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Biomedical subjects

J D Potter

Publications and source records attributed to J D Potter.

At least 307 records · Page 17Linked to original sources
PubMed1979

Large-scale purification of Na,K-ATPase and its protein subunits from lamb kidney medulla.

Procedures are described for the large-scale isolation of purified Na,K-ATPase (EC 3.6.1.3) from frozen lamb kidney outer medulla and for the separation of its two protein subunits by hydroxyapatite chromatography in sodium dodecyl sulfate (SDS). The methods described permit the routine isolation of up to 800 mg of purified Na,K-ATPase in one week, which can subsequently be separated into 500 mg of mr = 95,000 catalytic subunit and 200 mg of glycoprotein with four SDS-hydroxyapatite column runs.

Animals↗
PubMed1979

Iodization and thyroid status in relation to stillbirths and congenital anomalies.

Thyroid status is known to have an important bearing on the ability of a woman to conceive, and to bring a normal infant to term. Thyroid status has changed in a number of previously iodine deficient countries as a result of recent iodization programmes. In this study, examination has been made of changes in the rates of two pregnancy outcomes in Tasmania, a State of Australia, namely, stillbirth and infant death due to congenital anomalies. Tasmania began iodine supplementation in 1950. Stillbirth rates declined more rapidly than in Australia as a whole, but show a peak associated with the peak of iodine-induced thyrotoxicosis in 1966. Congenital anomalies causing death show an initial rise in the early 1950s. It is suggested that this is due to persistence of iodine deficiency in the foetus after maternal iodine status improved sufficiently to allow term delivery of a live infant. The later fall coincides with the reaching of reproductive age by the first cohort of Tasmanian women who had been iodine sufficient since childhood, who would therefore not deprive their own foetus of iodine. Examination of trends in New Zealand, Switzerland and Finland, which have each introduced iodization programmes, confirms the largely beneficial effect of iodine supplementation on the rate of infant death due to congenital anomalies.

Adolescent↗
PubMed1979

Time trends in colo-rectal cancer mortality in relation to food and alcohol consumption: United States, United Kingdom, Australia and New Zealand.

Recent epidemiologically and experimental research has implicated dietary factors, including alcoholic drinks, in cancers of the colon and rectum. Analysis of time trends in cancer mortality since 1921, in the United States, England and Wales, Australia, and New Zealand, in relation to changes in per capita consumption of foodstuffs and alcohol reveals some support for the protective effect of fibre, but an inconsistent role for fat and meat in colon cancer. For rectal cancer, and to a lesser extent colon cancer, the most consistent correlate in comparisons across time, and between place, sex, and age-group, is beer consumption. Possible reasons for this correlation within this data set are discussed.

Adult↗
PubMed1978

Calcium-induced conformational changes in a cyanogen bromide fragment of troponin C that contains one of the binding sites.

The increase observed in alpha helix from 20 to 38%, the spectral red shift of the absorption bands of the side chain chromophores, the increase of phenylalanine side chain optical activity and a decrease of cysteinyl side chain reactivity with 5,5'-dithiobis(2-nitrobenzoic acid) are consistent with a coil to helix change in the segment containing Cys-98 and Phe-99 and 102 when the CB9 fragment of troponin C (TnC) with an intact binding site III binds Ca2+. Since similar spectrophotometric observations have been made on the whole molecule the data with CB9 confirm our previous suggestion that binding site III of TnC is one of the strong binding sites for Ca2+. The estimate of the binding constant of Ca2+ for CB9 is about 2 orders of magnitude lower than in whole TnC, indicating further stabilization in the intact molecule.

Calcium↗
PubMed1978

Detection of two classes of Ca2+ binding sites in troponin C with circular dichroism and tyrosine fluorescence.

We have observed a biphasic increase in [theta]222 and the tyrosine fluorescence of troponin C (TnC) with increasing [Ca2+]. Approximately 62% of the total helical change and 81% of the total increase in tyrosine fluorescence occurred with Ca2+ binding to a class of sites with a KCa of approximately 2.7 X 10(7) M-1. The remaining spectral changes occurred with Ca2+ binding to a class of sites with KCa of approximately 3.1 X 10(5) M-1. We interpret these biphasic responses in terms of distinct structural changes occurring in TnC with Ca2+ binding to the high affinity (KCa approximately 2.1 X 10(7) M-1) Ca2+-Mg2+ sites and to the low affinity (KCa approximately 3.2 X 10(5) M-1) Ca2+-specific regulatory sites.

Binding Sites↗
PubMed1977

Determination of the amino acid sequence of troponon C from rabbit skeletal muscle.

The complete amino acid sequence of rabbit skeletal muscle troponin C (TnC) was determined from studies on overlapping peptides isolated from cyanogen bromide and tryptic digests. TnC was found to be a single polypeptide chain of 159 amino acid residues, including 2 residues of tyrosine and 1 each of cysteine, histidine, and proline. The amino end is acetylated, the calculated net charge at pH 7.0 is -29, and the calculated molecular weight is 17,965. There was no evidence for heterogeneity in the sequence. The previously proposed four apparent Ca2+ binding sites are located at residues 27 to 38, 63 to 74, 103 to 114, and 139 to 150.

