Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Via attachment of p-(omega-aminoethyl)phenylboronic acid to CH-Sepharose in the presence of water-soluble carbodiimide, a new sorbent for the biospecific chromatography of serine proteinases was obtained. The sorbent was shown to be suitable for the purification of subtilisn, alpha-chymotrypsin and trupsin. It is assumed that the serine hydroxyl group at the active site of the enzyme forms, with the boronic acid moiety of the ligand, a structure that imitates transition enzyme--substrage complex. The presence of glycerol selectively improves the binding of serine proteinases, presumably because of stabilization of the tetrahedral state of the boron atom. Direct isolation of subtilisin from a Bacillus subtilis cultural filtrate on phenylboronic acid-containing sorbent gives a virtually homogenous enzyme (42-fold purification) in a nearly-quantitative yield.
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PMID:Phenylboronic acid as a ligand for biospecific chromatography of serine proteinases. 9 36

A procedure for enzymatic determination of serum triglycerides [Clin. Chem. 19, 476 (1973)] has been adapted for use in continuous-flow analysis (Technicon AutoAnalyzer). A very simple manifold is used; serum is incubated at 37 degrees C with the lipase and alpha-chymotrypsin in potassium phosphate buffer (0.1 mol/liter, pH 7, containing 1.50 g of bovine serum albumin per liter). The liberated glycerol is dialyzed against the complete glycerol reagent. The change in absorbance at 340 nm resulting from oxidation of NADH is proportional to the dialyzed glycerol. The same manifold can be used to determine preformed glycerol if the hydrolyzing enzymes are omitted. The hydrolysis is complete, as shown by the use of equivalent glycerol standards. No prior treatment of the samples is necessary. Assays are run at 60 per hour in the AutoAnalyzer l, 80 per hour in the AutoAnalyzer ll. Results with both instruments for 150 samples correlated well with those obtained by the same enzymatic manual method and by the AutoAnalyzer fluorometric procedure.
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PMID:Mechanized enzymatic determination of triglycerides in serum. 16 59

The mobility of separate sites of the water-protein matrix depending on temperature and degree of hydration has been investigated by means of spin labels covalently attached to surface layers of proteins (alpha-chymotrypsin and human serum albumin) and also by a spin probe in a hydrophobic "pocket" of human serum albumin. The results obtained are compared with the data on the mobility of gamma-resonance labels (57Fe) firmly bound with the protein matrix in the same samples. At certain temperature and degree of hydration both spin and gamma-resonance label show an increase in mobility. With the degree of hydration increasing one may observe a simultaneous increase in energy and in entropy of activation: rotatory diffusion of spin labels, i. e., a compensation effect takes place which confirms the concept expressed earlier that cooperation of water-protein interactions is the main reason of CEF. It should be noted that at P/PS greater than 0.8 the values of delta E =7 divided by 10 kcal/mole, and delta S not equal to = 9 divided by 11 e. e. are specific to glycerol-like systems, i. e., under these conditions (P/Ps greater than 0.8) the water-protein layer has glycerol-like properties.
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PMID:[Effect of temperature and degree of hydration on the mobility of spin labels in surface layers of proteins]. 46 Feb 2

