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 binding of ADP to subfragment-1 was investigated by the gel filtration method. The amount of bound ADP was determined as a function of free ADP concentration. Linear Scatchard plots were obtained. The maximum binding number, 0.55 mole of ADP per 10(5) g of protein, and the dissociation constant, 1.6 x 10(-6) M, were obtained, using subfragment-1 prepared by tryptic digestion, in the presence of 0.083 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 25 degrees. Similar maximum numbers, about 0.5 mole per 10(5) g of protein, were obtained with subfragment-1 prepared by chymotryptic digestion of myosin or papain digestion of myofibrils. The maximum number did not depend on the KCl concentration or the temperature, while the dissociation constant decreased on decreasing either the KCl concentration or the temperature. Adenylyl imidodiphosphate binding to subfragment-1 prepared by chymotryptic digestion was also measured by the gel filtration method. The maximum binding number, 0.41 mole per 10(5) g of subfragment-1, and the dissociation constant, less than 10(-7) M, were obtained in the presence of 0.7 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 8 degrees. The difference absorbance at 288 nm of the difference absorption spectrum induced by ADP of subfragment-1 prepared by tryptic digestion was proportional to the amount of bound ADP. The steady-state ATPase rate of subfragment-1 prepared by tryptic digestion was inhibited competitively by ADP in the presence of MgCl2. The extent of the initial burst of ATPase [EC 3.6.1.3] decreased from 0.46 +/- 0.06 to 0.30 +/- 0.09 mole of Pi per 10(5) g of subfragment-1 on adding ADP to a level of 0.6 mM. Subfragment-1 prepared by tryptic digestion bound F-actin with a mole ratio of 1/0.96 of actin monomer. The binding was depressed by the addition of ADP. On the basis of these results, subfragment-1 preparations were assumed to be a half-and-half mixture of two kinds of protein, and properties of each protein are discussed.
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PMID:A study of the binding of adenosine diphosphate to myosin subfragment-1. 12 50

H-Meromyosin (HMM) was digested with insoluble papain [EC 3.4.22.2]. Neither the size of the initial burst of Pi liberation (0.5 mole/mole of myosin head) nor the Mg2+-ATPase [EC 3.6.1.3] activity of HMM in the steady state was affected by this treatment. Acto-S-1 was obtained by mixing F-actin with HMM digested with insoluble papain (HMM-S-1). The size of the initial burst of Pi liberation of acto-S-1 was 0.35 mole/mole of S-l at an ATP concentration of 0.5 mole/mole of S-1, and 0.5 mole/moleof S-1 at ATP concentrations above 1 mole/mole of S-1...
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PMID:Separation of subfragment-1 of H-meromyosin into two equimolar fractions with and without formation of the reactive enzyme-phosphate-ADP complex. 13 97

On the system methanethiol/imidazole/formaldehyde (modelling the active site of papain) we performed ab initio self-consistent-field molecular orbital calculations using a rather large basis of Gaussian-type functions. A point charge representation of the long central alpha-helix present in the enzyme, was added in order to establish the influence of the electric field of the helix (which amounts to 10(9) V m-1 in the active site region) on the equilibrium: RSH...Im in equilibrium RS-...ImH+, which is an essential step in a recently proposed mechanism for the catalytic action of papain. Our results show that the helix stabilizes the ion-pair by 15 kcal mole-1 more than the neutral form making the two configurations energetically equivalent and lowers the energy barrier in the reaction path by 8 kcal mole-1, thus shifting the equilibrium considerably towards the ionic situation and increasing the rate of proton transfer by several orders of magnitude. We conclude that "active site" helices, present in many enzymes, play a pertinent role in enzyme catalysis.
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PMID:On the role of the active site helix in papain, an ab initio molecular orbital study. 45 4

