Adil E. Shamoo

Further characterization of light and heavy sarcoplasmic reticulum vesicles. Identification of the ‘sarcoplasmic reticulum feet’ associated with heavy sarcoplasmic reticulum vesicles

Abstract Light and heavy sarcoplasmic reticulum vesicles were isolated from rabbit leg muscle using a combination of differential centrifugation and isophycnic zonal ultracentrifugation. Light sarcoplasmic reticulum vesicles obtained from the 30–32.5% and heavy sarcoplasmic reticulum vesicles obtained from the 38.5–42% sucrose regions of the linear sucrose gradient were determined to be free of surface and mitochondrial membrane contamination by marker enzyme analysis and electron microscopy. Thin sections of the light vesicles revealed empty vesicles of various sizes and shapes. Freeze-fracture replicas of the light vesicles showed an asymmetric distribution of intramembranous particles with the same orientation and distribution as the longitudinal sarcoplasmic reticulum in vivo. Heavy vesicles appeared as rounded vesicles of uniform size filled with electron dense material, similar to that seen in the terminal cisternae of the sarcoplasmic reticulum. The cytoplasmic surface of the membrane was decorated by membrane projections, closely resembling the ‘feet’ which join the sarcoplasmic reticulum to the transverse tubules in the intact muscle fiber. Freeze-fracture replicas of the heavy vesicles revealed an asymmetric distribution of particles which in some areas of the vesicles surface are larger and less densely aggregated than those of the light vesicles. In the best quality replicas, some regions of the luminal leaflet were not smooth but showed evidence of pits. These structural details are characteristic of the area of sarcoplasmic reticulum membrane which is covered by the ‘feet’ in the intact muscle. Heavy vesicles contained greater than six times the calcium content of light vesicles, 54 vs. 9 nmol Ca2+/μl of water space. After KCl washing both contained less than 4 nmol Ca2+/μl of water space. Although they transported at the same rate and the same total amount of calcium, the rate of passive Ca2+ efflux from the heavy vesicles was double that of light vesicles. The higher rate of calcium efflux from the heavy vesicles was inhibited by dantrolene, an inhibitor of Ca2+ release. High resolution sodium dodecyl sulfate gel electrophoresis showed that the light vesicles contained predominantly Ca2+-ATPase along with several approx. 55 000-dalton proteins and a 5000-dalton proteolipid, while the heavy vesicles contained Ca2+-ATPase and calsequestrin along with several approx. 55 000-dalton proteins, extrinsic 34 000- and 38 000-dalton proteins, intrinsic 30 000- and 33 000-dalton proteins and two proteolipids of 5000 and 9000 daltons. KCl washing of the heavy vesicles removed both the approx. 34 000- and 38 000-dalton proteins, and the ‘sarcoplasmic reticulum feet’ were no longer seen on the heavy vesicles. The KCl supernatant was enriched in the 34 000- and 38 000-dalton proteins, indicating that these proteins are possible components of the sarcoplasmic reticulum feet. The biochemical and morphological data strongly support the view that the light vesicles are derived from the longitudinal sarcoplasmic reticulum and that the heavy vesicles are derived from the terminal cisternae containing junctional sarcoplasmic reticulum membrane with the intact ‘sarcoplasmic reticulum feet’.

Anionic detergents as divalent cation ionophores across black lipid membranes.

SummaryThree 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 μm, 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 4×10−4m and 10−3m, 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 on the 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 5mm CaCl2, small concentrations of LaCl3 (<1 μm) 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 BLMs, and is not due to a direct effect of La3+ on detergent-mediated transport.

Adenosine 3′,5′-monophosphate-dependent phosphorylation of a 6000 and A 22000 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 characteristic 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 kinase 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.

ISOLATION OF IONOPHORES FROM ION‐TRANSPORT SYSTEMSfn1

In the report evidence is presented for the characterization and isolation of variety of ionophores from various sources. Evidence is also presented for the following: (1) the characterization of Na/sup +/-dependent ionophore from Na + K-ATPase; (2) the isolation of cation-selective ionophoric peptide fragment from Na/sup +/ + K/sup +/-ATPase; (3) the isolation of K/sup +/-dependent ionophore from Na/sup +/ + K/sup +/-ATPase; (4) the demonstration that a cholinergic-agent-sensitive cation-selective ionophore is part of the acetylcholine receptor; (5) the isolation and characterization of Ca/sup 2 +/-dependent ionophore as a peptide fragment (< 2,000 daltons) from sarcoplasmic reticulum Ca/sup 2 +/ + Mg/sup 2 +/-ATPase.

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.

