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)

A 110-kDa protein present in chicken intestinal brush-border microvilli is believed to laterally link the actin filament bundle that forms the structural core of the microvilli with the microvillar plasma membrane. We have purified a 110-kDa protein to greater than 95% homogeneity by extraction of brush borders with solution containing 0.6 M KCl and 5 mM ATP, followed by gel filtration chromatography, sedimentation as a complex with exogenous actin, and hydroxylapatite chromatography. The 110-kDa protein-calmodulin complex bound F-actin in the absence but not the presence of ATP and had K+,EDTA-ATPase (0.2 mumol/min/mg) and Ca2+-ATPase (0.2 mumol/min/mg) activities and Mg2+-ATPase activity (0.03 mumol/min/mg) that was not activated by F-actin. The actin-binding and ATPase activities of the complex were similar to those of purified brush-border myosin. However, immunoblot analysis showed no reactivity between the 110-kDa protein and polyclonal antibody against purified chicken brush-border myosin. Also, peptide maps of 110-kDa protein and myosin obtained by limited proteolysis with chymotrypsin and Staphylococcus aureus V8 protease had few, if any, peptides in common. Immunoblot analysis also showed that myosin heavy chain was stable under the conditions of the preparation.
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PMID:The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase. 609 41

Rat pancreases were minced and treated with collagenase or collagenase supplemented with chymotrypsin to yield a mixture of ducts, islets, acinar cell clusters, blood vessels, and nerves. Histologically and ultrastructurally, the isolated tissues resembled their in situ counterparts in most respects, the major difference being the destruction of the basement membranes (basal laminae). Ducts ranging in size from the common bile/main pancreatic duct to the intercalated ducts were identified in the digest, although interlobular ducts were most frequently observed. Acinar tissue fragments were separated from nonacinar structures either by flotation through discontinuous gradients of Ficoll or by sieving, the latter technique being the more efficient. Common bile/main ducts, interlobular ducts, and blood vessels were selected manually from the nonacinar fractions. Biochemical analyses showed that the entire nonacinar fraction, as well as isolated ducts and blood vessels, contained larger alkaline phosphatase, carbonic anhydrase, and Mg-ATPase specific activities than acinar tissue, whereas acinar tissue contained larger gamma-glutamyltranspeptidase and amylase activities. However, greater than 63% of the total recovered activity of each enzyme was associated with the acinar tissue. Both the association of the majority of each of these enzyme activities with the acinar tissue and the similarity in specific activities associated with ducts and blood vessels indicate that none of the enzymes tested is a unique marker for interlobular and larger ducts of the pancreas of the rat.
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PMID:Characterization of ducts isolated from the pancreas of the rat. 615 56

Rat and hamster pancreatic ducts were isolated by digestion with collagenase plus chymotrypsin and were cultured for eight weeks in an agarose matrix. Freshly isolated and cultured ducts were characterized morphologically and biochemically. The in vivo morphology of the ducts was maintained in vitro, although certain differences were noted. Both interlobular and intralobular ducts could be identified. gamma-Glutamyltranspeptidase and Mg-ATPase were stable enzymatic activities of the ducts of both species; alkaline phosphatase persisted only in the hamster ducts. Carbonic anhydrase and (Na + K)ATPase were minor activities of the rat ducts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the rat ducts suggested that actin was the major duct peptide and that the major zymogens were greatly diminished. These results demonstrate that pancreatic ducts can be maintained in vitro and can be used for biochemical studies of this minor pancreatic tissue type.
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PMID:Morphologic and biochemical characteristics of isolated and cultured pancreatic ducts. 616 52

