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Biomedical subjects

T E Ryan

Publications and source records attributed to T E Ryan.

6 recordsLinked to original sources
PubMed1978

Isolation of a low molecular weight Ca2+ carrier from calf heart inner mitochondrial membrane.

A protein was isolated from calf heart inner mitochondrial membrane with the aid of an electron paramagnetic resonance assay based on the relative binding properties of Ca2+, Mn2+, and Mg2+ to the protein. The molecular weight of this protein has been estimated to be about 3000 by urea/sodium dodecyl sulfate gel electrophoresis and amino acid analysis. The protein is shown to have two classes of binding sites for Ca2+ by flow dialysis studies and can extract Ca2+ into an organic phase. The selectivity sequence of this protein determined from the organic solvent extraction experiments shows that it favors divalent cations over monovalent cations. Also, the relative selectivity sequence for divalent cations is Ca2+, Sr2+ greater than Mn2+ greater than Mg2+. Ruthenium red and La3+ are shown to inhibit the protein-mediated extraction of Ca2+ into the organic solvent. The calcium translocation in a Pressman cell by this protein is selectively driven by a hydrogen ion gradient. Control experiments indicate that the Ca2+ trnsport properties of the protein are not due to the contaminating phospholipids. It appears that we have isolated from the inner mitochondrial membrane a calcium carrier, which we have named "calciphorin."

Amino Acids↗
PubMed1978

Potassium transference in Nitella.

Transmembrane movements of K+ and Cl- were studied under a variety of experimental conditions. Potassium was found to carry more than 50% of an externally applied inward positive current. The increase in K+ influx was much greater than that predicted by the purely passive model. The increase in Cl- efflux accounted for less than 10% of the applied current, in agreement with the value predicted for passive movement. 2,4-Dinitrophenol (DNP) caused an 80% reduction in K+ transference and a corresponding increase in the measured electrical resistance of the membrane. DNP also reduced the isotopically measured resting K+ influx and caused a substantial increase in both Cl- influx and efflux. Lowering of the pH from 5.7 to 4.7 also reduced the net K+ influx but without drastically altering the membrane resistance. It appears the major portion of an externally applied current does not travel through passive channels, but rather is shunted through a different membrane component. In conjunction with evidence previously establishing the H+ pump as the primary ion pump in Nitella, the data presented here are consistent with a K+/H+ exchange mechanism which can account for the observed net K+ accumulation and maintenance of the membrane potential above the electrochemical equilibrium potential of the major ions. This mechanism appears to be a likely candidate for the current shunt.

Biological Transport, Active↗
PubMed1977

Active calcium treatment transport via coupling between the enzymatic and the ionophoric sites of Ca2+ + Mg2+-ATPase.

The 20K dalton fragment of Ca2+ + Mg2+-ATPase obtained from th tryptically digested sarcoplasmic reticulum has been further purified using Bio-Gel P-100. This removed low-molecular-weight UV-absorbing and positive Lowry-reacting contaminants. The ionophoric activity of the 20K fragment in both oxidized cholesterol and phosphatidylcholine:cholesterol membranes is unaltered by this further purification. The 20K selectivity sequence in phosphatidylcholine:cholesterol membrane is Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ Mg2+. Digestion of intact sarcoplasmic reticulum vesicles with trypsin, which results in the dissection of the hydrolytic site (30K) from the ionophoric site (20K), is shown to disrupt energy transduction between ATP hydrolysis and calcium transport. This further implicates the 20K dalton fragment as a calcium transport site. These data and previous evidence are discussed in terms of a proposed model for the ATPase molecular structure and the mechanisms of cation transport in sarcoplasmic reticulum.

Adenosine Triphosphatases↗
PubMed1976

Localization of ionophore activity in a 20,000-dalton fragment of the adenosine triphosphatase of Sarcoplasmic reticulum.

The (Ca2+ + Mg2+)-dependent ATPase of sarcoplasmic reticulum has been shown to ast as a Ca2+-dependent and selective ionophore in artificial lipid bilayers. Four fragments of 55,000, 45,000, 30,000, and 20,000 daltons have been purified from tryptic digests of the enzyme and it has been shown that the 55,000- and 45,000-dalton fragments are obtained from a single cleavage of the 100,000-dalton ATPase, while the 30,000- and 20,000-dalton fragments are obtained subsequently by a cleavage of the 55,000-dalton fragment. The 55,000- and 20,000-dalton fragments have ionophore activity inhibited by ruthenium red and by mercuric chloride but not by methylmercuric chloride, an inhibitor of the hydrolytic site of the enzyme. Under standard conditions the 45,000-dalton fragment was not active as an ionophore, while the 30,000-dalton fragment acted as a nonselective ionophore. The 55,000- and 30,000-dalton fragments have been shown to contain the site of phosphorylation and of N-ethyl [2-3H]-maleimide binding indicative of the hydrolytic site in the enzyme, and this site is absent from the 20,000-dalton fragment. Therefore, the ionophoric and hydrolytic sites are localized in separate regions of the ATPase molecule and they have now been physically separated. The 20,000-dalton fragment was degraded with cyanogen bromide and fragments were separated by molecular sieving. Ionophore activity was found in fragments of molecular mass less than 2,000 daltons.

Adenosine Triphosphatases↗