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Biomedical subjects

J H Collins

Publications and source records attributed to J H Collins.

At least 127 records · Page 7Linked to original sources
PubMed1982

Effects of extracts of rat brain on the digitalis receptor.

There is a possibility that an endogenous substance exists which interacts with a ouabain binding site on Na+, K+-ATPase. Recently, several reports have appeared suggesting the presence of an endogenous digitalis-like substance in acid-acetone extracts of brain. We have demonstrated that in preparing an acid-acetone extract, peroxidized lipids and lysophospholipids are produced, both of which inhibit Na+, K+-ATPase, thereby complicating interpretation. Preliminary evidence suggests, however, that when rat brains are extracted with an aqueous-acetone mixture under nitrogen, a principle is obtained which specifically inhibits Na+, K+-ATPase.

Animals↗
PubMed1982

Purification and characterization of an (Na+ + K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain.

Highly purified lamb kidney (Na+ + K+)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na+ + K+)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (gamma 1 and gamma 2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that gamma 1 is an aggregated form of gamma 2. The total yield was 0.8-1.5 mol of gamma component per mol of catalytic subunit recovered. This indicates that the gamma component is present in stoichiometric amounts in the Na+ + K+)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.

Amino Acids↗
PubMed1982

Isolation of two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum.

We have isolated two proteolipids from rabbit skeletal muscle sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. One, PL-II, is identical to the proteolipid previously obtained by others using organic solvent extraction. The other, PL-I, has an amino acid composition very similar to those of proteolipids we previously isolated from canine cardiac SR and lamb kidney (Na,K)-ATPase.

Amino Acids↗
PubMed1981

Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II.

It has been previously demonstrated that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II is inhibited by phosphorylation of its two heavy chains (Collins, J. H., and Korn, E. D. (1980) J. Biol. Chem. 255, 8011-8014). In this paper, it is shown that a partially purified kinase preparation from Acanthamoeba catalyzes the incorporation of 3 mol of phosphate into each mole of myosin II heavy chain. Tryptic digestion of the 32P-myosin, followed by two-dimensional peptide mapping, indicates that two of the three sites phosphorylated by the kinase in vitro correspond to the two major phosphorylation sites on the myosin heavy chain in vivo. Phosphorylation of myosin II in vitro by the kinase fraction completely inhibits the actin-activated Mg2+-ATPase activity of myosin II. Myosin II can be isolated in a highly phosphorylated, enzymatically inactive form, then dephosphorylated to an active form, and finally rephosphorylated to an inactive form. The Acanthamoeba kinase fraction catalyzes the phosphorylation of all three sites on the heavy chain of myosin II at virtually the same rate. From a comparison of the decrease in actin-activated Mg2+-ATPase activity with the amount of phosphate incorporated into myosin II, and from the results obtained previously by dephosphorylating myosin II (Collins, J. H., and Korn, E. D., (1980) J. Biol. Chem. 255, 8011-8014), it can be inferred that two of the sites phosphorylated in vitro act in a synergistic manner to inhibit the actin-activated myosin II Mg2+-ATPase.

Adenosine Triphosphatases↗
PubMed1981

Interaction of calmodulin with skeletal muscle myosin light chain kinase.

Studies on myosin light chain kinase isolated from rabbit skeletal muscle show that the enzyme has a molecular weight of 80,000--84,000 with a sedimentation coefficient of 3.2 S and an apparent Stokes radius of 53 A. Gel filtration chromatography with a 3H-labeled calmodulin using a Hummel--Dryer technique shows that the enzyme will bind 1 mol of calmodulin per mol of enzyme, with an affinity of (1.9 +/- 0.5) x 10(7) M-1 in the absence of substrate. The calmodulin dependence of enzyme activation at limiting Mg2+ and light chain concentrations confirms this observation. The calcium dependence of activation of the enzyme--calmodulin complex is characterized by a Hill coefficient of 2.5, with half-activation occurring at 6.6 x 10(-7) M Ca2+. The amino acid composition shows a high percentage (9.1%) of proline, which may account for the large apparent Stokes radius and no clear resemblance to other skeletal muscle proteins. A comparison of the amino acid composition with that from turkey gizzard shows some resemblance.

Animals↗
PubMed1981

Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba.

