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1.
The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin
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The autolysis of trypsin and α-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in `inert' vessels) are second-order for both trypsin and α -chymotrypsin, with rate constants of 13.0mol−1·dm3·s−1 for trypsin (in 50mm-NaCl at pH8.0 at 25°C) and 10.2mol−1·dm3·s−1 for α-chymotrypsin (in 0.1m-glycine at pH9.2 at 30°C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and α-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of α-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH–activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the `microenvironment' of the enzyme associated with the negatively charged surface or macromolecule. 相似文献
2.
Frederick L. Gates 《The Journal of general physiology》1934,17(6):797-801
The ultraviolet absorption spectrum of Northrop''s pure crystalline pepsin has been determined. The curve of calculated molecular extinction coefficients is given. There is noted a general resemblance of the absorption curve for pepsin to that for urease and tyrosine; the absorption band is maximum at 2750–2800 Å.µ., minimum near 2500. A slight hump on either side of the peak of the extinction curve may be significant. 相似文献
3.
Involvement of an N-Acetylglucosaminidase in Autolysis of Propionibacterium freudenreichii CNRZ 725 总被引:1,自引:1,他引:0
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Riwanon Lemee Sylvie Lortal Bndicte Cesselin Jean van Heijenoort 《Applied microbiology》1994,60(12):4351-4358
Propionibacterium freudenreichii plays an important role in Swiss cheese ripening (it produces propionic acid, acetic acid, and CO2). Moreover, autolysis of this organism certainly contributes to proteolysis and lipolysis of the curd because intracellular enzymes are released. By varying external factors, we determined the following conditions which promoted autolysis of both whole cells and isolated cell walls of P. freudenreichii CNRZ 725: (i) 0.1 M potassium phosphate buffer (pH 5.8) at 40°C and (ii) 0.05 to 0.1 M KCl at 40°C. We found that early-exponential-phase cells possessed the highest autolytic activity. It should be emphasized that the pH of Swiss cheese curd (pH 5.5 to 5.7) is near the optimal pH which we determined. Ultrastructural observations by electron microscopy revealed a 16-nm-thick homogeneous cell wall, as well as degradation of the cell wall that occurred concomitantly with cell autolysis. In the presence of 0.05 M potassium chloride, there was a great deal of isolated cell wall autolysis (the optical density at 650 nm decreased 77.5% ± 7.3% in 3 h), and one-half of the peptidoglycan material was released. Finally, the main autolytic activity was due to an N-acetylglucosaminidase activity. 相似文献
4.
1. Synthesis of plastein from the products of peptic hydrolysis of small quantities of egg albumin can be demonstrated with amorphous or crystalline pepsin. 2. Synthesis of plastein from the products of peptic hydrolysis of amorphous or crystalline insulin can be demonstrated with amorphous or crystalline pepsin. 3. The plastein synthesised by pepsin from the products of peptic hydrolysis of insulin is physiologically inactive. 4. The plastein formed in the insulin experiments could not be crystallised by the methods used for the crystallisation of insulin. 5. The physiological activity of insulin is not destroyed by repeated freezing (at about –50°C.) and melting of an aqueous or an alcoholic solution of this hormone. 6. No marked decrease in the physiological activity of insulin after incubation at 37°C. with pepsin at pH 4.0, in dilute or concentrated solutions, was detected. 相似文献
5.
Specific contributions of tyrosyl and of tryptophanyl residues can be distinguished in the near-ultraviolet circular dichroism spectrum of porcine pepsin. Upon addition of the dipeptide substrate, N-acetylphenylalanyl-l-3,5-diiodotyrosine, at pH values below 4.0, a change in the circular dichroic spectrum results, suggesting that in the presence of substrate the asymmetric environment of certain aromatic amino acid residues of the enzyme is altered. The changes observed are discussed in relation to the enzymatic function of pepsin. 相似文献
6.
