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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 heavy chain fragmentation pattern of native myosin when digested by proteolytic enzymes is influenced by such conditions as the nature of the proteolytic agent, ionic strength and presence or absence of divalent cations. HMM and S-1 produced by digestion of 14CNEM-labelled myosin under various conditions were analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. Purified samples of these species were digested under controlled conditions by chymotrypsin and trypsin and a comparison of the observed heavy chain fragmentation patterns led to a sequential arrangement of the proteolytic fragments. The main features of this arrangement are the following: a 21K molecular weight tryptic peptide is found at the N-terminal side of myosin heavy chain. Adjacent to it is a 48K peptide, then a 19.5K peptide containing the two SH-1 and SH-2 thiols. These three peptides constitute the heavy chain of S-1. Adjacent to this S-1 heavy chain is a tryptic (and also chymotryptic) 40K peptide. The rest of the HMM heavy chain on the C-terminus is a sequence susceptible to both chymotrypsin and trypsin attack yielding an undefined number of small peptides.
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PMID:Proteolytic fragmentation of myosin: location of SH-1 and SH-2 thiols. 11 42

Subfragment-1 of HMM was prepared by tryptic [EC 3.4.21.4] digestion of HMM, which had been modified with 1 mole of CMB per mole of HMM at a specific SH group, SHr. S-1(T) obtained from CMB-HMM retained almost all the CMB, and the amount of bound CMB was about 0.8-0.9 mole per 2 moles of S-1(T). S-2 of CMB-HMM contained no bound CMB. The ATPase [EC 3.6.1.3] activity of HMM increased gradually with increase in the concentration of FA, and the acto-HMM ATPase was inhibited by excess substrate or removal of Ca2+ ions in the presence of RP. The ATPase activity of CMB-HMM increased to a maximum level on adding a small amount of FA, and the acto-CMB-HMM ATPase showed neither substrate inhibition nor Ca2+ sensitivity in the presence of RP. On the other hand, the dependence on the concentration of FA of the ATPase activity of acto-S-1(T) was unaffected by modification of S-1 with CMB. The Ca2+ sensitivity of the ATPase activity of acto-S-1(T) in the presence of RP was also unaffected by the modification. Acto-S-1(T) dissociated almost completely, while acto-CMB-S-1(T) was only 50% dissociated on adding ATP. More than 80% of the bound CMB was contained in S-1(T) undissociated from FA. Furthermore, superprecipitation of actomyosin induced by ATP was completely inhibited by adding about 2 moles of CMB-S-1(T) per mole of actin monomer. On the other hand, about 90% of the burst size of Pi liberation was retained in S-1(T) dissociated from FA. It was concluded that the two heads of the myosin molecule are different: one shows the initial burst of Pi liberation, and does not contain the SHr group which binds CMB (head B), and the other does not show the initial burst and contains the SHr group (head A). It was also concluded that modification of head A of HMM or myosin with CMB increases its binding strength to FA, and consequently the substrate inhibition and Ca2+ sensitivity of acto-HMM or actomyosin ATPase at head B are lost on modification of head A with CMB. CMB-S-1(CT) was prepared by chymotryptic [EC 3.4.21.1] digestion of CMB-myosin, and separated into two fractions by ultracentrifugation of acto-CMB-S-1(CT) in the presence of ATP. Three components of CMB-S-1(CT) with molecular weights of 9, 2.4, and 1.2 X 10(4) were separated by SDS-polyacrylamide gel electrophoresis. The ratios of the peak areas of the three components in electrophoretograms were the same in CMB-S-1(CT) and in the two fractions (1 : 0.18 : 0.09), indicating that heads A and B have the same subunit structure.
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PMID:Structure and function of the two heads of the myosin molecule. III. Cooperativity of the two heads of the myosin molecule, shown by the effect of modification of head A with rho-chloromercuribenzoate on the interaction of head B with F-actin. 13 79

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.
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PMID:Interaction of myosin subfragments with F-actin. 15 50

