11 results on '"B J, Pitts"'
Search Results
2. Guanine nucleotide regulation of a mammalian myocardial muscarinic receptor system. Evidence for homo- and heterotropic cooperativity in ligand binding analyzed by computer-assisted curve fitting
- Author
-
Mark L. Entman, Lutz Birnbaumer, R Mattera, and B J Pitts
- Subjects
chemistry.chemical_classification ,GTP' ,Stereochemistry ,Guanine ,Cooperative binding ,Cooperativity ,Cell Biology ,Biochemistry ,Adenylyl cyclase ,chemistry.chemical_compound ,chemistry ,Muscarinic acetylcholine receptor ,Nucleotide ,Binding site ,Molecular Biology - Abstract
Highly purified dog heart sarcolemmal membranes, with a content of approximately 5 pmol of muscarinic acetylcholine receptor (mAChR)/mg of protein, were analyzed for mAChR-mediated inhibition of adenylyl cyclase and ligand binding in the absence and the presence of guanine nucleotides. Adenylyl cyclase was found to be coupled to the mAChR, being attenuated approximately 30% in a GTP-dependent manner. Direct binding studies, using 3H-labeled oxotremorine M, showed high affinity binding (apparent KD = 10 nM) that was reduced on nucleotide addition. Dose-response curves for GDP, GTP, and guanyl-5'-yl imidodiphosphate showed them to be equipotent. On the basis of pirenzepine binding, only one type of mAChR, commonly referred to as M2, was detected. Direct binding of [3H]quinuclidinyl benzilate [( 3H]QNB) uncovered 50% more binding sites than 150 nM 3H-labeled oxotremorine M; addition of guanine nucleotides uncovered the existence of positive cooperativity in the binding of [3H]QNB. Agonist displacement curves of [3H]QNB binding, without and with guanine nucleotides, extended over several orders of magnitude, which is inconsistent with single site competitive kinetics. The results and their analysis by computer-assisted curve fitting indicated that the data are well fitted by a model in which a receptor is at least bivalent and exists in two states: one with and the other without cooperativity between its sites, with guanine nucleotides decreasing both the degree of cooperativity between the sites and the proportion of the receptor that is in the cooperative form. Since the guanine nucleotide effect is mediated by the Ni coupling protein, it is suggested that direct binding detects R'Ni complexes (cooperative), R"NiG complexes (cooperative but distinct from R'Ni), and R0 complexes (non-cooperative and unaffected by Ni or NiG), where R = mAChR, Ni = the inhibitory regulatory component of adenylyl cyclase unaffected by guanine nucleotide, and NiG = Ni affected by guanine nucleotide (G).
- Published
- 1985
3. Antigenic differences in (Na+, K+)-ATPase preparations isolated from various organs and species
- Author
-
J L McCans, George E. Lindenmayer, V P Butler, M V Ray, Arnold M. Schwartz, B D Raynor, and B J Pitts
- Subjects
chemistry.chemical_classification ,Antiserum ,biology ,ATPase ,Potassium ,chemistry.chemical_element ,Cell Biology ,Biochemistry ,Enzyme activator ,Enzyme ,chemistry ,Antigen ,biology.protein ,Na+/K+-ATPase ,Molecular Biology ,Incubation - Abstract
Antisera to purified (Na+, K+)-ATPase raised in rabbits and in sheep were purified by an absorption procedure employing purified canine kidney (Na+, K+)-ATPase. The antibodies were fractionated into two components, one which inhibited catalytic activity, and a second which inhibited ouabain binding. Under certain conditions, the fraction that inhibited ouabain binding also inhibited catalytic activity, and the effectiveness of both was dependent to some extent on the ligands present in the incubation medium. Thus, both antibody fractions appeared to detect conformations of the enzyme that depended upon ligand-induced perturbations. When the antibody raised against catalytic activity was incubated with erythrocyte membrane fragments, an inhibition of the (Na+, K+)-ATPase occurred, but only minimal or no effect on potassium influx or on digoxin-induced inhibition of potassium flux in intact erythrocytes was noted. In a similar experiment, however, the antibody against ouabain binding significantly inhibited potassium influx, suggesting specificity in terms of the macromolecular surfaces of the pump which were exposed to the external medium. We concluded that there may be organ and species differences among (Na+, K+)-ATPase preparations. Antibodies prepared in rabbits and sheep were fractionated by absorption to dog brain enzyme. Both the antibody fraction which bound to the brain enzyme and that which did not bind inhibited the dog kidney (Na+, K+)-ATPase, but only the former inhibited dog brain (Na+, K+)-ATPase. When the two fractions were recombined, inhibition was restored to the extent of the unfractionated antibody.
