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)

The temperature-dependence of local melting within the alpha-helical, coiled-coil structure of rabbit myosin rod has been investigated by following changes in the rate constants of proteolytic digestion. The kinetics of fragmentation of the rod by three different enzymes (alpha-chymotrypsin, trypsin and papain) over the temperature range 5 to 40 degrees C (pH 7, I = 0.5) has been monitored by electrophoresis of the digestion products on sodium dodecyl sulfate/polyacrylamide gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzyme by comparison with model substrates. Results from the three enzyme-probes are similar in showing that local melting within the rod occurs in two distinct stages. At temperatures between 5 and 25 degrees C, melting is confined to a restricted segment of the rod structure near the light meromyosin/heavy meromyosin junction. At temperatures between 25 and 40 degrees C, a wider segment of the rod lysing between the junction and the short subfragment-2 segment (the hinge domain) appears to be melting, judging from the broad spectrum of cleavage sites observed in this region. Results are compared with those from other physicochemical methods that measure the hinging or opening of the coiled-coil structure of the rod.
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PMID:An enzyme-probe method to detect structural changes in the myosin rod. 636 39

The folded 10 S conformation of turkey gizzard myosin is more resistant to proteolysis by papain than the extended 6 S conformation. These findings confirm those of Onishi and Watanabe (Onishi, H., and Watanabe, S. (1984) J. Biochem. (Tokyo) 95, 899-902). In addition, we suggest that the effect of phosphorylation on heavy-chain digestion by papain is related to the dependence of conformation on phosphorylation and not to a direct effect of phosphorylation itself. Proteolysis by Staphylococcus aureus protease and trypsin also is slower for 10 S compared to the 6 S conformation. Heavy chain hydrolysis by alpha-chymotrypsin is not dependent on myosin conformation. Filamentous myosin and heavy meromyosin are more resistant to papain proteolysis in the dephosphorylated compared to the phosphorylated states. The different sensitivities to proteolysis probably are caused by changes in the subfragment 1-subfragment 2 region of the molecule rather than at the heavy meromyosin-light meromyosin junction. These changes are induced as part of the 6 S-10 S transition and occur in monomeric and filamentous myosin and in heavy meromyosin. These more subtle alterations in the head-neck junctions of the molecule may be more important in modifying myosin enzymatic activity than the actual interaction of the tail and neck regions of the molecule.
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PMID:Conformation-dependent proteolysis of smooth-muscle myosin. 638 9

The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 Mr and 53,000 Mr per polypeptide chain) and a short subfragment-2 particle (34,000 Mr per polypeptide chain) by three different enzymes (alpha-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 degrees C. We followed the time-course of digestion at specific sites at high (I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 Mr) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 degrees C a restricted region at Mr = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 degrees C local melting was detected by all three enzymes at several specific sites within the hinge domain (Mr = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.
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PMID:An enzyme-probe study of motile domains in the subfragment-2 region of myosin. 639 18

Proteolytic fragments of 400 kD isolated from chymotrypsin-treated connectin, a muscle elastic protein, still retained the ability to cause aggregation of myosin filaments but lost the actin-bundling action. Tryptic digests of connectin showed similar effects. However, when connectin was hydrolyzed by pepsin to peptides smaller than approximately 40 kD, no such action was seen for both myosin and actin filaments. It is suggested that the actin bundling action of connectin filaments is due to topological restrictions. A modified reproducible procedure for the preparation of native connectin from chicken breast muscle is described in detail.
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PMID:Proteolytic fragments of connectin cause aggregation of myosin filaments but not of actin filaments. 644 94

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

Heavy chains of myosin rods and subfragment 1 were isolated from normal hearts and from mechanically overloaded hearts of young and older rats. These myosin heavy-chain fragments were cleaved by cyanogen bromide or partially proteolysed by pronase and by chymotrypsin after denaturation with sodium dodecyl sulfate. The peptides, analyzed by electrophoresis on a one-dimensional polyacrylamide slab gel, varied depending on the origin of the cardiac myosin heavy chains. Some bands present in the peptide patterns of the normal heart of young rats were missing from the pattern of greatly hypertrophied hearts and vice versa. We conclude that mechanical overloading of the heart stimulates the synthesis of cardiac myosin 'isozyme' with a heavy-chain primary structure which is different from that observed in the normal heart of young rat. The patterns from myosin heavy-chain peptides from the hearts of older rats were different from those for peptides from young rat hearts; these results also indicate the presence of a new myosin heavy chain specific to ageing. No difference was detected between the peptide patterns of heavy chains isolated from hypertrophied hearts of young and older rats, and those isolated from normal hearts of older rats.
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PMID:Evidence for new forms of cardiac myosin heavy chains in mechanical heart overloading and in ageing. 645 16