Thermal behavior of intact and LC-2 deficient myosin obtained from bovine heart was studied using EPR and DSC techniques. The reactive thiol sites (Cys 704) of myosin was labelled with 4-maleimidopiperidine-nitroxyl, and the measurements were taken in X-band in the conventional and saturation transfer EPR time domains. DSC scans were made from 5 degrees up to 60 degrees C with 0.25 degree C/min scan rate. Bovine heart myosin was isolated by standard methods. The LC-2 deficient myosin was prepared by cleaving myosin with alpha-chymotrypsin (400:1 molar ratio) for 1.5 min at 25 degrees. Our basic finding was a conformational change in LC-2 deficient myosin detected at 18 degrees C. It was not observed in intact myosin suggesting that the dissociation of the regulatory light chain resulted in a local structural change in the neighbourhood of the attached label in the 20 kD domain. The rotational correlation time of the label and the microwave saturation behavior of myosin at 25 degrees C exhibited no significant differences after removal of the LC-2 light chain. However, the mobility of the same label was significantly diminished in skeletal muscle. Studying the melting behavior of myosin, six endothermic peaks were detected at 19; 41.3; 43.3; 45.5; 48.5; and 54.3 degrees C (enthalpies: 708.4; 399; 773.8; 1089; 1612.8; and 3304.8 kJ/mol). They were assigned to the segment containing the essential thiols: HMM S-2, HMM S-1 (50kD and 20kD plus 27kD) and LMM. Removal of the LC-2 light chain was associated with the disappearance of the 18 degrees transition showing again a structural change in LC-2 deficient myosin which extended to a larger region.
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PMID:Conformational changes in bovine heart myosin as studied by EPR and DSC techniques. 196 39

Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80 degrees C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. Ki values for the complexes of papain and the inhibitors were estimated to be 2.8 x 10(-10) M for HMM-cystatin and 1.3 x 10(-9) M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.
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PMID:Cystatins from bovine brain: purification, some properties, and action on substance P degrading activity. 245 27

Limited digestion of caldesmon by alpha-chymotrypsin generates mainly 110, 80, 60, 38, and 28 kDa fragments. Affinity chromatography of these fragments on columns immobilized with myosin, HMM, or tropomyosin showed that the bound fraction from these columns was similar and it contained 110, 80, 60 and 28 kDa fragments. These fragments did not bind to myosin filaments, acto-HMM, actin or tropomyosin-actin in the solution, and they had no effect on the actin-activated ATPase of HMM. In contrast, the flow-through fraction from these affinity columns inhibited the actin-activated ATPase. Binding studies revealed that the 38 kDa fragment and its break down products bound to actin and tropomyosin-actin, and they were released partially from actin by calmodulin with a concomitant increase in the ATPase activity. These results indicate that, unlike the actin binding domain, the myosin and tropomyosin binding domains require the caldesmon molecule to be intact in order to exert their effects on the protein-protein interaction.
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PMID:Characteristics of the myosin and tropomyosin binding regions of the smooth muscle caldesmon. 252 36

We have developed a new method to prepare single-headed heavy meromyosin with high purity and a high yield. To examine whether the two heads on the same myosin molecule work cooperatively or not, it is important to prepare pure single-headed heavy meromyosin. Myosin was extracted from myofibrils treated with a solution containing CyDTA, a strong divalent cation chelator. CyDTA treatment was essential to the production of sHMM. Then such myosin was digested with chymotrypsin in the presence of divalent cations at high ionic strength. Crude sHMM was separated from double-headed HMM by affinity chromatography using an ADP-column. Contaminating S1 was removed by gel filtration. Heavy chain of sHMM obtained by the present method had no nick. Purified sHMM showed normal EDTA-ATPase and Ca-ATPase. It interacted with thin filament and its ATPase was activated by actin normally.
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PMID:New method to prepare single-headed heavy meromyosin with high purity and a high yield. 253 47