Amino Acid Sequence↗
PubMed1977

Ca2+-dependent regulation of cyclic-AMP phosphodiesterase by parvalbumin.

Carp parvalbumin has been shown to activate rat brain phosphodiesterase in a Ca2+-dependent manner. The concentration of Ca2+ required for half-maximal stimulation is 1.4 X 10(-7) M, whereas rat testis Ca2+-dependent regulator (CDR) of phosphodiesterase required 1.2 X 10(-6) M Ca2+. The difference in the slopes of the two curves demonstrated that the activation induced by parvalbumin was not the result of a small contamination by CDR. In addition, it has been shown that Ca2+ binding to parvalbumin parallels its activation of phosphodiesterase. These data suggest that Ca2+ must bind to a single specific metal binding site before phosphodiesterase can be fully activated.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1977

Biological cross-reactivity of rat testis phosphodiesterase activator protein and rabbit skeletal muscle troponin-C.

Phosphodiesterase activator protein and troponin-C have been purified from rat testis and rabbit skeletal muscle, respectively. The two proteins appear to be structurally distinct since the activator protein migrates faster than troponin-C on sodium dodecyl sulfate-polyacrylamide gels. Each of the calcium-binding proteins will, however, substitute for the other in their respective biological systems. Testis activator protein forms a complex with rabbit muscle troponin subunits TnI and TnT soluble in low salt. This hybrid complex (AIT) can regulate rabbit skeletal muscle actomyosin ATPase activity. AIT regulation, although influenced by free Aa2+ levels, is distinct from that of native troponin. Likewise, muscle troponin-C can substitute for activator protein in the stimulation of cyclic nucleotide phosphodiesterase. Troponin-C will fully stimulate phosphodiesterase although its affinity is 600-fold lower than that of activator protein. Ca2+ regulation studies demonstrate that both proteins require micormolar levels of free Ca2+ to induce phosphodiesterase activation. Activator protein requires 1.2 x 10(6) M and troponin-C, 1.9 X 10(6) M free Ca2+ for half-maximal stimulation of phosphodiesterase. The biological cross-reactivity of these proteins supports the sequence homology recently reported by Watterson et al. (Watterson, D.M., Harrelson, W.G., Keller, P.M., Sharief, F., and Vanaman, T.C. (1976) J.Biol. Chem. 251, 4501-4513). In addition, this preliminary study suggests that this nonmuscle troponin-C-like protein potentially may function in other Ca2+-regulated cellular events in addition to its moculation of cyclic nucleotide levels.

Adenosine Triphosphatases↗
PubMed1977

Thermodynamics of Ca2+ binding to troponin-C.

Troponin-C (TnC) from rabbit skeletal muscle contains two high affinity Ca2+ binding sites (sites 1 and 2) that bind Mg2+ competitively (Ca2+-Mg2+ sites) and two Ca2+ binding sites (sites 3 and 4) of lower affinity that do not bind Mg2+ (Ca2+-specific sites). The free energy (deltaG0i), enthalpy (delta H0i) and entropy (deltaS0i) of binding Ca2+ to each of the four sites (i = 1 to 4) on TnC have been evaluated from microcalorimetry and equilibrium dialysis. The enthalpy of Ca2+ binding to each site was identical (-7.7 kcal mol-1); the entropy of Ca2+ binding to sites 1 and 2 was deltaS01,2 approximately equal to 14.7 e.u. whereas delta S03.4 approximately equal to 8.0 e.u. The positive entropy associated with Ca2+ binding to sites 1 and 2 is probably due to displacement of water produced by the alpha-helix formation, known to accompany the binding of Ca2+ to the Ca2+-Mg2+ sites. Thus, Ca2+ binding to the Ca2+-Mg2+ sites is driven by both enthalpy and entropy and the lower Ca2+ affinity for sites 3 and 4 is reflected in the lower entropy of Ca2+-binding. The entropy associated with Ca2+ binding to sites 3 and 4 suggests that some change in protein conformation is occurring upon binding of Ca2+ to these sites.

Animals↗
PubMed1976

Effect of Ca2+ binding on troponin C. Changes in spin label mobility, extrinsic fluorescence, and sulfhydryl reactivity.