The Ca2+- and calmodulin-dependent myosin light chain kinase of rabbit skeletal muscle was converted to a Ca2+-independent form by limited proteolysis with alpha-chymotrypsin. The conditions prevailing during proteolysis are important and the loss of Ca2+-dependence was achieved best by hydrolysis of the Ca2+-calmodulin-kinase complex. The lack of Ca2+- and calmodulin-dependence was found using both myosin and isolated light chains as substrates. The specific activity of the Ca2+-independent form (Mr approximately 65,000) was similar to that of the native enzyme, i.e., 2 to 5 mumol phosphate transferred min-1 mg-1 kinase. The 65,000-dalton fragment was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and approximately 0.8 moles phosphate were incorporated per fragment.
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PMID:Conversion of a Ca2+-dependent myosin light chain kinase from skeletal muscle to a Ca2+-independent form. 668 81

During proteolytic digestion of myosin to prepare HMM or HMM-S-1 subfragments, myosin light chains are affected variously according to experimental conditions. In the presence of Ca2+ at low ionic strength trypsin rapidly degrades the DTNB light chain to a 18 K peptide. This new DTNB light chain is compared to a DTNB (17K) light chain obtained by chymotryptic digestion under similar conditions as shown here and in parallel studies. (Weeds and Pope (1977), J. Mol. Biol, 111, 129--157). Whereas the chymotryptic DTNB (17K) has lost its phosphorylation site (Ser-15), tryptic DTNB (18K) has lost only a strongly basic N-terminal peptide. A transitory (ca 14K) fragment is formed when digestion occurs in the presence of EDTA. A-1 light chain (20.7K) is cut to form a 20K species when myosin (of (CT)-HMM obtained ina high ionic strength medium) is digested with trypsin whether Me2+ is present or not. The new formed species has also lost its strongly basic N-terminal peptide and assumes a primary structure closer to that of A-2. Chymotrypsin was shown to have no effect on the A-1 light chain under the present conditions, whereas A-2 is not affected by chymotrypsin or trypsin under any of the conditions described in the present study.
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PMID:Fate of the light chains in the course of proteolytic digestion of rabbit fast skeletal myosin. 676 1

The patterns of myosin isozymes in embryonic and adult chicken pectoralis muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light chains and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, the predominant isozyme component in embryonic pectoralis myosin could be clearly distinguished from adult myosin isozymes. SDS-polyacrylamide gel electrophoresis indicated that the light chain composition of embryonic myosin was also different from that of adult myosin. The pattern of peptide fragments produced by myosin digestion with a-chymotrypsin differed significantly between embryonic and adult skeletal myosin. These results suggest that myosin in the embryonic pectoralis muscle is different in both light and heavy chain composition from myosin in the same adult tissue.
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PMID:Changes in myosin isozymes during development of chicken breast muscle. 680 70

Limited digestion of calmodulin (CaM)-dependent myosin light chain kinase from turkey gizzard with alpha-chymotrypsin in the presence of bound CaM generated an 80,000-dalton kinase fragment that was fully active in the absence of Ca2+. This kinase catalyzed specific Ca2+-independent phosphorylation of the 20,000-dalton light chain of myosin using isolated light chains, intact myosin, and actomyosin. Phosphorylation of myosin in the absence of Ca2+ allowed us to dissociate myosin phosphorylation from other potential Ca2+-dependent regulatory mechanisms, thus permitting an evaluation of the postulated central role of myosin phosphorylation in the regulation of smooth muscle contraction. Ca2+-independent myosin phosphorylation was found to cause loss of Ca2+ sensitivity of 1) actin-activated myosin ATPase activity in a crude actomyosin preparation, and 2) tension development in skinned smooth muscle fibers in the absence of Ca2+. Myosin phosphorylation is, therefore, the key event in actin activation of ATPase activity and initiation of contraction in skinned chicken gizzard fibers.
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PMID:Gizzard Ca2+-independent myosin light chain kinase: evidence in favor of the phosphorylation theory. 684 77


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