IgM rheumatoid factors (RF) were isolated from the sera of patients with rheumatoid arthritis and a serologically active Fabmicron RF fragment prepared by papain digestion. A radioimmunoassay was developed for the determination of interaction of 19S IgM RF and Fabmicron RF with human 7S IgG, heat-aggregated IgG, rabbit 7S IgG, and human pFc'. RF isolated under neutral conditions had a very low binding constant for human 7S IgG (of the order of 10(2) to 10(3) 1 mole-1) and a considerably higher value (ca. 10(5)) for the aggregated protein and monomeric rabbit IgG. RF obtained under acid conditions which dissociate the complexes with endogenous Ig, had a higher avidity for human IgG monomer as expected and also a comparable reactivity with rabbit IgG. Monovalent Fabmicron fragments of 'acid' RF had closely similar affinities for 7S and aggregated IgG suggesting that the enhanced binding with the aggregated protein is essentially dependent on its multivalency rather than the exposure of a new determinant lacking in the native molecule.
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PMID:The binding constants of IgM rheumatoid factors and their univalent fragments for native and aggregated human IgG;. 84 79

Cystatins are protein inhibitors of cysteine proteinases, which are believed to play an important role in the pathogenesis of periodontal disease. In this study, we report a new sensitive method for the quantitative analysis of cystatin activity in a small amount of crude sample such as gingival crevicular fluid. Cystatin activity in the crude sample was determined by using active site-titrated papain, which is a cysteine proteinase from the plant Carica papaya. Crude samples usually contain endogenous cysteine proteinases. These competed with the added papain for the active sites of the cystatins. The cystatin-cysteine proteinase complex was able to be dissociated by the addition of papain. This competition and dissociation could interfere with the determination of cystatin activity, since some of the cysteine proteinases, such as cathepsin B, hydrolyzed the specific substrate for papain during titration with the papain. In order to exclude this interference and measure total cystatin activity, the crude sample must be alkalinized (pH 11.0) for 5 min at 4 degrees C followed by 10 min at 40 degrees C before titration with papain. The minimum detectable amount of cystatins was 20 fmol/assay when it was calculated per mole of papain inhibitory sites. Using this method, significant levels of cystatin activity were detected in all the samples of gingival crevicular fluid taken from periodontal disease patients. These results suggest that cystatins could regulate the cysteine proteinases in gingival crevicular fluid and that this new method could be useful to clarify the role of cystatins in the pathogenesis of periodontal disease.
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PMID:Cystatin activity in gingival crevicular fluid from periodontal disease patients measured by a new quantitative analysis method. 153 1

An investigation has been made of the interactions of the enzyme papain with the polycations protamine, polybrene, poly(L-lysine), spermine, spermidine and the neutral polymer polyvinylpyrrolidone (PVP). At low concentrations, each behaves as an inhibitor of the enzyme. As their concentrations increased above a certain level, the activity of the systems increased, and their inhibition of the enzyme appeared to be less pronounced. When acting by themselves in the presence of the substrate haemoglobin, each of the polycations was a weak proteolytic catalyst with a ranking of catalytic effectiveness of protamine greater than polybrene greater than poly(L-lysine) greater than polyvinyl-pyrrolidone greater than spermidine greater than spermine. This effect could explain the anomalous inhibition of papain by these polycations. The interaction of papain with dansyl protamine (DNSP) and the extent of complex formation were studied using a fluorescence polarization technique and the results showed that there was a strong interaction occurred. The strength of binding was assessed by determination of the critical electrolyte concentration (0.2 M, NaNO3). The stoichiometry of the DNSP-papain complex was found to be 63 base moles of DNSP to one mole of papain.
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PMID:The interaction of papain with polycations. 256 59

