1. A quantitative method for the determination of pepsin is described depending on the change in conductivity of a digesting egg albumin solution. 2. The combination of pepsin with an insoluble substrate has been followed by this method. 3. The amount of pepsin removed from solution by a given weight of substrate is independent of the size of the particles of the substrate. 4. There is an optimum zone of hydrogen ion concentration for the combination of enzyme and substrate corresponding to the optimum for digestion. 5. It is suggested that the pepsin combines largely or entirely with the ionized protein. 相似文献
1. The peptidase activities of pig pepsins A and C and human pepsin and gastricsin were compared. 2. The peptides studied had the general formula A Leu Val-His-B. Hydrolysis at 37 degrees C and pH 2.07 occurred at the amino side of the leucine residue for all the enzymes and all the peptides. 3. When A was Ac-Ala the peptides were hydrolysed under these conditions slowly by pig pepsin C only. 4. Pig pepsin A and human pepsin were unable to hydrolyse the tyrosine-containing peptides under the conditions tested. Gastricsin (human pepsin C) had about one-third of the activity of pig pepsin C with these substrates. 5. The increase in the rate of hydrolysis caused by the extension of the chain by a single alanine residue was most marked for pig pepsin A and human pepsin. 相似文献
To investigate the active site structures of porcine pepsin and Rhizopus chinensis acid protease (RAP), spin label techniques were applied for these enzymes. Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6.2. 相似文献
A rapid gel filtration chromatography method is described for determination of the molecular weight distribution (MWD) of peptide mixtures by using calibrated Sephadex microbore columns. The method was applied to MWD analysis of peptide mixtures resulting from trypsin and pepsin digestion of glycinin—the major soybean storage protein—under different incubation conditions of pH, temperature, and time of hydrolysis. Possible sources of errors and suggestions for improvement are discussed. 相似文献
Cold-adapted organisms have developed a number of adjustments at the molecular level to maintain metabolic functions at low temperatures. Among other features, they can produce enzymes characterized by a high turnover number or a high catalytic efficiency. The present work is aimed at investigating the process of food digestion at low temperature through the study of pepsins in Antarctic notothenioids. For such a purpose, we have cloned and sequenced three forms of pepsin A and a single form of gastricsin from the gastric mucosa of Trematomus bernacchii (rock cod). Phylogenetic analysis has suggested that the three pepsin A isotypes arose from two gene duplication events leading to the most ancestral pepsin A3 and to the most recent forms represented by pepsin A1 and pepsin A2. Molecular modeling has unraveled significant structural differences in these enzymes with respect to their mesophilic counterparts. Hydropathy and flexibility determined on the substrate-binding subsites of Antarctic and mesophilic pepsins have shown for pepsin A2 reduced hydropathy and increased flexibility at the level of the substrate cleft, features typical of cold-adapted enzymes. Northern blot analysis of RNA from rock cod gastric mucosa hybridized with molecular probes designed on specific regions of different pepsin forms has shown that rock cod pepsin genes are expressed at comparable levels. The present results suggest that the Antarctic rock cod adopted two different strategies to accomplish efficient protein digestion at low temperature. One mechanism is the gene duplication that increases enzyme production to compensate for the reduced kinetic efficiency, the other is the expression of a new enzyme provided with features typical of cold-adapted enzymes. 相似文献
A method has been described for the isolation and crystallization of swine pepsin inhibitor from swine pepsinogen. Solubility experiments and fractional recrystallization show no drift in specific activity. The reversible combination of pepsin with the inhibitor was found to obey the mass law. The inhibitor is quite specific, failing to act on other proteolytic and milk clotting enzymes. The inhibitor is destroyed by pepsin at pH 3.5. Chemical and physical studies indicate that the inhibitor is a polypeptide of approximately 5,000 molecular weight with an isoelectric point at pH 3.7. It contains arginine, tyrosine, but no tryptophane and has basic groups in its structure. 相似文献