Human cardiac ventricular myosins were prepared from autopsy samples from nine adults, seven infants, and from surgical specimens from seven patients undergoing left ventricular septal myectomy for obstructive hypertrophic cardiomyopathy. Infant myosin differed from adult myosin in two important characteristics: (a) approximately 30% of the 27,000-dalton myosin light chain is replaced by a 28,000-dalton light chain, and (b) the actin-activated myosin MgATPase activity of infant myosin is significantly lower than that of adult myosin (64 nmol phosphate released/mg myosin per min vs. 124 nmol/mg per min at 37 degrees C). The K(+)-EDTA ATPase activity of the myosin measured in 0.5M KCl is also lower in infants (1,210 nmol/mg per min vs. 620 nmol/mg per min at 37 degrees C), but the Ca(++)-activated ATPase is not significantly different. There were no differences in enzymatic activity between the normal adult and cardiomyopathic myosins.A detailed study was performed to investigate possible variations in the structure of the myosin heavy chain in infant, adult, and cardiomyopathic samples. There were no significant differences between infant and normal adult, or between normal adult and cardiomyopathic myosins seen in pyrophosphate polyacrylamide gel electrophoresis, or peptide mapping using alpha-chymotrypsin, papain, or cyanogen bromide to generate peptides. These results suggest that isoenzymes of human ventricular myosin do not exist for the myosin heavy chain in the specimens examined from infants, adults, and patients with obstructive hypertrophic cardiomyopathy. The decreased actin-activated MgATPase activity found for infant myosin appears to be due solely to a partial replacement of the 27,000-dalton light chain of myosin with a 28,000-dalton light chain.
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PMID:Structural and enzymatic comparison of human cardiac muscle myosins isolated from infants, adults, and patients with hypertrophic cardiomyopathy. 621 Jul 10

Treatment of phosphorylated chicken gizzard myosin which had incorporated 1.5 mol of phosphate per 4.7 x 10(5) g of protein with 1-fluoro-2,4-dinitrobenzene resulted in the modification of the heavy and light chains when 5.8 mol of the reagent were bound to myosin. Concurrently, the K+-ATPase activity was inhibited and the modified myosin possessed actin activated-ATPase activity. Thiolysis of nearly 2 mol of the dinitrophenyl group mainly from the heavy chains (and some light chains) of the modified myosin with 2-mercaptoethanol restored the K+-ATPase activity. Digestion of phosphorylated gizzard myosin with chymotrypsin or papain occurred to a lesser extent than a control myosin. Chymotryptic fragments of phosphorylated and dinitrophenylated myosin were formed at a faster rate than those of dinitrophenylated myosin alone suggesting that phosphorylation of the light chain of Mr 20,000 altered the susceptibility of the heavy chains of myosin to proteolysis. Phosphorylation of dinitrophenylated gizzard myosin which had incorporated 5.5 mol of 1-fluoro-2,4-dinitrobenzene per 4.7 x 10(5) g of protein was the same as that of a control myosin; this was also the case for the thiolyzed dinitrophenylated myosin. In the absence of calcium, phosphorylation of control and dinitrophenylated myosins decreased by 73% suggesting that the phosphorylation reaction was calcium dependent. Phosphorylation and dinitrophenylation induced conformational changes in the light chains of gizzard myosin that may be involved in maintaining the structure of the heavy chain region.
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PMID:Effect of phosphorylation and dinitrophenylation on chicken gizzard myosin. 622 21

The calmodulin-sensitive Ca2+ -pumping ATPase was purified to virtual homogeneity from erythrocytes. The purified enzyme exists in two functional states, having low and high Ca2+ affinity. Transition from low to high affinity is induced by 1) calmodulin; 2) acidic phospholipids, long-chain polyunsaturated fatty acids, polyphosphoinositides; and 3) a controlled proteolytic treatment with trypsin or chymotrypsin. The ATPase can be reconstituted into liposomes, where it pumps Ca2+ in exchange for H+ with a stoichiometry to ATP approaching 1. The purified enzyme can be fragmented by trypsin into a number of transient and of limit polypeptides, of which the most interesting from the functional standpoint are the following: 1) a limit polypeptide of Mr 76,000 that contains the active site (i.e., the sequence where the acyl-phosphate is formed); 2) a limit polypeptide of Mr 33,500 that binds the hydrophobic photoactivable label 3-trifluoromethyl-3-(m-(125I-iodophenyl]-diazirine, and is thus presumably the most hydrophobic portion of the molecule; and 3) a transient polypeptide of Mr 90,000 and a limit polypeptide of Mr 25,000-28,000, which specifically bind azido-modified, 125 I-labeled calmodulin. The transient 90,000-dalton calmodulin receptor is rapidly degraded to the 81,000-76,000 limit polypeptide. It can be isolated from the other proteolysis products on calmodulin affinity chromatography columns. The isolated 90,000-dalton fragment is a fully competent, calmodulin-sensitive ATPase that pumps Ca2+ into reconstituted liposomes.
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PMID:Calmodulin-sensitive calcium-pumping ATPase of plasma membranes: isolation, reconstitution, and regulation. 623 47

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca++-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca++-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10 microns Ca++, free, but does not change the Km for Ca++. A possible physiological role for the activator is discussed.
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PMID:A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma. 624 48