We investigated the limited proteolysis of fast and slow myosins purified from rabbit psoas major and semimembranosus proprius muscles, respectively, by the main lysosomal proteinases: cathepsins B, H, L, and D. In EDTA containing buffer, cathepsin D cleaved both myosins only at the rod-S1 junction leading to the formation of two S1 fragments of slightly higher Mr than the three forms obtained with chymotrypsin. On addition of MgCl2 instead of EDTA, myosin hydrolysis was markedly reduced. In contrast, irrespective of the presence of either MgCl2 or EDTA, cathepsin B hydrolysed both myosins into HMM and LMM. Cathepsin L digested myosins more extensively than cathepsins B and D and the main fragments generated were, in decreasing order of importance, rod, S1, S2, HMM, and LMM. In the incubation conditions tested, cathepsin H displayed nondetectable action on myosins. As fast and slow myosin digest patterns were compared, the main differences observed concerned the size of the proteolytic products and the rate of hydrolysis, which was about 4-fold higher for the fast than for the slow isoform. This appeared consistent whatever enzyme was considered.
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PMID:Lysosomal proteinase-sensitive regions in fast and slow skeletal muscle myosins. 254 27

Two active forms (Mr 45,000 and 28,000) of a metalloendopeptidase that digest proteoglycans and other extracellular matrix components of connective tissues have previously been purified from rheumatoid synovial cells and characterized [Okada, Nagase & Harris (1986) J. Biol. Chem. 261, 14245-14255]. To study the mechanisms of activation the precursor of this metalloendopeptidase has now been purified. The final products are homogeneous on SDS/polyacrylamide-gel electrophoresis and identified as a set of zymogens of Mr 57,000 and 59,000, in which the latter form is probably the product of post-translational glycosylation of the Mr 57,000 zymogen, as it binds to concanavalin A. The zymogen can be activated by trypsin, chymotrypsin, plasma kallikrein, plasmin and thermolysin, but not by thrombin. Although the activated metalloendopeptidase is further degraded by trypsin, plasma kallikrein and thermolysin during a prolonged incubation, it is relatively stable against plasmin and chymotrypsin. Activation with 4-aminophenylmercuric acetate is dependent on its concentration. It requires the reaction with the zymogen, possibly through thiol groups, and the continued presence of the agent. During this treatment the zymogen undergoes a sequential processing; first it becomes active without changing its apparent molecular mass, and then it is processed to low-Mr species of Mr 46,000, 45,000 (HMM) and 28,000 (LMM). The rate of conversion of the precursor into an initial intermediate of Mr 46,000 follows first-order kinetics (t1/2 2.0 h with 1.5 mM-4-amino-phenylmercuric acetate at 37 degrees C) and is independent of the initial concentration of the zymogen or the presence of up to a 676-fold molar excess of substrate, whereas the generation of HMM and LMM species is affected by these parameters. These results indicate that activation of the prometalloendopeptidase by an organomercurial compound is initiated by the molecular perturbation of the zymogen that results in conversion into the 46,000-Mr intermediate by an intramolecular action; the subsequent processing of this intermediate in HMM and LMM species is a bimolecular reaction. In vivo it is probable that the precursor of this metalloendopeptidase is activated either by direct limited proteolysis by tissue or plasma endopeptidases, or, alternatively, by factors that cause certain conformational changes in the zymogen molecule.
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PMID:The precursor of a metalloendopeptidase from human rheumatoid synovial fibroblasts. Purification and mechanisms of activation by endopeptidases and 4-aminophenylmercuric acetate. 305 16

Pig heart myosins isolated from the free wall of the right ventricle and the free wall of the left ventricle were compared with respect to structural and enzymatic properties. The following parameters were studied (1) activation of myosin ATase by Ca2+ and K+j(2) molecular weight of the heavy and light chains of myosins as determined by electrophoretic migration in polyacrylamide sodium dodecyl sulfate (SDS) gels; (3) ability of the heavy chains to form aggregates at low ionic strength as revealed by electron microscopy; (4) sensitivity to the action of chymotrypsin. Differences were observed between left and right ventricular myosins (L-myosin and R-myosin) for all these parameters except for the molecular weight of heavy and light chains. The existence of large amounts of short synthetic filaments for R-myosin compared with L-myosin as revealed by the length repartition of the filaments, and the production of smaller quantities of HMM-S by chymotryptic digestion for R-myosin, strongly suggest the presence of different cardiac myosin heavy chain species.
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PMID:Pig cardiac myosin isoenzymes. 644 26


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