The separation of pepsin isoenzymes 1, 2, 3 and 5 (gastricsin) in human gastric juice was effected by chromatography on Mono Q ion-exchanger, and slow-moving proteinase was purified to homogeneity by using a modified procedure incorporating a novel affinity-chromatography step. The pH-activity profiles of these enzymes with mucus glycoprotein and basement-membrane substrates were determined; the profiles for pepsin 2 were noticeably different, and, in general, the pH optima for the hydrolysis of basement membrane were more acidic. Pepsin 1 expressed larger specificity constants (kcat./Km) than pepsin 3 with a series of synthetic peptide substrates, reflecting greater binding (smaller Km) by pepsin 1. Inhibitor studies at pH 1.7 and 4.5 with a series of P2-substituted lactoyl-pepstatins implied that valine at position P2 was optimal for inhibiting pepsins 1, 2 and 3 but detrimental for pepsin 5, whereas lysine at position P2 was tolerated well by pepsin 5 but not by pepsins 1, 2 and 3. The potency of lactoyl-pepstatin with lysine at position P2 did not increase as a function of pH. P2-substituted lactoyl-pepstatins failed to show any inhibitory selectivity among pepsins 1, 2 and 3. 相似文献
The substrates Z-XLeu-(Ala)2 and Z-Phe X-(Ala)2 (Z = benzyloxycarbonyl, X = various amino acid residues) were synthesized in order to investigate the primary specificity of acid proteinases from molds and yeasts. Since these peptides are mainly susceptible to cleavage by the enzymes at the peptide bonds shown by the arrows, it was possible to determine the specificity with respect to the amino acid residues on both sides of the splitting point. Pepsin was used for comparison. The results indicated that the microbial acid proteinases exhibit specificity for aromatic or hydrophobic amino acid residues on both sides of splitting point in peptide substrates, as does pepsin. However, the microbial enzymes showed somewhat broader specificity than pepsin. The former enzymes, which possess trypsinogen-activating ability, show specificity for a lysine residue, while pepsin or Mucor rennin-like enzyme does not. Although pepsin is very specific for a tyrosine residue on the imino side of the splitting point, the microbial enzymes do not show such stringency. 相似文献
A revised three-dimensional crystal structure of ethanol-inhibited porcine pepsin refined to an R-factor of 0.171 at 2.3 A resolution is presented and compared to the refined structures of the fungal aspartic proteinases: penicillopepsin, rhizopuspepsin, and endothiapepsin. Pepsin is composed of two nearly equal N and C domains related by an intra dyad. The overall polypeptide fold and active site structures are homologous for pepsin and the fungal enzymes. The weak inhibition of pepsin by ethanol can be explained by the presence of one or more ethanol molecules, in the vicinity of the active site carboxylates, which slightly alter the hydrogen-bonding network and which may compete with substrate binding in the active site. Structural superposition analysis showed that the N domains aligned better than the C-domains for pepsin and the fungal aspartic proteinases: 107-140 C alpha pairs aligned to 0.72-0.85 A rms for the N domains; 64-95 C alpha pairs aligned to 0.78-1.03 A rms for the C domains. The major structural difference between pepsin and the fungal enzymes concerns a newly described subdomain whose conformation varies markedly among these enzyme structures. The subdomain in pepsin comprises nearly 100 residues and is composed of two contiguous segments within the C domain (residues 192-212 and 223-299). the subdomain is connected, or "hinged," to a mixed beta-sheet that forms one of the structurally invariant, active site psi-loops. Relative subdomain displacements as large as a 21.0 degrees rotation and a 5.9 A translation were observed among the different enzymes. There is some suggestion in pepsin that the subdomain may be flexible and perhaps plays a structural role in mediating substrate binding, determining the substrate specificity, or in the activation of the zymogen. 相似文献
