Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q07644 (polypeptide)
 72,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A deoxyribonuclease has been purified more than 2000-fold from the green algae, Chlamydomonas reinhardii. The enzyme is most active on denatured DNA. Optimum activity is at pH 8.5, in 80 mM Tris-HCl buffer and 2 mM CaCl2. Other divalent cations can replace Ca2+ with varying lower efficiency. EDTA and inorganic phosphate are strongly inhibitory, while ATP and high concentrations of 2-mercaptoethanol are slightly inhibitory. The molecular weight is approximately 35 000, the Stokes radius is 2.7 nm, and the sedimentation coefficient 2.8 S. It is a single polypeptide chain, and the frictional ratio of 1.27 suggests it is only slightly asymetrical. The isoelectric point is 9.5. This enzyme has been termed exonuclease 1.
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PMID:A deoxyribonuclease from Chlamydomonas reinhardii. 1. Purification and properties. 1 43

High-molecular-weight (HMW) protein from human cataractous lenses, isolated by differential centrifugation, was deaggregated in 7M urea and then reaggregated in either the presence or absence of 10 mM CaCl2. Over 90% of the material reaggregated in the presence of calcium appears to have a size greater than 50 X 10(6) daltons. By contrast, only 20% to 25% of the material reaggregated in the absence of calcium has molecular weight greater than 50 X 10(6) daltons. Disulfide formation during reaggregation is unlikely in the latter experiment, since the addition of 50 mM mercaptoethanol caused no change in results. About 60% to 70% of the low-molecular-weight (LMW) protein fraction deaggregated in 7M urea buffer can be converted to HMW species in the presence of 10 mM CaCl2, when the deaggregating agent is removed. However, only 5% to 10% of this protein is converted to HMW species if the deaggregation step is eliminated. Experiments with 45 Ca indicate that whereas calcium is necessary for the formation of the HMW aggregates, only one calcium per approximately 5 X 10(5) daltons remains bound in the reaggregated material. The data suggest that although calcium may be required to induce aggregation to HMW species, it is not required to stabilize such macromolecules. SDS-polyacrylamide gel electrophoresis of the HMW species formed upon reaggregation of the dissociated HMW species with calcium indicates the presence of all the major polypeptide subunits of the original HMW species present in the lens; however, reaggregation in the absence of calcium yields HMW species lacking in the 9600 dalton component.
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PMID:Further investigation of the role of calcium in human lens protein aggregation. 10 12

Actin, myosin, and a high molecular weight actin-binding protein were extracted from rabbit alveolar macrophages with low ionic strength sucrose solutions containing ATP, EDTA, and dithiothreitol, pH 7.0. Addition of KCl, 75 to 100 mM, to sucrose extracts of macrophages stirred at 25 degrees caused actin to polymerize and bind to a protein of high molecualr weight. The complex precipitated and sedimented at low centrifugal forces. Macrophage actin was dissociated from the binding protein with 0.6 M KCl, and purified by repetitive depolymerization and polymerization. Purified macrophage actin migrated as a polypeptide of molecular weight 45,000 on polyacrylamide gels with dodecyl sulfate, formed extended filaments in 0.1 M KCl, bound rabbit skeletal muscle myosin in the absence of Mg-2+ATP and activated its Mg-2+ATPase activity. Macrophage myosin was bound to actin remaining in the macrophage extracts after removal of the actin precipitated with the high molecular weight protein by KCl. The myosin-actin complex and other proteins were collected by ultracentrifugation. Macrophage myosin was purified from this complex or from a 20 to 50% saturated ammonium sulfate fraction of macrophage extracts by gel filtration on agarose columns in 0.6 M Kl and 0.6 M Kl solutions. Purified macrophage myosin had high specific K-+- and EDTA- and K-+- and Ca-2+ATPase activities and low specific Mg-2+ATPase activity. It had subunits of 200,000, 20,000, and 15,000 molecular weight, and formed bipolar filaments in 0.1 M KCl, both in the presence and absence of divalent cations. The high molecular weight protein that precipitated with actin in the sucrose extracts of macrophages was purified by gel filtration in 0.6 M Kl-0.6 M KCl solutions. It was designated a macrophage actin-binding protein, because of its association with actin at physiological pH and ionic strength. On polyacrylamide gels in dodecyl sulfate, the purified high molecular weight protein contained one band which co-migrated with the lighter polypeptide (molecular weight 220,000) of the doublet comprising purified rabbit erythrocyte spectrin. The macrophage protein, like rabbit erythrocyte spectrin, was soluble in 2 mM EDTA and 80% ethanol as well as in 0.6 M KCl solutions, and precipitated in 2 mM CaCl2 or 0.075 to 0.1 M KCl solutions. The macrophage actin-binding protein and rabbit erythrocyte spectrin eluted from agarose columns with a KAV of 0.24 and in the excluded volumes. The protein did not form filaments in 0.1 M KCl and had no detectable ATPase activity under the conditions tested.
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PMID:Isolation and properties of actin, myosin, and a new actinbinding protein in rabbit alveolar macrophages. 12 34