A method is described for isolating a crystalline protein of high tryptic activity from beef pancreas. The protein has constant proteolytic activity and optical activity under various conditions and no indication of further fractionation could be obtained. The loss in activity corresponds to the decrease in native protein when the protein is denatured by heat, digested by pepsin, or hydrolyzed in dilute alkali. The enzyme digests casein, gelatin, edestin, and denatured hemoglobin, but not native hemoglobin. It accelerates the coagulation of blood but has little effect on the clotting of milk. It digests peptone prepared by the action of pepsin on casein, edestin or gelatin. The extent of the digestion of gelatin caused by this enzyme is the same as that caused by crystalline pepsin and is approximately equivalent to tripling the number of carboxyl groups present in the solution. The activity of the preparation is not increased by enterokinase. The molecular weight by osmotic pressure measure is about 34,000. The diffusion coefficient in ½ saturated magnesium sulfate at 6°C. is 0.020 ±0.001 cm.2 per day, corresponding to a molecular radius of 2.6 x 10–7 cm. The isoelectric point is probably between pH 7.0 and pH 8.0. The optimum pH for the digestion of casein is from 8.0–9.0. The optimum stability is at pH 1.8. 相似文献
7.
Emma A. Morrison Anne E. Robinson Yongjia Liu Katherine A. Henzler-Wildman 《The Journal of general physiology》2015,146(6):445-461
The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its “active-site” residues—glutamate 14 (Glu14) from each subunit—must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with 1H-15N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states. 相似文献
8.
pH and cell volume effects on H2O and phosphoryl resonance splitting in rapid-spinning NMR of red cells
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Two resonances are seen in the 1H-NMR spectrum of water in erythrocyte suspensions spun at the magic angle, a broad signal from water inside the cells and a sharp signal from extracellular water. The splitting is a result of a true chemical shift difference between the two populations, as bulk magnetic susceptibility effects are negated at the magic angle. The pH dependence of this chemical shift difference in erythrocyte suspensions was investigated. Splittings of 16.7 ± 0.1, 18.9 ± 0.9, and 21.0 ± 0.2 Hz were observed at pH 6.0, 7.0, and 8.5, respectively; however, this was accompanied by a change in the mean cell volume. To account for any contribution from the volume change, the osmolality of the pH 6.0 and 8.5 suspensions was adjusted to equalize the cell volume between samples at the three pHs. Under these conditions, the splitting was 18.3 ± 0.1 and 18.6 ± 0.1 Hz at pH 6.0 and 8.5, respectively. Thus the observed chemical shift difference between the two water resonances was independent of pH. Therefore the splitting of the water resonance was concluded to be directly proportional to the protein concentration within the cell. Measurements of the magnetic susceptibility difference between the two compartments were also carried out, yielding a value of 2.0 ± 0.2 × 10−7 (SI units) for erythrocytes in isotonic saline at pH 7.0. 相似文献
9.
Ben M. Dunn 《Archives of biochemistry and biophysics》1982,214(2):763-771
The binding between α-dimethylaminonaphthalenesulfonyl-(1–12) and porcine pepsin can be detected by the large changes that occur in the fluorescence spectra of the dimethylaminonaphthalenesulfonyl chromophore due to energy transfer from tryptophan residues of the protein. The interaction was previously shown to consist of two steps: a fast step leading to a greatly enhanced fluorescence followed by a slower rearrangement step which reduces the fluorescence but leads to tighter binding and inhibition of the catalytic activity of pepsin (1). The two steps have been studied over a wide range of values of pH, temperature, and ionic strength to gain additional insights into the physical events occurring during the interaction. Based on the pH and ionic strength dependence, the initial step most likely involves electrostatic interaction of the basic peptide inhibitor with the acidic surface of pepsin in a rapid collision process. The use of this fluorescent reporter group has also suggested that the equilibrium binding after the slower rearrangement may also be pH dependent with most effective binding at higher pH. The kinetics of the slow step were measured by monitoring the continuous fluorescence decay. The resulting rates are compared to the rates observed by others for binding of pepstatin to pepsin. From the pH dependence of fluorescence, pKapp values are obtained for the dansylated peptide (3.25), for the pH dependence of the initial binding step (4.87), and for the equilibrium position (4.75). 相似文献
10.