The Ca2+ binding component (TnC) of troponin has been selectively labeled with either a spin label, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, or with a fluorescent probe, S-mercuric-N-dansyl cysteine, presumably at its single cysteine residue (Cys-98) in order to probe the interactions of TnC with divalent metals and with other subunits of troponin. The modified protein has the same Ca2+ binding properties as native TnC (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628), viz. two Ca2+ binding sites at which Mg2+ appears to compete (Ca2+-Mg2+ sites, KCa = 2 X 10(7) M-1) and two sites at which Mg2+ does not compete (Ca2+-specific sites, KCa = 2 X 10(5) M-1). Either Ca2+ or Mg2+ alters the ESR spectrum of spin-labeled TnC in a manner that indicates a decrease in the mobility of the label, Ca2+ having a slightly greater effect. In systems containing both Ca2+ and Mg2+ the mobility of the spin label is identical with that in systems containing Ca2+ alone. The binding constants for Ca2+ and Mg2+ deduced from ESR spectral changes are 10(7) and 10(3) M-1, respectively, and the apparent affinity for Ca2+ decreases by about an order of magnitude on adding 2 mM Mg2+. Thus, the ESR spectral change is associated with binding of Ca2+ to one or both of the Ca2+-Mg2+ sites. Addition of Ca2+ to the binary complexes of spin-labeled TnC with either troponin T (TnT) or troponin I (TnI) produces greater reduction in the mobility of the spin label than in the case of spin-labeled TnC alone, and in the case of the complex with TnI the affinity for Ca2+ is increased by an order of magnitude. The fluorescence of dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-labeled TnC is enhanced by Ca2+ binding to both high and low affinity sites with apparent binding constants of 2.6 X 10(7) M-1 and 2.9 X 10(5) M-1, respectively, calculated from the transition midpoints. The presence of 2 mM Mg2+, which produces no effect on dansyl fluorescence itself, in contrast to its effect on the spin label, shifts the high affinity constant to 2 X 10(6) M-1. Spectral changes produced by Ca2+ binding to the TnC-TnI complex furnish evidence that the affinity of TnC for Ca2+ is increased in the complex. The reactivity of Cys-98 to the labels and to 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is decreased by Ca2+ or Mg2+ both with native TnC and in 6 M urea. The reaction rate between Cys-98 and Nbs2 decreases to one-half the maximal value at a Ca2+ concentration that suggests binding to the Ca2+-Mg2+ sites. Formation of a binary complex between TnI and TnC reduces the rate of reaction, and there is a further reduction by Ca2+. The effect of Ca2+ takes place at concentrations that are 1 order of magnitude lower than in the case of TnC alone. These results suggest that the Ca2+ binding site adjacent to Cys-98 is one of the Ca2+-Mg2+ binding sites.

Binding Sites↗
PubMed1975

The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase.

Purified troponin (Tn), the complex of the Ca-2+ binding subunit (TnC), the inhibitory subunit (TnI), and the tropomyosin binding subunit (TnT) binds 4 mol of Ca-2+ per mol. Two sites bind Ca-2+ with a binding constant of 5 times 10-8 M- minus 1, and two with a binding constant of 5 times 10-6 M- minus 1. In the presence of 2 mM MgCl2 the binding to four sites can be characterized with a single affinity constant of 5 times 10-6 M- minus 1. Purified TnC also binds 4 mol of Ca-2+ per mol; two sites have a binding constant of 2 times 10-7 M- minus 1 and two have one of 2 times 10-5 M- minus 1. In the presence of 2 mM MgCl2 the binding constant of the sites of higher affinity is reduced to 2 times 10-6 M- minus 1, while Ca-2+ binding to the sites of lower affinity is unaffected. Assuming competition between Mg-2+ and Ca-2+ for the high affinity sites on TnC and Tn, the changes in Ca-2+ binding can be accounted for with KMg values of 5 times 10-3 M- minus 1 and 5 times 10-4 M- minus 1, respectively. Tn and TnC bind 4 mol of Mg-2+ per mol in the absence of Cs-2+. The fact that at [Ca-2+] similar to 10- minus 5 M four Ca-2+ and only two Mg-2+ are bound per mol of TnC in the presence of 2 mM Mg-2+ further supports the view that there is direct competition between Mg-2+ and Ca-2+ for the high affinity Ca-2+ binding sites on TnC and Tn. These results then suggest that Tn and TnC contain six divalent cation binding sites: two high affinity Ca-2+ binding sites that also bind Mg-2+ competitively (Ca-2+-Mg-2+ sites); two sites with lower affinity for Ca-2+ that do not bind Mg-2+ (Ca-2+-specific sites); and two sites that bind Mg-2+ but not Ca-2+ (Mg-2+-specific sites). The complex of TnC and TnI (1:1 molar ratio) has the same binding properties as Tn, suggesting a conformational change in TnC upon interaction with TnI. Studies on myofibrillar ATPase activity as a function of free Ca-2+ concentration at two different free Mg-2+ concentrations suggest that full activation by Ca-2+ occurs only upon binding of Ca-2+ to the two Ca-2+-specific binding sites in Tn but does not require binding of Ca-2+ to the Ca-2+-Mg-2+ sites.

Adenosine Triphosphatases↗
PubMed1975

The regulatory system of the actin-myosin interaction.

According to recent views the active troponin (Tn) complex consists of three components distinguishable by their ability to bind Ca2+ (TnC), to combine with troponyosin (TnT), and to inhibit actomyosin ATPase with ot without Ca2+ (TnI). Addition of Tn to an actin-myosin-tropomyosin system produceas a Ca2+ requirement for ATPase activity. Recent developments bearing on the mechanism both in vitro and in vivo are reviewed in order to correlate biochemical, physiochemical, electron microscopic, and physiological findings. The use of various combination of isolated components considerably helps in pinpointing changes that occur within components (conformational changes) and in the relative positions of interacting moieties. Available data on differences between the skeletal and cardiac system are also discussed.

Actins↗