The physicochemical properties of nuclear and cytosolic glucocorticoid-binding components from corticoid-sensitive (CS) and corticoid-resistant (CR) mouse lymphoma P1798 cells have been compared. Nuclei or cytosol fractions were prepared from lymphocytes that had been labeled at 37 or 4 degrees, respectively, with 30 nM [3H]triamcinolone acetonide ([3H]TA). [3H]TA was extracted with 0.6 M KCl, 10 mM spermidine, or 4.5 mM MgCl2 from CS nuclei and with 0.6 M KCl or 10 mM spermidine from CR nuclei. As reported previously, nuclear-associated [3H]TA in CR cells was resistant to extraction with mM concentrations of MgCl2. Loss of bound steroid during extraction with 0.6 M KCl was minimized by including the chymotrypsin inhibitor, carbobenzoxy-L-phenylalanine, in the extraction buffer. The inhibitor was not required during extraction with spermidine or MgCl2. Nuclear and cytosolic extracts were examined by analytical agarose gel filtration and glycerol density gradient centrifugation under high salt (0.6 M KCl) conditions. The glucocorticoid-binding component in KCl, spermidine, and MgCl2 extracts from CS nuclei was considerably larger and more asymmetrical [Stokes radius, 57 to 59 A; sedimentation coefficient, 3.64 to 3.70S; molecular weight, 90,000 daltons; frictional ratio, 1.8; axial ratio (prolate ellipsoid), 15] than the [3H]TA-macromolecular complex in KCl and spermidine extracts from CR nuclei[Stokes radius, 29 A; sedimentation coefficient, 3.23 to 3.30S; molecular weight, 40,000 daltons; frictional ratio, 1.25; axial ratio (prolate ellipsoid), 5]. Control experiments showed that the smaller size of the glucocorticoid-binding component in CR nuclei was probably not due to cleavage of a larger, CS-like complex during the extraction procedure. The larger size of the CS [3H]TA complex did not appear to result from aggregation of s a smaller species. No difference in physicochemical parameters of the binding component was observed if cells were labeled with [3H]dexamethasone instead of [3H]TA. However, [3H]dexamethasone complexes were less stable than those formed with [3H]TA as indicated by considerable dissociation of [3H]dexamethasone during gel filtration and gradient centrifugation. This may be due to the 3- to 5-fold lower relative binding affinity of [3H]dexamethasone. Analysis of [3H]TA-labeled cytosol by gel filtration and gradient centrifugation revealed the presence of a single binding component with physicochemical properties similar to those of nuclear [3H]TA complexes from the same strain of tumor. These results suggest that previously described differences in extractability of nuclear-associated [3H]TA between the CS and CR strains of mouse lymphoma P1798 and the lack of response of CR P1798 to glucocorticoid administration may be due, at least in part, to the presence of an altered glucocorticoid-binding component in the resistant tumor cells.
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PMID:Physicochemical differences between glucocorticoid-binding components from the corticoid-sensitive and -resistant strains of mouse lymphoma P1798. 47 39

ATP citrate lyase was purified by two different procedures from the livers of rats first starved and then fed with a fat-deficient and high carbohydrate-glycerol diet. These enzyme preparations were judged homogeneous by sedimentation equilibrium and polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was around 4.4 X 10(5) as determined by sedimentation equilibrium. On sodium dodecyl sulfate gel electrophoresis the enzyme usually showed a single protein band with an estimated molecular weight of 1.2 X 10(5). A similar value for the molecular weight of the subunit was obtained by gel filtration on 6% agarose in the presence of 6 M guanidinium chloride. The molecular weight of this polypeptide chain was estimated by sedimentation equilibrium to be around 1.1 X 10(5). These results indicated that ATP citrate lyase has a subunit structure of four polypeptides of similar size. The extinction coefficient of the dry protein and its amino acid composition are also reported. Some batches of fully active enzyme, judged to be homogeneous by sedimentation equilibrium and polyacrylamide gel electrophoresis, showed two additional major polypeptides (Mr approximately 7.1 X 10(4) and 5.5 X 10(4)) on sodium dodecyl sulfate gel electrophoresis. Studies on the polypeptides produced by proteolytic modification of the native enzyme by trypsin indicated that the additional protein bands observed on sodium dodecyl sulfate gel electrophoresis with some of the batches of enzyme could have been formed by limited proteolysis ("nicking") of the original 1.1 X 10(5) subunit. Trypsin treatment of the native enzyme did not affect the enzyme activity, whereas chymotrypsin and pronase treatment inactivated the enzyme. The trypsin-treated enzyme, which contained only the two smaller polypeptides, did not differ significantly from the untreated enzyme with respect to sedimentation behavior, phosphorylation by ATP, Km for citrate, and immunoreactivity, but it was more heat-labile than the untreated enzyme. The phosphate group on the phosphorylated "nicked" enzyme was located on the larger polypeptide fragment.
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PMID:Structure of ATP citrate lyase from rat liver. Physicochemical studies and proteolytic modification. 82 50