Fluorescein 5'-isothiocyanate (FITC) has been shown to specifically inactivate the Na+- and K+-stimulated adenosine triphosphatase ((Na,K)-ATPase) at low concentrations (Karlish, S. J. D. (1979) Na+,K+ATPase Structure and Kinetics 115-128). The site of modification of purified dog kidney (Na,K)-ATPase by FITC has been investigated by enzymatic cleavage and fluorescence resonance energy transfer. The binding of FITC, which occurs at a stoichiometry of approximately one site per ATP binding site, causes an ATP-protectable inactivation of ATPase activity suggesting that it is reacting at the ATP hydrolysis site. The FITC reaction site apparently is located near the center of the COOH-terminal 77,000-dalton peptide fragment obtained by chymotryptic cleavage of the alpha subunit. Addition of ouabain to the native enzyme in the presence of chymotrypsin enhances cleavage at this site and releases the fluorescein moiety from the membrane. It is further shown that the distance from the FITC reaction site to the ouabain binding site, as judged by fluorescence resonance energy transfer from anthroyl ouabain to FITC, is approximately 74 A. These results demonstrate that ouabain inhibits the (Na,K)-ATPase by causing a protein conformational change which extends an unusually large distance across the membrane.
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PMID:The active site structure of Na+- and K+-stimulated ATPase. Location of a specific fluorescein isothiocyanate reactive site. 627 7

We have found that opiate receptors in smooth microsomal fractions differ from synaptic membrane-associated receptors in proteolytic sensitivity. With 3 proteases of different substrate specificities (trypsin, chymotrypsin and S. griseus protease) smooth microsomal opiate receptors from rat brain were consistently less sensitive to limited proteolysis than were synaptic membrane receptors. Thiamine pyrophosphatase, a luminal Golgi membrane marker enzyme, exhibited a similar resistance to S. griseus protease in microsomal preparations, while microsomal Na+/K+-ATPase (ouabain-sensitive) was readily destroyed by trypsin. We also discovered that smooth microsomal opiate receptors co-migrate with both Golgi membrane and endoplasmic reticulum marker proteins on equilibrium density gradients under isopycnic conditions. Electron microscopic examination of the Golgi-enriched fraction showed the typical cisternae frequently associated with isolated Golgi membranes. Synaptic junctions, presynaptic membranes, myelin and mitochondria were conspicuously absent from this fraction. Since the microsomes isolated in vitro showed similar topography to those in vivo, the binding sites for opiates could be localized on the luminal surface membranes of the microsomal fractions. The exquisite sensitivity of synaptic membrane opiate receptors to proteolysis suggests that these receptors are found on the extracellular surface of the synaptic junction.
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PMID:Microsomal opiate receptors differ from synaptic membrane receptors in proteolytic sensitivity. 629 19

The relationship has been studied of structural changes in the alpha-subunit (Mr approximately equal to 100 000) of Na+,K+-ATPase to the binding and translocation of Rb+ and Na+. Two conformations, E1 and E2, are distinguished by controlled proteolysis of the alpha-subunit and fluorescence techniques. The de-phospho forms, E1Na and E2K, are stabilized by binding of Na+ and K+ or Rb+ to cytoplasmic sites on pure Na+,K+-ATPase in membrane fragments. In phospholipid vesicles reconstituted with pure Na+,K+-ATPase, the transitions between E1Na and E2K are coupled to translocation of cation in ouabain- or vanadate-sensitive passive fluxes along gradients for K+ and Na+. The direction of these fluxes is opposite to that of the active Na+-K+ transport. Coupling of transitions between the phospho forms, E1P and E2P, and the cation translocation was studied after selective proteolysis of the alpha-subunit. Cleavage with trypsin at the carboxyl-terminal side (bond 1) of the aspartyl phosphate residue or with chymotrypsin at the amino-terminal side (bond 3) blocks Na+, K+-ATPase and Na+-K+ transport, but the two splits have widely different effects on partial reactions. Cleavage of bond 3 blocks transition from E1P to E2P and abolishes both (ADP + ATP)-Na+/Na+ exchange and (ATP + Pi)-Rb+/Rb+ exchange reactions. Cleavage of bond 1 interferes neither with the transitions nor with the exchange reactions. Thus, both the cation-induced transitions between E1Na and E2K and the transitions between the phospho forms, E1P and E2P, of the alpha-subunit are coupled to flipping of cation sites between the inside-exposed state (E1) and the outside-exposed state (E2).
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PMID:Conformational changes in the alpha-subunit, and cation transport by Na+, K+-ATPase. 630 21


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