- Published
- 1975
4. Stoichiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. Coupling to the sodium pump
- Author
-
B J Pitts
- Subjects
Sarcolemma ,Vesicle ,Sodium ,Cardiac muscle ,chemistry.chemical_element ,Cell Biology ,Calcium ,Biochemistry ,Ouabain ,Coupling (electronics) ,medicine.anatomical_structure ,chemistry ,medicine ,Efflux ,Molecular Biology ,medicine.drug - Abstract
Vesicles isolated from cardiac muscle exhibited Na,Ca exchange activity which can be measured by 45Ca influx or efflux of by 22Na efflux. The stoichiometry of Na,Ca exchange was 3 Na:1 Ca. These vesicles also exhibited ATP-dependent 22Na transport which was inhibited by ouabain indicating that this activity is due to the sodium pump, an activity which is thought to reside only in the sarcolemma. The addition of calcium caused rapid efflux of 22Na from vesicles loaded by ATP-dependent 22Na uptake indicating that the Na,Ca exchange is located in the same vesicles as the sodium pump and is thus also a sarcolemmal activity.
- Published
- 1979
5. Stoichiometry of sodium-calcium exchange in cardiac sarcolemmal vesicles. Coupling to the sodium pump
- Author
-
B J, Pitts
- Subjects
Kinetics ,Dogs ,Sarcolemma ,Heart Ventricles ,Myocardium ,Sodium ,Animals ,Biological Transport, Active ,Biological Transport ,Calcium - Abstract
Vesicles isolated from cardiac muscle exhibited Na,Ca exchange activity which can be measured by 45Ca influx or efflux of by 22Na efflux. The stoichiometry of Na,Ca exchange was 3 Na:1 Ca. These vesicles also exhibited ATP-dependent 22Na transport which was inhibited by ouabain indicating that this activity is due to the sodium pump, an activity which is thought to reside only in the sarcolemma. The addition of calcium caused rapid efflux of 22Na from vesicles loaded by ATP-dependent 22Na uptake indicating that the Na,Ca exchange is located in the same vesicles as the sodium pump and is thus also a sarcolemmal activity.
- Published
- 1979
6. Recent advances in cardiac glycoside-Na+,K+-ATPase interaction
- Author
-
E T, Wallick, G E, Lindenmayer, L K, Lane, J C, Allen, B J, Pitts, and A, Schwartz
- Subjects
Adenosine Triphosphatases ,Sheep ,Sodium ,Biological Transport, Active ,Kidney ,Ligands ,Choline ,Cardiac Glycosides ,Kinetics ,Structure-Activity Relationship ,Potassium ,Animals ,Ouabain ,Protein Binding - Abstract
Na+,K+-ATPase has been purified from lamb kidney and consists of two polypeptide peaks on polyacrylamide gel electrophoresis with an enzyme activity of 1,000 mumole Pi/mg pro per hr. A scheme depicting the interaction of cardiac glycoside with the enzyme and ligand effects on binding has been constructed. Under all ligand conditions, ouabain binding tends to reach the same maximum if sufficient ouabain is present. Initial rates vary with ligand conditions. Using a chase method, the rate of dissociation of the glycoside from the enzyme is not influenced by the ligands present, although with separation of the enzyme-glycoside complex from the binding medium, differences are noted. The effect of ouabain on Na binding demonstrated two classes of sites, KD = 0.2 mM and KD = 18 mM. Denaturation decreased the high affinity sites. There was also a good correlation between ouabain binding and inhibition of Na binding. Clearly, ligands are critical in regulating cardiac glycoside interaction with the enzyme.
- Published
- 1977
7. Antigenic differences in (Na+, K+)-ATPase preparations isolated from various organs and species
- Author
-
J L, McCans, G E, Lindenmayer, B J, Pitts, M V, Ray, B D, Raynor, V P, Butler, and A, Schwartz
- Subjects
Adenosine Triphosphatases ,Digoxin ,Erythrocytes ,Sodium ,Biological Transport, Active ,Brain ,Cross Reactions ,Kidney ,Antigen-Antibody Reactions ,Enzyme Activation ,Dogs ,Species Specificity ,Organ Specificity ,Potassium ,Animals ,Humans ,Ouabain ,Immunoelectrophoresis ,Protein Binding - Abstract
Antisera to purified (Na+, K+)-ATPase raised in rabbits and in sheep were purified by an absorption procedure employing purified canine kidney (Na+, K+)-ATPase. The antibodies were fractionated into two components, one which inhibited catalytic activity, and a second which inhibited ouabain binding. Under certain conditions, the fraction that inhibited ouabain binding also inhibited catalytic activity, and the effectiveness of both was dependent to some extent on the ligands present in the incubation medium. Thus, both antibody fractions appeared to detect conformations of the enzyme that depended upon ligand-induced perturbations. When the antibody raised against catalytic activity was incubated with erythrocyte membrane fragments, an inhibition of the (Na+, K+)-ATPase occurred, but only minimal or no effect on potassium influx or on digoxin-induced inhibition of potassium flux in intact erythrocytes was noted. In a similar experiment, however, the antibody against ouabain binding significantly inhibited potassium influx, suggesting specificity in terms of the macromolecular surfaces of the pump which were exposed to the external medium. We concluded that there may be organ and species differences among (Na+, K+)-ATPase preparations. Antibodies prepared in rabbits and sheep were fractionated by absorption to dog brain enzyme. Both the antibody fraction which bound to the brain enzyme and that which did not bind inhibited the dog kidney (Na+, K+)-ATPase, but only the former inhibited dog brain (Na+, K+)-ATPase. When the two fractions were recombined, inhibition was restored to the extent of the unfractionated antibody.
