Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

High-affinity binding sites for the potent 1,4-dihydropyridine calcium channel blocker [3H]-nimodipine were solubilized from guinea-pig skeletal muscle microsomes with digitonin and CHAPS [3-(3-cholamidopropyl)-dimethyl-ammonio-l-propanesulfonate]. Detergent-solubilized binding sites could not be sedimented by centrifugation (50,000 X g, 4 h), passed freely through 0.2 micron nitrocellulose filters and were stable at 4 degrees C with half-lives of greater than 60 h. The solubilized 1,4-dihydropyridine binding sites were precipitable with polyethyleneglycol 6000 on Whatman GF/C filters. Saturation analysis of solubilized microsomes with [3H]-nimodipine revealed a single class of binding sites (Bmax = 0.5 to 1.7 pmol per mg of protein) with a KD of 2.2-3.6 nmol/l at 37 degrees C. Specific binding of the 1,4-dihydropyridine calcium channel label was fully reversible (k-1 = 1.5 min-1, at 37 degrees C). The solubilized drug receptors discriminated between the optical enantiomers of chiral 1,4-dihydropyridine calcium channel blockers, (-)- and (+)D-600 as well as between l-cis and d-cis-diltiazem. d-cis-Diltiazem stimulated the binding of [3H]-nimodipine (ED50:3.6 mumol/l), by increasing the Bmax and slowed the dissociation rate of the labelled 1,4-dihydropyridine calcium channel blocker. The solubilized binding sites were sensitive to pronase, alpha-chymotrypsin and phospholipases A and C indicating their protein nature as well as their lipid requirement. Chelation of endogeneous divalent cations by EDTA, EGTA or CDTA inhibits high-affinity [3H]-nimodipine binding, demonstrating that divalent cations are required for high affinity [3H]-nimodipine binding. Detergent-solubilized binding sites are adsorbed by several sepharose-immobilized lectins, including concanavalin A, wheat germ agglutinin and lentil-lectin but not by helix pomatia lectin. Preparative chromatography on concanavalin A sepharose was performed and the adsorbed [3H]-nimodipine binding sites were selectively eluted by alpha-methylmannoside; NaCl (1 mol/l) being completely ineffective as elutant. The purification factors by this method were 17-40-fold. The binding sites could be also purified (up to 10-fold) by sucrose density centrifugation. The S20, w value of the drug receptors is 12.9 s. It is concluded that the 1,4-dihydropyridine binding sites of the putative calcium channel are intimately associated with carbohydrate containing structures. Since the detergent-solubilized material shows allosteric regulation of 1,4-dihydropyridine binding, interaction with chemically different classes of calcium channel blockers, metalloprotein nature and a S20, w value which is indicative of structure large enough to span the membrane, we conclude that we have solubilized and partially purified the putative calcium channel.
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PMID:Solubilization and partial purification of putative calcium channels labelled with [3H]-nimodipine. 631 49