Hydrolysis of beta A2-casein by bovine chymosin and pepsin A was performed in order to compare the hydrolysis of the two enzymes on this protein. Different conditions have been tested: pH 5.5 for 116h and pH 3.5 for 7 h [E/S = 1/100 (w/w)] for chymosin. pH 3.0 for 24 h [E/S = 1/1000 (w/w)] for pepsin A. Under these conditions 17 peptides were obtained after the action of chymosin and 23 after the action of pepsin A. They corresponded respectively to the cleavage of 14 and 15 peptide bonds for chymosin and pepsin A. However, six of the peptide bonds were only hydrolyzed by chymosin and seven other bonds only by pepsin A. Our results showed a preferential splitting at the Leu-X, Ser-X, and Trp-X bonds for chymosin and Leu-X, Met-X, and Thr-X, for pepsin A. Some of the identified peptides contained sequences with possible physiological roles. 相似文献
1. The results of conductivity experiments with alkali caseinate solutions are given and a graphical method of extrapolation, which gives a straight line, is described. The results of the conductivity experiments are shown to be in accord with the results of the previous transference experiments. 2. The change of conductivity of the alkali caseinate solutions with temperature is shown to follow a straight line relationship. 3. The high value of the mobility which was obtained for the casein ion and the high temperature gradient are discussed in relation to McBain''s theory of colloidal electrolytes. 相似文献
A 15 amino acid synthetic peptide, which spanned the dibasic cleavage site C-terminal to neurotensin (NT), in its 170-residue canine precursor, was synthesized by solid-phase methods. Using this substrate in combination with a radioimmunoassay specific for the C-terminal region of NT, a simple assay was developed to monitor protease-mediated cleavage of the Leu8-Lys9 bond in the substrate. Hog pepsin and the related enzymes, rhizopus pepsin, bovine cathepsin D, and mouse renin, were found to be effective in this assay, pepsin cleaving only this bond to liberate the NT-like sequence. The pH dependence of the reaction indicated that pepsin, cathepsin D, and renin exhibited significant activity at pH's characteristic for secretory vesicles (pH 5.5-6.5). In addition, pepsin and cathepsin D were shown to process the native precursor at pH's as high as 5.5. These results, although not proof, are consistent with the idea that endoproteases with pepsin-like specificity may be involved in the processing of the NT precursor in neural/endocrine cells. 相似文献
The determination of the three dimensional structure of chymosin at 3 A resolution by molecular replacement method is described. The rotation functions for various aspartic proteases were calculated and combined results were used for the refinement of orientational parameters of chymosin molecules in the unit cell. The interpretation of Crowther-Blow translation function map with packing consideration enable to place correctly the molecules in the chymosin unit cell. Several difference Fourier syntheses for chymosin were calculated and differences between pepsin and chymosin structures were detected. 相似文献
Affinity chromatography on immobilized Fe(3+) ions--immobilized-metal-ion affinity chromatography (IMAC) method--was used for the determination of pepsin and pepsinogen phosphorylation. IMAC is a very powerful method for detailed studies of proteins. Dephosphorylation of the pepsinogens and pepsins has no effect on their proteolytic ability. For this reason, the determination of proteolytic activity was used for the detection of pepsinogen (pepsin) presence in the collected fractions as a very suitable and specific method. Pepsins and their zymogens probably have the same amounts of phosphate ions in their molecule. The exact definition of conditions is very important for the prepurification of the proteinases and for their analysis. 相似文献
Proteolysis of two purified recombinant enzymes, namely, the Aspergillus niger phytase (r-PhyA) and the Escherichia coli pH 2.5 acid phosphatase (r-AppA), by pepsin and trypsin was investigated in this study. After r-PhyA and r-AppA were incubated with different concentrations of pepsin or trypsin, their residual phytase activities and amounts of inorganic phosphorus released from soybean meal were determined. Both enzymes retained more than 85% of their original activities at the trypsin/phytase ratios (w/w) 0.001 and 0. 005, while r-AppA and r-PhyA lost 60 and 20% of the original activity at the ratio of 0.01 or 0.025, respectively. In contrast, there was a 30% increase in phytase activity after r-AppA was incubated with pepsin at the ratios of 0.005 or 0.01. Meanwhile, r-PhyA lost 58 to 77% of its original activity under the same conditions. Trypsin and pepsin affected the hydrolysis of phytate phosphorus from soybean meal by r-AppA and r-PhyA in a similar way to their residual phytase activities. All of these in vitro proteolyses were confirmed by SDS-PAGE analysis. Our results demonstrate different sensitivities of r-AppA and r-PhyA to trypsin and pepsin, suggesting active trypsin resistant r-PhyA and pepsin resistant r-AppA polypeptides. 相似文献