- Published
- 1975
8. Rapid purification of canine cardiac sarcoplasmic reticulum Ca2+-ATPase
- Author
-
W B, Van Winkle, B J, Pitts, and M L, Entman
- Subjects
Molecular Weight ,Kinetics ,Sarcoplasmic Reticulum ,Dogs ,Species Specificity ,Myocardium ,Animals ,Calcium-Transporting ATPases ,Rabbits - Abstract
A pure, enzymatically active Ca2+-dependent adenosine triphosphatase (Ca2+-ATPase) has been isolated from canine ventricular sarcoplasmic reticulum. In contrast to that derived from skeletal muscle, the Ca2+-ATPase from cardiac sarcoplasmic reticulum was more active when solubilization and subsequent purification took place in the presence of its substrates, Ca2+ and ATP. Cholate- or deoxycholate-solubilized Ca2+-ATPase is recovered following rapid glycerol dilution and centrifugation. The Ca2+-ATPase is stable and possesses hydrolytic capacities up to 4 mumol/mg/min. Sodium dodecyl sulfate-polyacrylamide gels reveal the presence of one protein in the range of 95,000 to 100,000 daltons. This method also yields purified Ca2+-ATPase from fast skeletal muscle of similar activities to those reported by other laboratories.
- Published
- 1978
9. Modulation of calcium in the heart
- Author
-
A, Schwartz, G S, Levey, M L, Entman, E G, Ezrailson, W B, Van Winkle, E P, Bornet, and B J, Pitts
- Subjects
Adenosine Triphosphatases ,Dogs ,Myocardium ,Cell Membrane ,Sodium ,Potassium ,Animals ,Calcium ,Models, Biological ,Adenylyl Cyclases - Published
- 1976
10. Guanine nucleotide regulation of a mammalian myocardial muscarinic receptor system. Evidence for homo- and heterotropic cooperativity in ligand binding analyzed by computer-assisted curve fitting
- Author
-
R, Mattera, B J, Pitts, M L, Entman, and L, Birnbaumer
- Subjects
Guanylyl Imidodiphosphate ,Heart Ventricles ,Myocardium ,Oxotremorine ,Ligands ,Guanosine Diphosphate ,Models, Biological ,Receptors, Muscarinic ,Guanine Nucleotides ,Quinuclidinyl Benzilate ,Kinetics ,Dogs ,Sarcolemma ,Animals ,Guanosine Triphosphate ,Mathematics ,Software ,Adenylyl Cyclases - Abstract
Highly purified dog heart sarcolemmal membranes, with a content of approximately 5 pmol of muscarinic acetylcholine receptor (mAChR)/mg of protein, were analyzed for mAChR-mediated inhibition of adenylyl cyclase and ligand binding in the absence and the presence of guanine nucleotides. Adenylyl cyclase was found to be coupled to the mAChR, being attenuated approximately 30% in a GTP-dependent manner. Direct binding studies, using 3H-labeled oxotremorine M, showed high affinity binding (apparent KD = 10 nM) that was reduced on nucleotide addition. Dose-response curves for GDP, GTP, and guanyl-5'-yl imidodiphosphate showed them to be equipotent. On the basis of pirenzepine binding, only one type of mAChR, commonly referred to as M2, was detected. Direct binding of [3H]quinuclidinyl benzilate [( 3H]QNB) uncovered 50% more binding sites than 150 nM 3H-labeled oxotremorine M; addition of guanine nucleotides uncovered the existence of positive cooperativity in the binding of [3H]QNB. Agonist displacement curves of [3H]QNB binding, without and with guanine nucleotides, extended over several orders of magnitude, which is inconsistent with single site competitive kinetics. The results and their analysis by computer-assisted curve fitting indicated that the data are well fitted by a model in which a receptor is at least bivalent and exists in two states: one with and the other without cooperativity between its sites, with guanine nucleotides decreasing both the degree of cooperativity between the sites and the proportion of the receptor that is in the cooperative form. Since the guanine nucleotide effect is mediated by the Ni coupling protein, it is suggested that direct binding detects R'Ni complexes (cooperative), R"NiG complexes (cooperative but distinct from R'Ni), and R0 complexes (non-cooperative and unaffected by Ni or NiG), where R = mAChR, Ni = the inhibitory regulatory component of adenylyl cyclase unaffected by guanine nucleotide, and NiG = Ni affected by guanine nucleotide (G).
- Published
- 1985
11. The function of the non-specific adenylyl aminohydrolase of Aspergillus oryzae
- Author
-
B J Pitts and B A Orsi
- Subjects
History ,biology ,Adenine Nucleotides ,biology.organism_classification ,Computer Science Applications ,Education ,Aspergillus ,Non specific ,Aspergillus oryzae ,Biochemistry ,Aminohydrolases ,Enzyme Induction ,RNA ,Enzyme Repression ,Aminohydrolase ,Function (biology) ,Research Article - Published
- 1969
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