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A E Shamoo

Publications and source records attributed to A E Shamoo.

15 recordsLinked to original sources
PubMed1979

Anionic detergents as divalent cation ionophores across black lipid membranes.

Three ionic detergents commonly used in membrane-bound protein isolation and reconstitution experiments, SDS, cholate, and DOC, are shown to act as divalent cation ionophores when incorporated into black lipid membranes made from either oxidized cholesterol or a mixture of phosphatidylcholine and cholesterol (PC/cholesterol = 5:1 mg). At a concentration greater than or equal to 1 microM, SDS shows large selectivity differences between cations and anions and among the different cations tested (Ba2+, Ca2+, Sr2+, Mg2+, and Mn2+). Deoxycholate and cholate at concentrations greater than 4 X 10(-4) M and 10(-3) M, respectively, also act as divalent cation ionophores. The selectivity sequence measured for these two detergents is evidence for a strong ionic interaction between the divalent cation and the anionic charged groups on the detergent. In the case of cholate, the conductance depends on the third or fourth power of the cholate concentration and shows a linear dependence on CaCl2 concentration. The conductance for deoxycholate depends onthe sixth or seventh power of the DOC concentration and is also linearly dependent on the CaCl2 concentration. In an oxidized cholesterol black lipid membrane in the presence of 5 mM CaCl2, small concentrations of LaCl3 (less then 1 microM) inhibit the ionophoric activity of each of the detergents tested. Evidence is presented to show that this inhibitory effect is a nonspecific effect on oxidized cholesterol BLM's, and is not due to a direct effect of La3+ on detergent-mediated transport.

Cations, Divalent↗
PubMed1978

Purification and characterization of the 45,000-Dalton fragment from tryptic digestion of (Ca2+ + Mg2+)-adenosine triphosphatase of sarcoplasmic reticulum.

Tryptic digestion of (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle has previously been shown to cleave the enzyme initially into a 55,000-dalton fragment and a 45,000-dalton fragment. In the present study the two fragments are solubilized in sodium dodecyl sulfate (SDS) and separated by preparative polyacrylamide gel electrophoresis. The 45,000-dalton fragment is found to be a relatively nonselective, divalent cation-dependent ionophore when incorporated into an oxidized cholesterol membrane (BLM). Ionophoric activity of this fragment is inhibited by low concentrations of LaCl3, HgCl2, and various reducing agents. There appears to be one or two relatively inaccessible disulfide bonds in the 45,000-dalton fragment that are essential for transport. Addition of reducing agents inhibits the ionophoric activity of the succinylated undigested enzyme and the 45,000-dalton fragment, but has no effect on the 55,000-dalton fragment. These experiments imply that the 45,000-dalton fragment and the 55,000-dalton fragment are in a series arrangement in the membrane.

Animals↗
PubMed1978

Ionophorous properties of the 20,000-dalton fragment of (Ca2+ + Mg2+)-ATPase in phosphatidylcholine: cholesterol membranes.

The purified 20,000-dalton fragment of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase has been shown by us (A.E. Shamoo, T.E. Ryan, P.S. Stewart, D.H. MacLennan, 1976. J. Biol. Chem. 251:4147) to have Ca2+-selective ionophoric activity. The Ca2+-ionophoric fragment has been purified by either SDS-column chromatography or SDS-preparative gel electrophoresis. The Ca2+-ionophoric fragment has been subjected to prolonged dialysis to insure the removal of bound SDS from the fragment. The selectivity sequence of this fragment in black lipid membranes (BLM) formed from either oxidized cholesterol or phosphatidylcholine/cholesterol is the same, PBa greater than PCa greater than PSr greater than PMg greater than PMn. This selectivity sequence is the same as that for the intact (Ca2+ + Mg2+)-ATPase. Treatment of the fragment with cholate to absolutely insure the removal of bound SDS resulted in the fragment having a selectivity sequence as above except that PMn greater than PMg. This and other data indicate that the 20,000-dalton fragment is the site containing the Ca2+-ionophoric activity of the (Ca2+ + Mg2+)-ATPase.

Animals↗
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↗
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↗
PubMed1976

Differential effects of mercurial compounds on excitable tissues.