The proteolytic specificities of chymopapain and papaya proteinase omega were investigated by using the alpha-chains of manatee and mole haemoglobin, whose primary structures are known, as substrates. The resulting peptides from each enzymatic cleavage were isolated by gel filtration on Sephadex G-25, followed by reversed-phase HPLC of the separated peaks and, in some cases, further purified by preparative thin-layer electrophoresis. The purified peptides were then identified on the basis of their amino-acid composition. The proteolytic specificities of chymopapain and papaya proteinase omega, deduced from the experimental cleavage patterns, are compared to that of papain. As in the case of papain, the specificity-determining factor is the amino-acid residue of the substrate that will be bound in subsite S2 (the next but one from the scissible bond). Aromatic residues in this position, preferred by papain, are not important for chymopapain and papaya proteinase omega. Cleavages preferentially occur when S2 is occupied by leucine, valine or threonine. For chymopapain, proline in position S2 also causes cleavage.
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PMID:The thiol proteinases from the latex of Carica papaya L. IV. Proteolytic specificities of chymopapain and papaya proteinase omega determined by digestion of alpha-globin chains. 268

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.
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PMID:Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase. 352 70

Fc intermediate (Fci) is a papain-generated fragment of human IgG which is intermediate in charge, mol. wt and state of cleavage between the Fc and Fc' fragments of IgG. It is composed of two polypeptide chains of unequal mol. wt held together by non-covalent bonds between the C gamma 3 regions. The larger polypeptide chain has both a C gamma 2 and C gamma 3 domain and its N-terminus is at Leu 235 (60%) and Leu 234 (40%) (IgGl Eu numbering). The smaller polypeptide chain is composed of a C gamma 3 domain with its N-terminus at Gly 341. The carboxy-termini obtained by carboxypeptidase digestion and by a computer program which determined the most probable sequences by fitting the amino acid compositions to the sequence of IgG Eu Fc were heterogeneous involving residues 440-446 for the large polypeptide chain and 429-436 for the small one. The calculated mol. wt of the large polypeptide chain was 26,183, assuming the N-terminus at Leu 234 and C-terminus at 446 and including the carbohydrate moiety. The calculated mol. wt for the small polypeptide chain was 10,682, with the N-terminus at 341 and assuming the C-terminus at 434, for a combined mol. wt of 36,865 for the Fci fragment. Sedimentation equilibrium ultracentrifugation of Fci under non-dissociating conditions showed an Mn of 36,200 +/- 1200, an Mw of 36,400 +/- 600 and an Mz of 37,000 +/- 300 g/mole. The best yields of Fc were obtained with a 6-hr digestion and the best yields of Fcl and Fc' were obtained with digestion for 18 hr in phosphate buffer. Digestion in Tris buffer for 18 hr gave results similar to the 18-hr digestion in phosphate buffer except the yields of Fc' were less. This fragment may be useful for exploring biological functions of human IgG Fc. In addition, some Fc fragments obtained by papain digestion of human IgG either in phosphate or Tris buffer are not covalently bonded and are probably cleaved on the carboxy-terminal side of the interchain disulfide bonds at Leu 234 or Leu 235, the N-termini for the large polypeptide chain of Fci. This indicates that, if disulfide bonded Fc fragments are needed, gel filtration under dissociating conditions will be necessary to remove non-covalently bonded Fc.
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PMID:Fc intermediate (Fci), a papain-generated fragment of human IgG, intermediate in charge, molecular weight and cleavage between the Fc and Fc' fragments of IgG. 402 18

Human erythrocyte acetylcholinesterase is an amphipathic enzyme whose hydrophobic membrane-binding domain can be selectively labeled with a lipophilic photoreagent and removed by digestion with papain. In this paper we demonstrate that methanolysis releases covalently bound fatty acids from the hydrophobic domain and thus confirm that this domain is a covalently linked glycolipid at the enzyme subunit C-terminus. About one mole of saturated and one mole of unsaturated fatty acids were released per mole of domain. Since the predominant unsaturated fatty acids (22:4 and 22:5) are minor components of the esterified fatty acid pool in human erythrocyte membranes, assembly of the glycolipid must involve a selected unsaturated fatty acid pool.
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PMID:Identification of covalently attached fatty acids in the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase. 408 90


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