Bovine plasma factor V has been isolated by a preparative procedure involving barium sulfate adsorption, QAEC extraction, poly(ethylene glycol) precipitation, and finally chromatography on a desulfated Sepharose 6B column. Factor V was recovered as a single peak in yields of 35-40% with a specific activity of 50-70 representing a purification of 1000-2000-fold relative to the starting plasma. The apparent molecular weight of the purified factor V was 439,000 +/- 5000. On sodium dodecyl sulfate gel and analytical gel electrophoresis, this factor V preparation showed multiple bands, but results are inconclusive with regard to a possible subunit structure for this factor. The purified factor V was stable for at least 1-2 weeks when stored at 4 degrees C in 0.2 M Tris-acetate, 50 mM CaCl2, 10% glycerol, pH 7.5. When stored at -20 degrees C in 50% glycerol, this preparation was stable for several months. Treatment of the purified factor V with bovine factor Xa, RVV-V, thrombin, or chymotrypsin (but not trypsin) led to a seven- to ten-fold increase in clotting activity and a concomitant decrease in apparent molecular weight. The latter was comparable for each activation system yielding the following average molecular weight values: factor VaSa, 246,000-, factor Va RVV-V, 251,500; Factor Vathr, 239,000; alpha-chymotrypsin, but not trypsin, can activate plasma factor V yielding a product similar to that observed with the above activators. The molar quantities of each of the activators required varied considerably with thrombin having the highest specific activity and factor Xa the lowest. Activation by factor Xa was greatly facilitated by the addition of phospholipid. In the presence of a mixture of phosphatidylcholine/phosphatidylserine (1:1, w/w), the activation of factor V by factor Xa plus Ca2+ required one-third the amount of factor Xa protein as that required in the absence of phospholipid. Even though each of these activators appears to act in an enzymatic manner, the chemical nature of the conversion is unknown at this time.
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PMID:The activation of factor V by factor Xa or alpha-chymotrypsin and comparison with thrombin and RVV-V action. An improved factor V isolation procedure. 126 97

Light microscopic observations using Nomarski optics on the aldehyde-fixed hypothalamus of normal adult cats, monkeys and rabbits revealed the presence of cells in the supraoptic, paraventricular and periventricular nuclei which possessed yellow birefringent inclusions. Immunogold labelling showed that in each species the cells displayed oxytocin-like immunoreactivity, both in electron-dense inclusions within some (but not all) cisterns of rough endoplasmic reticulum and in secretory granules. The cells in cats and rabbits were in all respects indistinguishable from the homologous 'birefringent' cells previously described in rats, but in monkeys, cells frequently contained additional inclusions in cisterns of rough endoplasmic reticulum which did not display oxytocin or vasopressin-like immunoreactivity, even after trypsin, pepsin or chymotrypsin treatment of sections. Observations on cats and rabbits using fluorescence microscopy revealed that the birefringent cells possessed bright autofluorescence which facilitated the identification of more cells than were seen using Nomarski optics alone. Autofluorescence was abolished when sections were mounted in glycerol, or when exposed to light for protracted periods of time. Attempts to label for monoamines in these cells were not successful, suggesting that the fluorescence is not due to aldehyde-induced amine fluorescence. It is not clear why neuropeptides are retained in some rough endoplasmic reticulum cisterns. It is possible that these birefringent cells contain a peptide, or peptides, which are abnormal in some manner, or which may be other members of the oxytocin gene family. Alternatively, the processing of neuropeptides to permit their export to the Golgi apparatus may be deficient. Acetylcholinesterase (AChE) histochemistry revealed that, unlike other oxytocin neurons, cells with intracellular accretions lacked detectable acetyl cholinesterase. As AChE is a known peptidase, it may be involved in regulating peptide export from the rough endoplasmic reticulum.
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PMID:Neuropeptide accretions in the endoplasmic reticulum of oxytocinergic neurons in cats, monkeys and rabbits: a widespread phenomenon. 129 66

