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Biomedical subjects

A E Shamoo

Publications and source records attributed to A E Shamoo.

At least 55 records · Page 3Linked to original sources
PubMed1983

Ionophorous properties of the 13 000-Da fragment from sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

The 25 000-Da tryptic fragment from rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase was subjected to cyanogen bromide digestion, and the four fragments isolated. Only the 13 000-Da fragment induced ionophorous activity in planar thin lipid membranes made with 5:1 (w/w) phosphatidylcholine/cholesterol in decane. The membranes became cation selective, with a selectivity sequence among divalent of Mn2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. This is different from that of the 25 000-Da fragment (A.E. Shamoo, 1978, J. Memb. Biol. 43, 227-242), it's 'parent' 55 000-Da fragment, and the intact enzyme, all of which have the same selectivity sequence. The inhibitory effects of Hg2+, Cd2+ and Zn2+ were also examined. All were inhibitory, with Zn2+ being the most effective of these. The heavy-metal-induced inhibition of Ca2+ conductance could be reversed by selective chelation of the heavy metals by EDTA. From changes in the selectivity as well as changes in heavy-metal-induced inhibition behavior, we conclude that the ion transport site of the 13 000-Da fragment may not be the same site as that of the parent fragment. It is either a different site altogether or has been physically modified by peptide cleavage.

Animals↗
PubMed1983

Ca2+-cardiolipin interaction in a model system. Selectivity and apparent high affinity.

The interaction of cardiolipin with Ca2+ was assessed by measuring the cardiolipin-mediated extraction of 45Ca2+ from an aqueous to an organic (methylene chloride) phase. Cardiolipin binds Ca2+ with high affinity [Kd(apparent) = 0.70 +/- 0.17 microM (S.D.)]. Cation-cardiolipin interactions are selective. Interaction of cardiolipin with Ca2+ is insensitive to Na+, but is inhibited by divalent cations with Mn2+ greater than Zn2+ greater than Mg2+. In addition La3+ and Ruthenium red are particularly potent inhibitors of Ca2+ binding by cardiolipin. Cardiolipin-mediated extraction of Ca2+ into an aqueous phase is also inhibited by phosphatidylcholine. Inhibition of Ca2+-cardiolipin interaction by phosphatidylcholine (a phospholipid known to stabilize the bilayer conformation) may implicate inverted, non-bilayer lipid structures in the binding.

Calcium↗
PubMed1980

Adenosine 3',5'-monophosphate-dependent phosphorylation of a 6000 and a 22,000 dalton protein from cardiac sarcoplasmic reticulum.

In canine cardiac sarcoplasmic reticulum, adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase specifically phosphorylates two proteins, as seen by sodium dodecyl sulfate-slab gel electrophoresis and autoradiography. One protein has a molecular weight ranging between 22,000 and 24,000 daltons and has previously been identified and named phospholamban (Tada, M., Kirchberger, M.A. and Katz, A.M. (1975) J. Biol. Chem. 250, 2640-2647). The other protein that the 32P label incorporates into has a molecular weight of approximately 6000. Like the 22,000 dalton protein, the 6000 dalton protein has characteristics of phosphoester bonding. The time-dependent course of phosphorylation shows that initially the 32P label is incorporated more rapidly into the 22,000 dalton protein than the 6000 dalton protein, with both proteins reaching a steady-state level of phosphorylation after 10 min of incubation. When both protein kinase and cyclic AMP are eliminated from the incubation medium, both the 22,000 and the 6000 dalton protein are still phosphorylated, but only to about a quarter of the activity found when cyclic AMP and protein kinases are included in the incubation mixture. The addition of phosphodiesterase completely eliminates the phosphorylation of both proteins. Treating the microsomes with trypsin prevents subsequent phosphorylation of either protein. Phosphorylating the microsomes first, then treating with trypsin, renders both the 22,000 and the 6000 dalton proteins resistant to even prolonged trypsin attack. Unphosphorylated, both proteins are solubilized by a very low concentration of deoxycholate. After phosphorylation the proteins cannot be solubilized by deoxycholate. Phosphorylation appears to alter greatly the physical properties of these proteins. Control experiments exclude the possibility that a lipid is being phosphorylated. After phosphorylation the phosphorylated 22,000 dalton protein is separated from the 6000 dalton protein by proteolipid extraction. After first treating the microsomes with methanol, the 22,000 dalton protein is then soluble in acidified chloroform/methanol, while the 6000 dalton protein remains insoluble. The finding that both proteins have much different biochemical properties when phosphorylated than when not, may be relevant in how they regulate calcium transport in the sarcoplasmic reticulum.

Animals↗
PubMed1980

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

A protein has been isolated from calf heart inner mitochondrial membrane with the aid of an electron paramagnetic resonance (EPR) 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 isolated protein has been shown to have high affinity and high specificity for Ca2+ (Jeng, A. Y., Ryan T. E., and Shamoo, A. E. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2125-2129). However, the protein was found to be contaminated with a large amount of phospholipids. There are 150 mol of phospholipids associated with each mole of the protein. The protein is delipidated using Sephadex LH-20 column chromatography. The contaminating phospholipids can be reduced to 0.1 mol of phospholipids/mol of protein. There are no detectable free fatty acids, hexosamines, or sialic acids associated with the delipidated protein. This protein is named "calciphorin," meaning calcium ionophore protein.

Animals↗
PubMed1980

ATP synthesis by Ca2+ + Mg2+-ATPase in detergent solution at constant Ca2+ levels.

ATP has been synthesized by the purified Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum (SR) solubilized in nonionic detergent dodecyloctaoxyethylenglycol-monoether in a solution containing inorganic phosphate and glycerol by changing pH upon addition of ADP. The Ca2+ concentration is kept constant during the experiment. Optimum synthesis is found at CaCl2 = 0.6 mM and the delta pH = 2.9 +/- 0.2. The enzyme has been digested by trypsin for 1 and 20 min, and it is found that synthesis of ATP is correlated with the Ca2+-uptake into SR. The data indicate that the enzyme alone is responsible for active transport of Ca2+ in SR. The driving force for the ATP synthesis of the process may be due to various ion-protein interactions. H+ cannot substitute for Ca2+ in the synthesis of ATP but acts probably through a modification of the Ca2+ binding sites. The data give support that the integrity of the enzyme molecule between its hydrolytic site and the Ca2+-binding sites is essential for the overall Ca2+ transport.

Adenosine Triphosphate↗
PubMed1980

Ion pathways in proteins of the sarcoplasmic reticulum.

In summary, we have begun to characterize three different ion pathways in the sarcoplasmic reticulum. Ca2+-ionophoric activity has been traced to a 13,000-dalton CNBr fragment localized at the amino terminus of the ATPase molecule... The pathway involved in Ca2+ release can be distinguished from the pathway involved in Ca2+ uptake by its insensitivity to quercetin. An anion pathway is sensitive to DIDS and appears to be localized in the ATPase molecule

Amino Acid Sequence↗