Approximately 8-10 mg of highly actin-activatable, CA2+-sensitive Acanthamoeba myosin II can be isolated in greater than 98% purity from 100 g of amoeba by the new procedure described in detail in this paper. The enzyme isolated by this procedure can be activated by actin because its heavy chains are not fully phosphorylated (Collins, J. H., and Korn, E. D. (1980) J. Biol Chem. 255, 8011-8014). We now show that Acanthamoeba myosin II Mg2+-ATPase activity is more highly activated by Acanthamoeba actin than by muscle actin. Also, actomyosin II ATPase is inactive at concentrations of free Mg2+ lower than about 3 mM and fully active at Mg2+ concentrations greater than 4 mM. Actomyosin II Mg2+-ATPase activity is stimulated by micromolar Ca2+ when assayed over the narrow range of about 3-4 mM Mg2+ but is not affected by Ca2+ at either lower or higher concentrations of Mg2+. The specific activity of te actomyosin II Mg2+-ATPase also increases with increasing concentrations of myosin II when the free Mg2+ concentration is in the range of 3-4 mM but is independent of the myosin II concentration at lower or higher concentrations of Mg2+ . This marked effect of the Mg2+ concentration on the Ca2+-sensitivity and myosin concentration-dependence of th specific activity of actomyosin II ATPase activity does not seem to be related to the formation of myosin filaments, and to be related to the formation of myosin filaments, and myosin II is insoluble only at high concentrations of free Mg2+ (6-7 mM) were neither of these effects is observed. Also, the Mg2+ requirements for actomyosin II ATPase activity and myosin II insolubility can be differentially modified by EDTA and sucrose.

Actins↗
PubMed1980

A fluorescent probe study of Ca2+ binding to the Ca2+-specific sites of cardiac troponin and troponin C.

Cardiac troponin C (C-TnC) was labeled with the sulfhydryl-specific fluorescent probe molecule 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid at cysteine 35 and 84 to produce C-TnCIA. This modified protein binds Ca2+, undergoes Ca2+-induced increases in alpha helix, and forms a complex with other troponin subunits as does unlabeled C-TnC. C-TnCIA undergoes a small fluorescence decrease with Ca2+ or Mg2+ binding to the two high affinity Ca2+-Mg2+ sites of C-TnC and a large biphasic approximately 2.1-fold fluorescence increase with Ca2+ binding to two lower affinity Ca2+-specific sites with KCa of approximately 4.5 X 10(5) M-1 and approximately 5 X 10(2) M-1. C-TnCIA was formed in a complex with troponin I (TnI) and troponin T to form C-TnIA. This fluorescent reconstituted whole troponin undergoes a 25% decrease with Ca2+ binding to a Ca2+-specific site of KCa approximately 3 X 10(6) M-1. C-TnC, therefore, contains a single Ca2+-specific site of approximate equal affinity as the two Ca2+-specific regulatory sites of skeletal TnC. This Ca2+-specific site in C-TnC (like its two corresponding sites in S-TnC) undergoes an approximate 10-fold increase in affinity in whole troponin or when TnC is complexed with TnI. Since the two Ca2+-specific sites in skeletal troponin have been shown to be the regulatory sites of skeletal muscle contraction we suggest that this single Ca2+-specific site, of equal affinity, in C-TnC is the regulatory site of cardiac muscle contraction.

Animals↗
PubMed1979

Peptide maps of the myosin isoenzymes of Acanthamoeba castellanii.

Extracts of Acanthamoeba castellanii contain four myosin-like ATPases (Maruta, H., Gadasi, H., Collins, J.H., and Korn, E.D. (1979) J. Biol. Chem. 254, 3624-3630): double-headed Acanthamoeba myosin II and single-headed Acanthamoeba myosins IA, IB, and IC, which have heavy chains of 170,000, 130,000, 125,000, and 130,000 daltons, respectively, as well as different light chains. In the accompanying paper, evidence is presented that suggests that Acanthamoeba myosin IC is the same molecule as Acanthamoeba myosin IA plus a regulatory 20,000-dalton peptide. This conclusion is confirmed by the identity of the peptide maps obtained by limited proteolysis of the heavy chains of Acanthamoeba myosins IA and IC by Staphylococcus aureus V8 protease. However, peptide maps of the heavy chains of Acanthamoeba myosins IA, IB, and II obtained by limited proteolysis by the Staphylococcus protease and chymotrypsin and by chemical cleavage by cyanogen bromide and cyanylation have few, if any, peptides in common. From this evidence, and the enzymatic and subunit data in the accompanying paper, it is concluded that the three Acanthamoeba myosin isoenzymes, IA (IC), IB, and II, are products of different genes.

Amoeba↗