High molecular weight, multicatalytic proteinases (named proteasomes) have been for the first time found, on the basis of different protein patterns, in the cytoplasmic soluble fractions of both non-hormone-treated (premature) and progesterone-treated (mature) oocytes of a frog (Rana pipiens). These enzymes, pooled separately as two fractions sedimenting between around 19S and the bottom (over 27S) on glycerol density gradient centrifugation, were composed of several molecular forms with apparent high molecular weights ranging from over 700 kDa, as judged on Sepharose 6B gel filtration. In addition, both the fractions hydrolyzed distinctly a Tyr-containing substrate in the presence of SDS as an activator, and exhibited higher activities toward Arg-containing substrates in the absence of SDS, and activity toward a Glu-containing substrate in the presence and absence of SDS. Immunological experiments using antibodies against proteasomes purified from ovaries of Xenopus laevis clearly revealed characteristic cross-reactivity with both the fractions found in Rana. These data suggest that these enzymes in the two fractions from the respective oocytes in Rana are very similar or identical to the proteasomes of Xenopus. The enzymes in premature oocytes eluted at 0.15-0.18M NaCl on a DEAE-cellulose column disappeared on treatment with TPCK, a well-known chymotrypsin inhibitor, suggesting that the 0.15-0.18M NaCl-eluate contained chymotrypsin-like proteinases probably latent in ovo. The enzymes in mature oocytes had not similar chromatographical patterns to those in premature oocytes. These results suggest that the enzymes already present in premature oocytes may be involved through conformational alterations as to the protein pattern in oocyte maturation following induction by progesterone.
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PMID:High molecular weight-multicatalytic proteinases in premature and mature oocytes of Rana pipiens. 200 78

Two different types of diacylglycerol kinase (DGK) have been purified 10,455-fold (DGK I) and 7,410-fold (DGK IV) from the cytosol and membrane fractions of rat brain, respectively. The cytosolic DGK was purified by successive chromatographies on Affi-Gel Blue, Q-Sepharose F.F., Mono Q, hydroxylapatite, and ATP-agarose. The membrane-bound DGK was purified from the 2 M NaCl extract of membranes by chromatography on Affi-Gel Blue, phenyl-Superose, hydroxylapatite, and ATP-agarose. The resultant preparations contained homogeneous enzymes with a Mr of 110,000 (DGK I) and 150,000 (DGK IV) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These enzymes both phosphorylate 1,2-dioleoyl glycerol at rates of 11.5 mumol/min/mg protein for DGK I and 5.2 mumol/min/mg protein for DGK IV. Both enzymes require divalent cations and ionic detergents for activity. Magnesium is the most potent cation for both enzymes, but Ca2+ was also found to be fairly effective. Manganese is less effective than Mg2+ or Ca2+. Anionic detergents such as sodium deoxycholate or sodium cholate stimulate the activities of both enzymes, although DGK IV is stimulated more markedly than DGK I at lower concentrations. The optimal pH for the two enzymes was found to be the same, pH 7.4. Some phospholipids such as phosphatidylserine and phosphatidylinositol elevate the kinase activities of these kinases even in the absence of detergents. DGK IV is activated more significantly than DGK I by low amounts of phospholipids. The two enzymes also show structural differences. DGK I and DGK IV give different peptide maps after digestion with Staphylococcus aureus V8 protease or alpha-chymotrypsin. The results suggest that these enzymes are different forms of DGK and may be involved in different biological processes.
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PMID:Purification and characterization of membrane-bound and cytosolic forms of diacylglycerol kinase from rat brain. 215 14

Complexes (2:1) of chymotrypsin with human alpha 2-macroglobulin have been prepared in the presence of 200 mM methylamine such that 90% of the chymotrypsin remains noncovalently bound to the alpha 2-macroglobulin. Reaction of this complex with the active-site-directed spin-labeling reagent 4-[(ethoxyfluorophosphinyl)oxy]-2,2,6,6-tetramethylpiperidinyl+ ++-1-oxy results in nitroxide labeling of the active-site serine residue of the complexed chymotrypsin. Electron spin resonance (ESR) spectra of this complex were recorded at 275 K in buffer and at 263 K in 50% glycerol. At 263 K in 50% glycerol the spectrum is that expected for a rigid glass, whereas at room temperature the ESR spectrum shows that the chymotrypsin is only slightly immobilized compared with free spin-labeled chymotrypsin. These findings are discussed in relation to possible models of inhibition of protease activity by alpha 2-macroglobulin. It is concluded that the trap mechanism of Barrett and Starkey [Barrett, A. J., & Starkey, P. M. (1973) Biochem. J. 133, 709-724] is the only model currently considered that can account for the present findings.
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PMID:In support of the trap hypothesis. Chymotrypsin is not rigidly held in its complex with human alpha 2-macroglobulin. 244 56


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