Roger M. Herriott 《The Journal of general physiology》1938,21(4):501-540
1. A method is described for the preparation of pepsinogen from swine gastric mucosae which consists of extraction and fractional precipitation with ammonium sulfate solutions followed by two precipitations with a copper hydroxide reagent under particular conditions. Crystallization as very thin needles takes place at 10°C., pH 5.0 and from 0.4 saturated ammonium sulfate solution containing 3–5 mg. protein nitrogen per milliliter. 2. Solubility measurements, fractional recrystallization, and fractionation experiments based on separation after partial heat or alkali denaturation and after partial reversal of heat or alkali denaturation failed to reveal the presence of any protein impurity. 3. The properties of the enzymatically inactive pepsinogen were studied and compared with the properties of crystalline pepsin. The properties of pepsinogen which are similar to those of pepsin are: molecular weight, absorption spectrum, tyrosine-tryptophane content, and elementary analysis. The properties in which they differ are: enzymatic activity, crystalline form, amino nitrogen, titration curve, pH stability range, specific optical rotation, isoelectric point, and the reversibility of heat or alkali denaturation. 4. Conversion of pepsinogen into pepsin at pH 4.6 was found to be autocatalytic; i.e., the pepsin formed catalyzes the reaction. Conversion of pepsinogen into pepsin is accompanied by the splitting off of a portion of the molecule containing 15–20 per cent of the pepsinogen nitrogen. 相似文献
11.
Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured. 相似文献
12.
Clarence A. Morrell Henry Borsook Hardolph Wasteneys 《The Journal of general physiology》1927,8(6):601-617
1. No destruction of pepsin by heat is demonstrable at pH 1.6 until a temperature of 40°C. is exceeded. 2. The influence of the backward reaction in peptic hydrolysis is shown in the diminishing rate at which increasing concentrations of protein are hydrolyzed. 3. The backward reaction causes the optimum for the hydrolysis of higher concentrations of protein to be attained at a lower temperature than with more dilute solutions. 4. The proteose and peptone associated with commercial pepsin retard hydrolysis in the same sense as the products due to the action of the enzyme. 相似文献
13.
The structure and functions of the M2 protein from Influenza A are sensitive to pH, cholesterol, and the antiinfluenza drug Amantadine. This is a tetrameric membrane protein of 97 amino-acid residues that has multiple functions, among them as a proton-selective channel and facilitator of viral budding, replacing the need for the ESCRT proteins that other viruses utilize. Here, various amino-acid-specific-labeled samples of the full-length protein were prepared and mixed, so that only interresidue 13C-13C cross peaks between two differently labeled proteins representing interhelical interactions are observed. This channel is activated at slightly acidic pH values in the endosome when the His37 residues in the middle of the transmembrane domain take on a +2 or +3 charged state. Changes observed here in interhelical distances in the N-terminus can be accounted for by modest structural changes, and no significant changes in structure were detected in the C-terminal portion of the channel upon activation of the channel. Amantadine, which blocks proton conductance by binding in the aqueous pore near the N-terminus, however, significantly modifies the tetrameric structure on the opposite side of the membrane. The interactions between the juxtamembrane amphipathic helix of one monomer and its neighboring monomer observed in the absence of drug are disrupted in its presence. However, the addition of cholesterol prevents this structural disruption. In fact, strong interactions are observed between cholesterol and residues in the amphipathic helix, accounting for cholesterol binding adjacent to a native palmitoylation site and near to an interhelix crevice that is typical of cholesterol binding sites. The resultant stabilization of the amphipathic helix deep in the bilayer interface facilitates the bilayer curvature that is essential for viral budding. 相似文献
14.
The cleavage specificities of typical aspartic proteinases: pepsin A, gastricsin, cathepsin D and rhizopuspepsin, were examined at different pH values with oxidized insulin B chain as a substrate with special attention to the specificities near neutral pH. Significant differences in relative specificity for scissile bonds were observed between pH 2.0 and 5.5-6.5, which may be partly related with the changes in dissociation states of the His and Glu residues in the substrate and the ionizable residues in the active site of each enzyme. 相似文献
15.
