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Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The statistical thermodynamic model of protein structure proposed in paper I is developed with special attention to the hydrophobic interaction. Calorimetric measurements of the thermal denaturation of five globular proteins, ribonuclease A, lysozyme, alpha-chymotrypsin, cytochrome c, and myoglobin, are quantitatively analyzed using the model. The thermodynamic parameters obtained by the least squares method reflect the global, average properties of proteins and are in good agreement with the expected values estimated from experimental and theoretical studies for model peptides. The average bond energy epsilon is well related to the tertiary structure of each protein. However, the difference in the parameters between different proteins is not observed for the cooperative energy ZJ and the chain entropy alpha. The individuality of a protein as far as its structural stability is concerned, is mainly reflected by the parameter gamma specifying the hydrophobic nature of a protein. The model is further applied in the analysis of several aspects of the structural stability of globular proteins. Denaturation induced by denaturants, salts, and pH are also explained by the model in a unified manner.
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PMID:Structural changes and fluctuations of proteins. II. Analysis of the denaturation of globular proteins. 1 68

A protein methylase III responsible for specifically methylating the cytochrome c in Neurospora crassa was partially characterized by using unmethylated horse heart cytochrome c as a substrate. This enzyme utilizes S-adenosyl-L-methionine as the methyl donor. An analysis of the distribution of [14C]methyl groups in the peptides obtained by chymotrypsin digestion of the enzymically methylated cytochrome c showed that all of the radioactivity could be recovered within a single peak after chromatography. This indicates that the enzyme methylates a specific amino acid sequence within cytochrome c. On hydrolysis of the radioactive chymotryptic peptide, Me-14C-labelled epsilon -N-mono-methyl-lysine, epsilon-N-dimethyl-lysine and epsilon-N-trimethyl-lysine were identified. The enzyme can easily be extracted from the N. crassa mycelial pads and was purified approx. 30-fold.
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PMID:Cytochrome c-specific protein methylase III from Neurospora crassa. 19 92

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.
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PMID:Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases. 24 Jul 15

1. Incubation of NADH-ubiquinone oxidoreductase (Complex I) with chymotrypsin caused loss of rotenone-sensitive ubiquinone-1 reduction and an increase in rotenone-insensitive ubiquinone reduction. 2. Within the same time-course, NADH-K(3)Fe(CN)(6) oxidoreductase activity was unaffected. 3. Mixing of chymotrypsin-treated Complex I with Complex III did not give rise to NADH-cytochrome c oxidoreductase activity. 4. Gel electrophoresis in the presence of sodium dodecyl sulphate revealed selective degradation of several constituent polypeptides by chymotrypsin. 5. With higher chymotrypsin concentrations and longer incubation times, a decrease in NADH-K(3)Fe(CN)(6) oxidoreductase was observed. The kinetics of this decrease correlated with solubilization of the low-molecular-weight type-II NADH dehydrogenase (subunit mol.wts. 53000 and 27000) and with degradation of a polypeptide of mol.wt. 30000. 6. Phospholipid-depleted Complex I was more rapidly degraded by chymotrypsin. Specifically, a subunit of mol.wt. 75000, resistant to chymotrypsin in untreated Complex I, was degraded in phospholipid-depleted Complex I. In addition, the 30000-mol.wt. polypeptide was also more rapidly digested, correlating with an increased rate of transformation to type II NADH dehydrogenase.
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PMID:Effects of proteolytic digestion by chymotrypsin on the structure and catalytic properties of reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase from bovine heart mitochondria. 41 83

A new variety of affinity chromatography of enzymes is described which consists of building up an affinity adsorbent composed of a real substrate. The chromatography is performed at a sub-zero temperature where the turnover of the enzyme is very low or stopped. As a model system Sepharose-bound L-trialanine p-nitroanilide was for used the affinity binding of porcine pancreatic elastase, which was adsorbed to the column in a hypersaline medium at--14 degrees and eluted from the column at the same temperature using 50% (v/v) ethylene glycol. The affinity adsorbent proved to be vary specific as it did not retain trypsin, chymotrypsin and ovalbumin and retained only 20% of cytochrome c.
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PMID:Affinity chromatography at sub-zero temperatures. A model study with porcine pancreatic elastase. 76 28

The process of reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i.e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the well-known ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/water-immiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.
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PMID:Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis. 164 49