Native cuticle collagen, obtained from Nereis virens, was incubated with purified bacterial collagenase (EC 3.4.4.19). The kinetics of proteolysis were monitored by viscometry, in parallel with similar digestions of calf skin collagen. Comparison of the kinetics of digestion of the two collagens, at similar enzyme to substrate ratios (w/w), showed that the native cuticle collagen was relatively refractory to digestion by bacterial collagenase. Characterization of the cuticle collagen digest by sodium dodecyl sulfate-polyacrylamide electrophoresis and agarose gel filtration in CaCl2 showed a large polypeptide, of about 300,000 daltons, to be a major product. The native form of this product, a unique fragment, was isolated from the digest by ethanol precipitation. It was found to have an intrinsic viscosity of 120 dl/g, to have an optical rotary dispersion curve characteristic of collagen, to undergo a typical collagenous thermal transition with a Tm of 23.2 degrees, and to have a calculated molar mass of 900,000 g with molecular dimensions of 9,000 X 13 A. It had an amino acid composition which was similar, but not identical with the native cuticle collagen. Although the original substrate contained two dissimilar chains, A and B, in a molar ratio of 1:2, the collagenase-resistant product appeared to be composed of only one type of polypeptide fragment. Possibly, the original subunits contain similar, if not identical collagenase-resistant regions.
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PMID:Nereis cuticle collagen. Isolation and properties of a large fragment resistant to proteolysis by bacterial collagenase. 19 99

Synthesis of cellular protein was substantially inhibited within 1 h of infection with herpes simplex virus, type 2, strain G (HSV-2). The inhibition also occurred, although no virus-specific protein synthesis was detected, after infection with u.v. irradiated virus and in cytoplasts that had been enucleated before infection. The inhibitory activity could not be distinguished from infectivity by dilution, sedimentation or reaction with gamma-globulin. HSV-2 also suppresssed the synthesis of Sendai virus proteins, but not those specified by HSV-1. Host protein synthesis was no more sensitive than virus protein synthesis to an increased concentration of NaCl in the medium, nor could the suppression of host synthesis be prevented by adding excess MgCl2 to the medium or by omitting CaCl2 or NaCl. It was accompanied by the breakdown of polyribosomes, which also occurred in the presence of cycloheximide but not at 4 degrees C. The breakdown yielded ribosomes that were sensitive to a high salt concentration, unlike those produced by treatment of polyribosomes with RNase. The synthesis of cellular DNA and RNA was also inhibited following infection with u.v.-inactivated virus. It is concluded that the suppression of host protein synthesis (and probably also of host DNA and RNA synthesis) is caused by a constituent of the infecting virus particles. The mechanism is obscure but probably does not depend on the leakage out of the cell of Mg2+ or into the cell or Ca2+ or Na+ ions, nor on the specific inhibition of initiation of host polypeptide chains, nor on RNase-like attack on host polyribosomes.
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PMID:Suppression of the synthesis of cellular macromolecules by herpes simplex virus. 21 20

Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3--CaCl2, and K2HPO4-KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3-CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (greater than 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to action were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris--HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3--CacCl2 appeared to be the best buffer and Tris--HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.
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PMID:Studies on the preparation of rat liver plasma membrane fractions and on their polypeptide patterns. 68 61

Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Epitope mapping of the human biliary amphipathic, anionic polypeptide: similarity with a calcium-binding protein isolated from gallstones and bile, and immunologic cross-reactivity with apolipoprotein A-I. 138 60

A protein liquid membrane composed of coacervated alpha-elastin, a chemical fragmentation product of the biological elastic fiber protein, functioned as an amphoteric liquid ion-exchange membrane. Ionic permselectivities of the alpha-elastin coacervate membrane to a series of metal chlorides were investigated for the concentration-cell systems by the ordinary electrochemical measurements. Effects of pH on the transmembrane potential responses for NaCl, CaCl2, and MgCl2 systems were examined. Only in the Ca(2+)-containing system did potential responses stay at constant levels against the pH changes, whereas in the other systems, increasing pH caused potential changes, indicating an improvement of cationic permselectivity across the alpha-elastin coacervate membrane. It was suggested that the characteristic Ca2+ transport mechanisms across the alpha-elastin coacervate membrane are related in some way to the polypeptide backbone interactions specific and selective to Ca2+ ions.
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PMID:Alpha-elastin coacervate as a protein liquid membrane: effect of pH on transmembrane potential responses. 142 Sep 86

Because freezing samples decreases calpastatin activity and the application of exogenous calcium activates the calpain proteolytic system, thereby improving tenderness, the objective of this study was to determine whether freezing would enhance the effects of CaCl2 marination on the tenderness of beef steaks. Longissimus steaks were obtained from 10 beef steers 6 d postmortem. One-half of the steaks were frozen at -30 degrees C for 6 wk. The remaining steaks were treated fresh; one-half were subjected to a 150 mM CaCl2 marinade for 48 h. Frozen steaks were thawed and subjected to the same treatment. Treatments consisted of 1) fresh control, 2) fresh marinated, 3) frozen control, and 4) frozen marinated. Samples were taken before and after treatment (6 and 8 d) for calpastatin activity determination and d 8 for SDS-PAGE. Warner-Bratzler shear force values were measured 8 d postmortem. Data were analyzed using a paired comparison t-test procedure. Results showed that freezing and marination significantly decreased calpastatin activity. A .35-kg improvement (P = .07) in Warner-Bratzler shear force was observed with freezing, whereas a .78-kg improvement (P less than .01) in tenderness was observed with marination. However, prior freezing enhanced the effects of marination. Therefore, the decrease in calpastatin activity seemed to allow greater proteolysis by the calpains with the application of Ca2+. The SDS-PAGE of myofibril preparations indicated that more small polypeptide fragments (28 to 32 kDa) appeared and a 95-kDa fragment was more intense in the marinated samples than in control samples, indicating that proteolysis was enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Freezing and calcium chloride marination effects on beef tenderness and calpastatin activity. 142 84

The requirement of extracellular Ca2+ for insulin action has been indicated by past studies. With a view to understand the interaction of insulin with Ca2+ in the vicinity of the cell membrane, we have examined the ability of insulin and its constituent polypeptide chains A and B to translocate Ca2+ and Mg2+ across the lipid bilayer in two sets of synthetic liposomes. The first were unilamellar vesicles made of dimyristoylphosphatidylcholine and contained the Ca2+ sensor dye arsenazo III. Peptide-mediated Ca2+ and Mg2+ transport in these vesicles was monitored at 37 degrees C in a neutral buffer containing CaCl2 or MgCl2 using a difference absorbance method. In the second set, multilamellar vesicles of egg lecithin containing trapped fura-2 were employed and the cation transport was followed at 20 degrees C by fluorescence changes in the dye. Control experiments indicated that the hormonal peptides caused no appreciable perturbation of the vesicles leading to leakage of contents or membrane fusion. In both liposome systems, substantial Ca2+ and Mg2+ transport was observed with insulin and the B chain; the A chain was less effective as an ionophore. Quantitative analysis of the transport kinetic data on the B chain showed a 1:1 peptide-Ca2+ complex formed inside the membrane. In light of the available structural data on Ca2+ binding by insulin and insulin receptor, our results suggest the possibility of the hormone interacting with the receptor with the bound Ca2+.
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PMID:Induction of Ca2+ transport in liposomes by insulin. 158 68


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