Conformational changes induced in pepsin and pepsinogen by iodination of tyrosine residues and the possible role of lysine residues on conformational stability of pepsinogen are investigated by circular dichroism (CD) studies in solution. At low degrees of iodination (6 I/molecule) the pepsin molecule denatured, with complete loss of β-structure at pH 5.5. Pepsinogen showed greater resistance to conformational change on iodination (10 I/molecule) and about 30% of its ordered structure is retained. In the aromatic region, the tyrosyl CD bands of iodinated pepsin decreased in intensity, indicating a change in the environment of tyrosine residues. A comparison with the CD spectra of expanded structures of pepsin in 6 m guanidine hydrochloride or alkaline solutions (pH 9.75) indicated retention of a significant amount of tertiary structure in iodinated pepsin. Changes in tertiary structures were marginal on iodination of pepsinogen. Less than 1% (residue moles) of poly-l-lysine, a known inhibitor, was found to destabilize the secondary and tertiary structure of pepsin at pH 6.75, although the lysine-rich 1–44 segment of pepsinogen tends to stabilize the conformation of the pepsin chain. This seems to suggest that the inhibitory effects of polylysine on pepsin occur by a mechanism different from that of the activity-limiting effect of the lysine-rich 1–44 segment of pepsinogen. 相似文献
16.
Monkey pepsinogens and pepsins. VI. One-step activation of Japanese monkey pepsinogen to pepsin 总被引:1,自引:0,他引:1
When Japanese monkey pepsinogen was activated at pH 2.0 in the absence of pepstatin, the activation segment of the amino(N)-terminal 47 residues was released as a single intact polypeptide. This clearly shows that the pepsinogen was activated to pepsin directly. This direct activation was called a 'one-step' process. On the other hand, when pepsinogen was activated at pH 2.0 in the presence of pepstatin, an appreciable amount of pepsinogen was converted to an intermediate form between pepsinogen and pepsin, although a part of pepsinogen was activated directly to pepsin. The intermediate form was generated by releasing the N-terminal 25 residues of pepsinogen. This activation through the intermediate form is thought to be a 'two-step' or 'stepwise-activating' process involving a bimolecular reaction between pepstatin-bound pepsinogen and free pepsin. 相似文献
17.
Summary Known methods for the acceleration of yeast autolysis have been investigated and new methods have been developed. It is shown that autolysis can be induced by plasmolysis with a number of solvents. The efficiency of this treatment is dependent on the nature of the solvent, its concentration and the duration of the treatment. Plasmolysis generally does not cause release of molecules of high molecular weight (MW) such as enzymes. However, addition of water initiates autolysis and the enzyme carboxypeptidase Y (MW 64000), for example, is released. The rate of this process is very dependent on pH; at the optimal pH (around 8.0) essentially complete autolysis is achieved within 20 h using the best solvents. Control of pH through the process is required. Straight-chain alcohols of medium chain length, i.e. C6–C9 appear to function efficiently in amounts of only 1.2 ml/100 g yeast. In amounts of 2.5–10 ml solvent/100 g yeast trichloroethane, chloroform and in particular ether also provide efficient plasmolysis. Furthermore, it was shown that treatment of an aqueous suspension of yeast cells with a variety of non-ionic as well as ionic detergents caused autolysis. The influence of pH corresponds to that observed with organic solvents, i.e. a pH around 8.0 is optimal. This autolysis process was most efficient when the compressed yeast had been initially plasmolysed by treatment with sodium chloride followed by addition of water. The inexpensive detergents Triton X-100 and N-lauroylsarcosine appeared to be among the most efficient. The methods described in this paper are inexpensive and can be employed on a large scale. In addition, cell debris is easily removed, which is very important for subsequent down-stream processing. In the alternative method using physical breakage by homogenization this step is highly problematic.
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18.