The amino acid sequence of endothiapepsin, the aspartic protease from Endothia parasitica has been determined. The enzyme consists of 330 residues. The sequence determination was performed exclusively at the protein level. The homology of this fungal milk-clotting enzyme with aspartic proteases is demonstrated by alignment with pepsin, chymosin, gastricsin, renin, and cathepsin D from various vertebrates and proteinase A from Saccharomyces cerevisiae showing 25-30% identity. The identity with mucor rennin from Mucor pucillus was 21% and with penicillopepsin from Penicillium janthinellum 53%, the fungal enzymes thus representing the lowest as well as the highest degree of homology. 相似文献
Fluorescence polarization has been used to study the interaction of dansylated protamine with the enzymes: pepsin, α-chymotrypsin, alkaline phosphatase and invertase. These interactions have been compared with those between dansylated protamine and polyacrylate, or polyvinylsulphate. Each of the various complexes was found to be dissociated by the addition of sodium nitrate and a critical electrolyte concentration (CEC) was determined for each system, to allow assessment of the relative order of binding to the dansylated protamine. This order was: polyvinylsulphate >pepsin >polyacrylate >alkaline phosphatase >α-chymotrypsin. The strength of binding was also assessed by determination of a binding constant, Ka. The values of Ka showed the same relative order of binding as the CEC values. Invertase behaved similarly to the other enzymes, but it was not possible to obtain an unambiguous assessment of the comparative strength of binding. In each case, the stoichiometry of the complex was also determined. 相似文献
The three-dimensional crystal structure of human pepsin and that of its complex with pepstatin have been solved by X-ray crystallographic methods. The native pepsin structure has been refined with data collected to 2.2 A resolution to an R-factor of 19.7%. The pepsin:pepstatin structure has been refined with data to 2.0 A resolution to an R-factor of 18.5%. The hydrogen bonding interactions and the conformation adopted by pepstatin are very similar to those found in complexes of pepstatin with other aspartic proteinases. The enzyme undergoes a conformational change upon inhibitor binding to enclose the inhibitor more tightly. The analysis of the binding sites indicates that they form an extended tube without distinct binding pockets. By comparing the residues on the binding surface with those of the other human aspartic proteinases, it has been possible to rationalize some of the experimental data concerning the different specificities. At the S1 site, valine at position 120 in renin instead of isoleucine, as in the other enzymes, allows for binding of larger hydrophobic residues. The possibility of multiple conformations for the P2 residue makes the analysis of the S2 site difficult. However, it is possible to see that the specific interactions that renin makes with histidine at P2 would not be possible in the case of the other enzymes. At the S3 site, the smaller volume that is accessible in pepsin compared to the other enzymes is consistent with its preference for smaller residues at the P3 position. 相似文献
A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ~35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca2+, Mg2+, Cu2+, Co2+) on proteolytic activities of these enzymes studied. 相似文献
An account of x-ray crystallographic studies of monoclinic porcine pepsin crystals is presented. The chain fold specific for aspartyl proteases is described in detail. As the results of 2-A refinement have shown, the actual structure is that of ethanol-inhibited pepsin. The structure, although close to those of fungal aspartyl proteases, has some specific features: one of them is an insertion near the S'1 site which restricts the position of dipeptide substrates and makes their productive binding more probable than in the fungal enzymes. 3-A resolution data on the binding of the dipeptide phenylalanyl-diiodotyrosine methyl ester are discussed. 相似文献