Reversible denaturation of several proteins (alpha-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was studied. The regularities of this process were analyzed, employing both experimental and literary data based on the results of kinetic and spectroscopic measurements. In all the systems under study denaturation proceeded in a threshold manner, i. e., an abrupt change in the catalytic and/or spectroscopic properties of the dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of reversible protein denaturation by organic solvents was proposed. This model is based on the widely accepted viewpoint that the undisturbed water shell around the protein globule is necessary for maintaining the dissolved protein in the native state. Quantitative analysis of the model led to an equation establishing a relationship between the threshold concentration of an organic solvent and its physico-chemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation fits well in the experimental data for all the proteins tested. Based on the above thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents (termed as the denaturation capacity or DC) was proposed. Different organic solvents arranged according to their DC values form the DC scale of organic solvents which permits to predict theoretically the threshold concentration of any organic solvent for a given protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Interconnection of physico-chemical characteristics of organic solvents and their denaturing ability in relation to proteins]. 166 45

Infrared spectra have been obtained for 12 globular proteins in aqueous solution at 20 degrees C. The proteins studied, which vary widely in the relative amounts of different secondary structures present, include myoglobin, hemoglobin, immunoglobulin G, concanavalin A, lysozyme, cytochrome c, alpha-chymotrypsin, trypsin, ribonuclease A, alcohol dehydrogenase, beta 2-microglobulin, and human class I major histocompatibility complex antigen A2. Criteria for evaluating how successfully the spectra due to liquid and gaseous water are subtracted from the observed spectrum in the amide I region were developed. Comparisons of second-derivative amide I spectra with available crystal structure data provide both qualitative and quantitative support for assignments of infrared bands to secondary structures. Band frequency assignments assigned to alpha-helix, beta-sheet, unordered, and turn structures are highly consistent among all proteins and agree closely with predictions from theory. alpha-Helix and unordered structures can each be assigned to only one band whereas multiple bands are associated with beta-sheets and turns. These findings demonstrate a method of analysis of second-derivative amide I spectra whereby the frequencies of bands due to different secondary structures can be obtained. Furthermore, the band intensities obtained provide a useful method for estimating the relative amounts of different structures.
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PMID:Protein secondary structures in water from second-derivative amide I infrared spectra. 215 34

Helical regions in many tetrapyrrole proteins are highly amphiphilic, one side interacting with a hydrophobic core and another side interacting with the polar solvent. The mean helical hydrophobic moment is a measure of amphiphilicity of a helix. Helical regions in myoglobin, the alpha and beta subunits of C-phycocyanin, and cytochrome c can be distinguished from nonhelical regions by use of a hydrophobic moment analysis. 24 of 27 (89%) of the helical regions in these proteins were located by this analysis. Calculations were also performed on chymotrypsin, ribonuclease, and papain, which do not possess as pronounced a hydrophobic core as the tetrapyrrole-containing proteins. Less than 50% of the helical regions were correctly located, indicating a lack of amphiphilicity in the helices of these proteins. The hydrophobic moment analysis was also used to predict helical regions in phytochrome, the ubiquitous photoreceptor in plants. Additionally, this analysis is used to quickly locate internal hydrophilic residues which may be functionally important. The distribution of hydrophobic moments from a random sequence was determined so that qualitative and to some extent quantitative comparisons between different amphiphilic helices may be made.
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PMID:Location of helical regions in tetrapyrrole-containing proteins by a helical hydrophobic moment analysis. Application to phytochrome. 217 Mar 85

The two-subunit cytochrome c oxidase from Paracoccus denitrificans has been sequentially digested with chymotrypsin and Staphylococcus aureus V8 protease. The smaller subunit of the enzyme (apparent Mr 32,000) was split into numerous peptides that were removed by anion-exchange HPLC. The larger subunit was only digested to a limited extent (from an apparent Mr 45,000 to Mr 43,000), and the spectral properties were preserved relative to the native enzyme (a reduced minus oxidized difference spectrum with maxima at 447 and 607 nm in the Soret and alpha region, respectively). As judged from CO-reduced spectra this proteolytically digested, one-fragment oxidase was found to contain an equal amount of cytochromes a and a3. The enzymatic activity with reduced cytochrome c as substrate in the presence of Triton X-100 proceeded with equal affinity (apparent Km = 0.5-1.0 microM) and with a Vmax of approximately 20% (40 s-1) of that found with the native enzyme (200 s-1). When the assay system was supplemented with soybean phospholipids, the Km became 2 microM for both enzymes and the Vmax became 730 and 170 s-1 for the native and the digested enzyme, respectively. Thus subunit I of P. denitrificans oxidase, and most probably of the other cytochrome c oxidases as well, contains both hemes and at least one Cu atom and has significant enzymatic activity.
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PMID:Cytochrome c oxidase from Paracoccus denitrificans: both hemes are located in subunit I. 284 84


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