The action of semicarbazide on the aggregation of the tropocollagen macromolecule 总被引:1,自引:1,他引:0
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1. A difference in conformation was found between the collagen in solutions treated with semicarbazide hydrochloride and those treated with sodium chloride. This difference could be correlated with the difference in extent of aggregation between the fibrils precipitated from these solutions. 2. The action of semicarbazide hydrochloride depended on the pH and temperature of treatment in a complex manner. At constant temperature semicarbazide enhanced aggregation at pH values less than 4·3, but decreased aggregation was observed at pH values greater than 5·0. At pH 4·3 the effect of semicarbazide on aggregation varied with temperature, the tendency to increased aggregation being more pronounced at 34° and 36–37°. Similar increased aggregation tendencies superimposed on an overall decreased aggregation were observed at these temperatures at pH8·9. 3. A specific binding of semicarbazide to the collagen molecule was indicated. 相似文献
19.
Acid inactivation of and incorporation of phosphate into alkaline phosphatase from Escherichia coli 总被引:3,自引:3,他引:0
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1. Alkaline phosphatase of Escherichia coli undergoes below pH 6·0 a reversible acid inactivation that has been studied and related to the extent of uptake of inorganic phosphate occurring below pH 6·0. 2. The rate of inactivation is rapid in the first few minutes but later it decreases markedly. Temperature, pH, composition of buffer and other factors have an important effect on the inactivation. 3. About 60% of the activity lost at pH values above 3·5 is rapidly recovered when the enzyme is taken back to pH 8·0, independently (within certain limits) of the extent of the inactivation. 4. Phosphate and Zn2+, although very good protectors of the inactivation by acid, are not by themselves able to reverse the acid inactivation. 5. Inorganic phosphate seems not to be incorporated into the acid-inactivated enzyme. 6. Incorporation of more than one mole of phosphate/mole of enzyme has been obtained, but the phosphate residues seem to be incorporated to serine residues with a common sequence, suggesting two identical active serine residues/molecule of active enzyme. 相似文献
20.
Molecular and crystal structures of monoclinic porcine pepsin refined at 1.8 A resolution 总被引:7,自引:0,他引:7
A R Sielecki A A Fedorov A Boodhoo N S Andreeva M N James 《Journal of molecular biology》1990,214(1):143-170
The molecular structure of the archetypal aspartic proteinase, porcine pepsin (EC 3.4.23.1), has been refined using data collected from a single monoclinic crystal on a twin multiwire detector system to 1.8 A resolution. The current crystallographic R-factor (= sigma parallel to Fo/-/Fc parallel to/sigma/Fo/) is 0.174 for the 20,519 reflections with /Fo/ greater than or equal to 3 sigma (Fo) in the range 8.0 to 1.8 A (/Fo/ and /Fc/ are the observed and calculated structure factor amplitudes respectively). The refinement has shown conclusively that there are only 326 amino acid residues in porcine pepsin. Ile230 is not present in the molecule. The two catalytic residues Asp32 and Asp215 have dispositions in porcine pepsin very similar to the dispositions of the equivalent residues in the other aspartic proteinases of known structure. A bound solvent molecule is associated with both carboxyl groups at the active site. No bound ethanol molecule could be identified conclusively in the structure. The average thermal motion parameter of the residues that comprise the C-terminal domain of pepsin is approximately twice that of the residues in the N-terminal domain. Comparisons of the tertiary structure of pepsin with porcine pepsinogen, penicillopepsin, rhizopus pepsin and endothia pepsin reveal that the N-terminal domains are topographically more similar than the conformationally flexible C-terminal domains. The conformational differences may be modeled as rigid-body movements of "reduced" C-terminal domains (residues 193 to 212 and 223 to 298 in pepsin numbering). A similar movement of the C-terminal domain of endothia pepsin has been observed upon inhibitor binding. A phosphoryl group covalently attached to Ser68 O gamma has been identified in the electron density map of porcine pepsin. The low pKa1 value for this group, coupled with unusual microenvironments for several of the aspartyl carboxylate groups, ensures a net negative charge on porcine pepsin in a strongly acid medium. Thus, there is a structural explanation for the very early observations of "anodic migration" of porcine pepsin at pH 1. In the crystals, the molecules are packed tightly into a monoclinic unit cell. There are 190 direct contacts (less than or equal to 4.0 A) between a central pepsin molecule and the five unique symmetry-related molecules surrounding it in the crystalline lattice. The tight packing in this cell makes pepsin's active site and binding cleft relatively inaccessible to substrate analogs or inhibitors. 相似文献