolated after trypsin digestion of U-CD59. The asparagine-linked sugar chains and the glycan portion of the GPI anchor were isolated from these peptides following hydrazinolysis or deamination and dephosphorylation, respectively. Their structures were analyzed by sequential exoglycosidase digestion and methylation analyses. The structures of the asparagine-linked sugar chains of U-CD59 were biantennary complex type, only 4.2% of which are monosialylated. The backbone structure of the GPI anchor was similar to that of Try-panosoma brucei variant surface glycoprotein, but showed significant variations in its side-chain moieties. This is the first detailed structural analysis of the human GPI anchor and the first detailed analysis of the carboxyl-terminal structure of the soluble-form GPI-anchored protein. The results indicate that the backbone structure of the GPI anchor is conserved from parasites to human and that at least a part of the soluble-form GPI-anchored protein has the structure produced by the action of glycan-phosphatidylinositol-specific phospholipase D.
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PMID:Structural study on the glycosyl-phosphatidylinositol anchor and the asparagine-linked sugar chain of a soluble form of CD59 in human urine. 751 86

The interaction between bovine lactoferrin (bLf) and ascorbate (Asc) was investigated through malondialdehyde (MAD) formation in a solution containing DNA, bleomycin (BLM), and Fe2+ or Asc. The inhibition by bLf on MDA formation in the presence of Asc was not changed even by adding carbonate or oxalate ions to the solution. The percentage inhibition by the hydrolysates of bLf treated with pepsin, trypsin, and both enzymes on MDA formation was almost the same as that by the untreated bLf in the presence of Asc. The inhibition of MDA formation also occurred with the filtrate obtained from a solution containing bLf and Asc, but not with that from a solution of bovine serum albumin and Asc. The interaction of bLf and Asc was observed by gel filtration in a Sephadex G75 column. The binding amount of Asc was estimated to be 87 mol per mole of bLf.
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PMID:Interaction of lactoferrin with ascorbate and the relationship with bleomycin-dependent DNA damage. 753 54

Active proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1) were generated by trypsin digestion of the native protein. Brief proteolysis produced a 77-kDa fragment that contained the highly conserved X and Y regions but lacked the amino-terminal domain (amino acids 1-60). Prolonged digestion of PLC-delta 1 produced two fragments, one of 45 kDa that contained the entire X region and another of 32 kDa that consisted of the entire Y region and COOH-terminal domain. The 45- and 32-kDa fragments were isolated as an active heterodimeric complex. The 77-kDa fragment and the complex catalyzed calcium-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in detergent/phospholipid mixed micelles. When compared with the native enzyme, both the 77-kDa fragment and the complex exhibited a reduced capacity to processively hydrolyze PIP2; increasing the mole fraction of PIP2 in the mixed micelle surface greatly increased the rate of PIP2 hydrolysis catalyzed by the native enzyme but not the fragments. Both fragments also exhibited a reduced affinity for substrate; the native enzyme bound to bilayer vesicles consisting of phosphatidylcholine and PIP2 with high affinity (Ka approximately 10(6) M-1), whereas the fragments bound weakly (Ka < 10(4) M-1). These results demonstrate that the X, Y, and COOH-terminal regions form a calcium-dependent catalytic core that is resistant to proteolysis. The amino-terminal domain appears to be essential for high affinity binding to PIP2 but not catalysis. These observations are consistent with the idea that the amino-terminal domain forms part of a PIP2 binding site, which anchors PLC-delta 1 to the membrane surface during processive hydrolysis of its substrate.
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PMID:Proteolytic fragments of phosphoinositide-specific phospholipase C-delta 1. Catalytic and membrane binding properties. 768 17

Staphylococcus hyicus lipase is a serine hydrolase. In order to identify the active site histidine of S. hyicus lipase we have chemically modified S. hyicus lipase with 1-bromo-octan-2-one. The enzyme is rapidly inactivated by this inhibitor with a half-time of 578 s at pH 6.5 and 30 degrees C. Addition of the enzyme's cofactor calcium increases the inactivation rate approx. 2-fold. When n-hexadecylphosphocholine, a non-hydrolysable substrate analogue, is added the inactivation rate decreases about 3-fold, suggesting that a residue in the active site of S. hyicus lipase is involved in the inactivation reaction. Inactivation of S. hyicus lipase with 14C-labelled 1-bromo-octan-2-one shows that 1.4 moles of inhibitor per mole of lipase are incorporated. The results of an electrospray mass spectrometric study of the inactivated enzyme are consistent with this finding. In order to identify the modified residue, both the inactivated and the unmodified lipase were digested with cyanogen bromide followed by trypsin. The resulting peptides were analysed using HPLC and fast atom bombardment mass spectrometry. The results allow the modified residue to be assigned to the peptide Gly597-Lys612. Collision induced dissociation mass spectrometry allowed the modified residue to be identified as His-600. From these results we conclude that this residue forms part of the catalytic triad of S. hyicus lipase.
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PMID:Identification of the active site histidine in Staphylococcus hyicus lipase using chemical modification and mass spectrometry. 771 Oct 54

Bovine trypsinogen was used as a model protein for studying changes in the conformational stability induced by pH or binding of the calcium ion. Spectrophotometrically monitored thermal unfolding of trypsinogen and beta-trypsin in the acidic pH range yielded substantial differences in the stability parameters. Compared to beta-trypsin, trypsinogen exhibits lower enthalpy of denaturation delta Hden, higher denaturational heat capacity change delta Cp,den, but very similar temperature of denaturation Tden. pH-dependence of the conformational stability of the ligand-free trypsinogen, measured also by GdnCl-induced unfolding, is bell shaped with the maximum free energy of unfolding delta Gden = 10.9 kcal/mole at pH 5.5 (4.5 pH units below its isoelectric point). At pH 8.3 the conformational stability of the zymogen drops to delta Gden = 3.2 kcal/mole, but increases by delta delta Gden = 6.1 kcal/mole in the presence of Ca2+. This significant stabilization of the zymogen by the calcium ion is also pH-dependent. To assess the effect of Ca2+ on the trypsinogen molecule, the spectrophotometric titrations and NOESY spectra were carried out. Based on the structural analysis, the long range effects between Ca2+-->Ile73-->Trp141 and the interdomain His40-Asp194 ion pair are proposed to be partially responsible for trypsinogen stabilization. Additionally, the steady-state parameters for hydrolysis of the oligopeptide amide substrate catalysed by free trypsinogen, its complexes with Ca2+ and the IleVal dipeptide and by beta-trypsin were measured. It appears that in the pH range 5.5 to 8.3 the stability and the catalytic activity/ligand binding properties are fully separated. Whereas the deprotonation of His57 accounts for the increase of kcat/km parameter, deprotonation of His40 is involved in the huge decrease of the conformational stability. Similarly, a large stabilization by the calcium ion is not accompanied by changes in enzymatic activity. Presented data are encouraging for an enzyme design directed toward improved stability.
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PMID:Ligand-induced changes in the conformational stability of bovine trypsinogen and their implications for the protein function. 772 25


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