Sarcoplasmic reticulum (SR), Ca2+ plus Mg2+-ATPase, and Ca2+-ionophore were obtained from white rabbit skeletal muscles. Methylmercury inhibited the Ca2+ plus Mg2+-ATPase and Ca2+-transport but had no effect on the Ca2+-ionophore. Mercuric chloride inhibited all three functions (i.e., ATPase, transport and ionophoric activity). The mechanism of HgCl2 inhibition of the Ca2+-ionophore was by competition with Ca2+ for Ca2+-ionophoric site whereas its inhibition of the enzyme and Ca2+-transport was due to the blockage of essential sulfhydryl (--SH) groups. Ca2+ plus Mg2+-ATPase and Ca2+-transport were more sensitive to methylmercury than to HgCl2. Acetylcholine receptor (AChR) was obtained for the electric organ of T. californica. Methylmercury inhibited the ACh binding to AChR WITH Ki = 5.7 - 10(-6) M. This effect was not due to mercuric ion alone since mercuric chloride up to 10(-4) M did not affect ACh binding to AChR. It is concluded that: the Ca2+ plus Mg2+-ATPase and Ca2+-transport contain --SH groups essential for their activity, and that the two functions are tightly coupled; the Ca2+-ionophore contains no --SH groups essential for its activity; CH3HgCl inhibition of Ca2+ plus Mg2+-ATPase and Ca2+-transport is partly due to its reactivity with --SH groups in hydrophobic environment; the Ca2+-transport is inhibited by HgCl2 through two processes, one which is the blockage of --SH groups and another which is the inhibition of the Ca2+-ionophoric site; and the inhibition of ACh binding to AChR is due to the blockage of --SH groups in hydrophobic environment, which is inaccessible to Hg2+. Our data present for the first time a molecular basis for the myopathy associated with mercurial compounds toxicity.

Acetylcholine↗
PubMed1975

Carbamylcholine and acetylcholine-sensitive, cation-selective ionophore as part of the purified acetylcholine receptor.

Black lipid membranes were formed with oxidized cholesterol in the presence of either the acetylcholine receptor, purified from the electric organ of the electric ray Torpedo californica or its tryptic digest. In both cases, conductance of cations increased and was dependent on the concentration of the receptor protein. Conductance of Ca++ was dependent on the concentration, but addition of carbamylcholine gave no reproducible of consistent effects. Only in the case of the tryptic digest of the acetylcholine receptor did carbamylcholine and acetylcholine consistently induce monovalent cation selective conductance (PNa,K: PCl=4.4). The induced monovalent cationic conductance due to carbamylcholine (10 muM) varied from 10- to over 100-fold. Curare (10muM) prevented the action of carbamylcholine. Na-dodecyl sulfate gel electrophoresis of the acetylcholine receptor, before and after tryptic digestion, indicated that this mild enzyme treatment hydrolyzed the receptor molecule subunits. Nevertheless, the receptor molecule retained its full binding of [acetyl(-3)H]acetylcholine; and analytical gel electrophoresis indicated that it remained intact possibly through hydrogen, hydrophobic and disulfice bonding.

Acetylcholine↗
PubMed1975

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca++ +Mg++)-ATPase of sarcoplasmic reticulum.

We have shown that a Ca++-ionophore activity is present in the (Ca++ +Mg++)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A. E. Shamoo & D. H. MacLennan, 1974. Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca++ +Mg++)-ATPase and Ca++ transport, but had no effect on the activity of the Ca++ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca++ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca++ transport by blocking essential -SH groups. However, it appears that there are no essential -SH groups in the Ca++ ionophore and that mercuric chloride inhibited the Ca++ ionophore activity by competition with Ca++ for the ionophoric site. Blockage of Ca++ transport by mercuric chloride probably occurs both at sites of essential -SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca++ ionophoric activity.

Adenosine Triphosphatases↗
PubMed1975

Mechanism of action of "ruthenium red" compounds on Ca2+ ionophore from sarcoplasmic reticulum (Ca2+ + Mg2+)- adenosine triphosphatase and lipid bilayer.

Sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase was previously shown to have Ca2+-dependent and -selective ionophoric activity when tested in oxidized cholesterol lipid bilayer membranes (Shamoo, A. E., and MacLennan, D. H. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 3522). ruthenium red, a known inhibitor of (Ca2+ + Mg2+)-ATPase, is found to inhibit the Ca2+-ionophoric activity associated with (Ca2+ + Mg2+)-ATPase. Furthermore, ruthenium red alone acts as an anion-selective ionophore in lipid bilayers with the the following selectivity sequence for anions: l- greater than Cl-, Br- greater than F- greater than NO3-. The PCl-/PNa+ ratio was approximately 4/l. The presence of ruthenium red in excess of Ca2+ ionophore in lipid bilayer experiments converts the cation selectivity of the bilayer due to Ca2+ ionophore into anion selectivity.

Adenosine Triphosphatases↗
PubMed1975

Vitamin D -- induced changes in the renal membrane ATPase system.

Ca-dependent and Mg-dependent ATPase activity was found to be depressed significantly in renal membrane preparations from vitamin D-deficient as compared to normal rats. Administratiin of 2000 i.u. vitamin D2 to deficient rats 30 hours before sacrifice restored the membrane enzyme activity to normal. ATP binding by enzymes was also reduced in vitamin D deficiency and raised by repletion. Vitamin D may therefore be required for normal Ca-and Mg-dependent ATPase activity.

Adenosine Triphosphatases↗