Chloride-Induced Release of Actively Loaded Calcium from Light and Heavy Sarcoplasmic Reticulum Vesicles

SummaryLight and heavy sarcoplasmic reticulum vesicles (LSR, HSR) isolated from rabbit leg muscle have been used in a study of chloride-induced Ca2+ release. The biochemical and morphological data indicate that LSR is derived from the longitudinal reticulum and HSR is derived from the terminal cisternae of the sarcoplasmic reticulum. LSR and HSR were both able to accumulate Ca2+ in the presence of ATP to amounts greater than 100 nmol Ca2+/mg of protein in less than 1 min. LSR and HSR each had a biphasic time course of Ca2+ uptake. The initial uptake was followed by a rapid release, after approximately 1 min, of 30–40% of the accumulated Ca2+, which was then followed by a slower phase of Ca2+ accumulation. Ca2+ taken up by the SR vesicles could be released from both the LSR and HSR by changing the anion outside the vesicles from methanesulfonate to chloride. Due to the difference in permeability between methanesulfonate and chloride, this change should result in a decreased positivity inside the vesicles with respect to the exterior. It could also result in osmotic swelling of the vesicles. Changing the ionic medium from chloride to methanesulfonate caused no release of Ca2+. The amount of accumulated Ca2+ released in 6 sec by changing the anion outside the vesicles from methanesulfonate to chloride was 30–35 nmol/mg membrane protein for LSR and HSR, respectively. Osmotic buffering with 200mm sucrose caused a slight inhibition of chloride-induced Ca2+ release from HSR (17%→15%) but it greatly reduced the release of Ca2+ from LSR (32%→15%). The specificity of Ca2+ release was measured using SR vesicles which were passively loaded with 10mm22Na+. LSR released five times more22Na+ than HSR under same conditions as chloride-induced Ca2+ release occurred. Na dantrolene (20 μm) had no effect on the release of Ca2+ from LSR but it inhibited the chloride-induced Ca2+ release from HSR by more than 50%. Na dantrolene also increased the Ca2+ uptake in the HSR by 20% while not affecting LSR Ca2+ uptake. Our results indicate the presence of a chloride-induced, Na dantrolene inhibited, Ca2+ release from HSR, which is not due to osmotic swelling.

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

SummaryBlack lipid membranes were formed with oxidized cholesterol in the presence of either the acetylcholine receptor, purified from the electric organ of the electric rayTorpedo 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 its concentration, but addition of carbamylcholine gave no reproducible or 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 μm) varied from 10- to over 100-fold. Curare (10 μm) 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-3H]acetylcholine; and analytical gel electrophoresis indicated that it remained intact possibly through hydrogen, hydrophobic and disulfide bonding.

Phosphorylation of Heavy Sarcoplasmic Reticulum Vesicles: Identification and Characterization of Three Phosphorylated Proteins

SummaryHeavy sarcoplasmic reticulum vesicles derived from the terminal cisternae of the sarcoplasmic reticulum have been shown to contain endogenous protein kinase activity and associated substrate proteins. Heavy vesicles were phosphorylated at room temperature in 5mm MgCl2, 1mm EGTA, 10mm HEPES (pH 7.4) and 10 μm γ-32P-ATP.32P-phosphoproteins were determined by sodium dodecyl sulphate gel electrophoresis and autoradiography. In the absence of ethylene glycol bis (β-aminoethyl ether) N,N,N′,N′-tetraacetic acid (EGTA), there was little phosphorylation due to the high level of ATPase activity. Phosphorylation of three proteins of 64,000 daltons (E1), 42,000 daltons (E2), and 20,000 daltons (E3) was observed in the presence of 1mm EGTA. Phosphorylation of these proteins wascAMP-independent, hydroxylamine-resistant, and was seen without the addition of protein kinase. In the presence of HgCl2 (2.5mm) or sodium deoxycholate (1%) no protein phosphorylation was observed. ProteinE1 was heavily phosphorylated in the presence of 200mm KCl, while its phosphorylation was inhibited by 20 μm sodium dantrolene, an inhibitor of Ca2+ release. PhosphoproteinE3 was found in light and heavy sarcoplasmic reticulum vesicles whileE1 andE2 were found only in heavy vesicles. The phosphoproteinE2 had the properties of an intrinsic membrane protein while the proteinE1 bejaved as an extrinsic membrane protein. ProteinsE2 andE3 corresponded in mobility to minor sarcoplasmic reticulum proteins whileE1 had the same mobility as calsequestrin. The presence of high calcium (5mm) during electrophoresis caused calsequestrin to run at a lower molecular weight (≈56,000 instead of 64,000 daltons), and correspondingly the phosphoproteinE1 ran at a lower molecular weight. Finally, calsequestrin purified by a double gel electrophoresis method has been shown to be phosphorylated.

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

SummaryWe 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. USA71:3522). 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.

Na+-dependent ionophore as part of the small polypeptide of the (Na++K+)-ATPase from eel electroplax membrane

SummaryThe (Na++K+)-ATPase from eel electroplax membranes is resolved into two polypeptides by means of sodium dodecyl sulfate (SDS) preparative gel electrophoresis. From the literature, the larger polypeptide has been known to have a molecular weight in the range of 85,000 to 135,000, while the molecular weight of the smaller polypeptide is known to be between 40,000 and 60,000. When the two polypeptides are combined with a large/small molar ratio of 1∶2, a very Na+-dependent voltage-in-dependent ionophoric activity is observed. The Na+-specificity is manifested as a near absolute requirement for Na+ in order for the two polypeptides to cause an increase in conductance. Further work with tryptic digests of the two polypeptides suggests that the Na+-dependent ionophoric activity is associated with the smaller polypeptide of (Na++K+)-ATPase.