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The sarcoplasmic reticulum of rabbit skeletal muscle contains a small “proteolipid,” i.e., a protein which is soluble in acidic CHCl 3 CH 3 OH . We propose the name sarcolipin for this small protein, to signify its lipid-like solubility and association with the sarcoplasmic reticulum. We have determined the following amino acid sequence for sarcolipin, using protein chemistry methods: M E R S T R E L C L N F T V V L I T V I L I W L L V R S Y Q Y. This 31-residue sequence includes a 19-residue hydrophobic segment which probably spans the sarcoplasmic reticulum membrane. The molecular weight calculated from the sequence, 3733, agrees with that measured by fast atom bombardment mass spectrometry, showing that sarcolipin contains no attached fatty acyl or other prosthetic groups.

Treatment of sarcoplasmic reticulum vesicles with ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA), Chelex-100, 1,10-phenanthroline, 8-hydroxyquinoline, or 8-hydroxyquinoline sulfonic acid increases the reactivity of SH groups with N-(1-pyrene)maleimide (PMI). The effect of Chelex treatment can be reversed by the addition of 10−6−10−5M Zn2+ to the Chelex-treated microsomes. The activation of the PMI reaction by EGTA was not reversed by subsequent addition of calcium, although the presence of excess calcium during EGTA treatment abolished the effect. Analysis of the elemental composition of sarcoplasmic reticulum by plasma emission spectroscopy indicates the presence of Zn, Cu, Fe, and Hg in amounts of 1–2 nmol/mg protein; of these only the Zn content is reduced significantly by treatment of microsomes with EGTA or Chelex-100. These observations suggest that Zn2+ may play a role in the regulation of the reactivity of SH groups in sarcoplasmic reticulum either by direct interaction with cysteinyl residues or by an effect upon the conformation of a subpopulation of ATPase molecules.

Quench-flow kinetic studies of the rate of formation and decomposition of phosphoprotein by the [Mg 2+ + Ca 2+ ] activated ATPase of sarcoplasmic reticulum indicate that the rate limiting step of ATP cleavage is the hydrolysis of phosphoprotein. The rapid initial burst of ADP liberation during ATP hydrolysis is consistent with this interpretation. The dependence of the rate of phosphoprotein formation upon free calcium concentration suggests cooperativity with a Hill coefficient greater than 2. The pH dependence of phosphoprotein hydrolysis and its inhibition by photooxidation implicate the involvement of histidine residues in the process.

The effects of Mg2+, Mn2+, Zn2+, Co2+, Ni2+, Ca2+, and Sr2+ upon the equilibrium constant (K) and formation rate constant (k1) of H-meromyosin-ADP complex were studied. The affinity constant of ADP binding decreases in the following order Mn > Mg > Zn > Ni > Co > Sr ∼- Ca. The calculated dissociation rate constant (k2) of the enzyme-ADP complex was similar to the rate of ATP hydrolysis with all divalent metals tested, except Sr2+. With Mg as predominant metal ion the following dissociation constants were obtained for the binding of various nucleoside diphosphates to H-meromyosin: ADP 1–2 × 10−6 m , UDP 2 × 10−5 m , CDP 3 × 10−5 m , IDP 10−4 m . The results are compatible with the suggestion that at 6 °C the dissociation of ADP from the active site limits the rate of ATP hydrolysis with Mg2+, Mn2+, Co2+, and Ca2+.

The mode of involvement of phospholipids in the hydrolysis of ATP and the transport of Ca 2+ by sarcoplasmic reticulum membranes was studied using various phospholipases for the controlled degradation of microsomal membrane phospholipids. The hydrolysis of microsomal phospholipids by phospholipase A from Crotalus terr. terr . (EC 3.1.1.4) and by phospholipase C enzymes isolated from Clostridium welchii (EC 3.1.4.3) and from Bac. cereus inhibits the hydrolysis of adenosine-5′-triphosphate without comparable inhibition of the formation of phosphoprotein intermediate. The observations are consistent with a strict requirement for membrane phospholipids in the decomposition of phosphoprotein.

The Ca permeability of phosphatidyl choline vesicles of diverse fatty acid composition was measured. The rate of 45 Ca release from liposomes equilibrated with 1 m m 45 CaCl 2 was found to be about 8 × 10 −18 moles of Ca/cm 2 /sec for egg lecithin and about 5.3 × 10 −17 moles of Ca/cm 2 /sec for dioleyllecithin at 30 °. Incorporation of cholesterol into dioleyllecithin micelles reduced the rate of Ca release. The Ca permeability of the phosphatidyl choline micelles was insensitive to changes in the pH, calcium or sodium concentration of the medium but increased with increasing temperature. The effect of temperature was most marked with dioleyl lecithin dispersions, but was clearly apparent with dipalmitoyl, plant, bovine, and egg lecithins as well. The activation energy of Ca release fell in the range of 4.2–9.6 kcal/mole. Macrocyclic antibiotics (valinomycin, tyrocidin, and gramicidin) at relatively high concentration increased the rate of Ca release similarly to their effects on fragmented sarcoplasmic reticulum membranes.

The fluorescence of 8-anilino-1-naphthalene sulfonate (ANS) 3 is enhanced by skeletal muscle microsomes and micellar dispersions of phospholipids. The magnitude of the enhancement is a unique function of the ionic composition, rising with the concentration of cations according to a titration curve. The effect of cations might reflect the increased hydrophobic character of the membrane or the increased binding of ANS, induced by cations. The optimum pH for the ANS fluorescence in the presence of microsomes or lecithin is at pH 1–4 and at pH 7.3 a complex temperature dependence was observed with indications of a transition at 35–40 °. Treatment of microsomes or lecithin with phospholipase C causes a decrease of ANS fluorescence while trypsin digestion had little or no effect. Polymyxin B, a circular polypeptide, inhibits the ATPase activity and Ca 2+ transport of muscle microsomes accompanied by a large increase in fluorescence enhancement in the presence of ANS. Tyrocidine also inhibited the biochemical functions while gramicidin was less effective. Polyene antibiotics were generally without effect. These observations indicate that caution is required in the evalution of ANS fluorescence data in terms of conformational changes in membrane proteins alone, since the contribution of phospholipids to the fluorescence is significant.

The rate of incorporation of [ 32 P]orthophosphate, [ 14 C]- or [ 3 H]leucine and [ 14 C]acetate into the phospholipids and proteins of Sarcoplasmic reticulum membranes of rat skeletal muscle was measured after intraperitoneal injection of a single dose of radioactive precursor. The [ 32 P]orthophosphate incorporated into microsomal membranes was recovered almost exclusively in the phospholipids, while the radioactivity from [ 14 C]- or [ 3 H]leucine was found in the microsomal proteins. Acetate labeled to roughly similar extent the protein and lipid components of microsomal membrane. The halftimes of turnover calculated from the decay of radioactivity of proteins and phospholipids after injection of the different precursors were in the range of 10–18 days for all particulate fractions.

Reaction of myosin or H-meromyosin with 10–20 moles of N-ethylmaleimide (NEM) per mole of enzyme inhibits the Mg++, Ca++ or EDTA-moderated ATPase activity, while the affinity of the enzyme for ADP increases. Inhibition of ADP binding requires 40–200 moles of NEM per mole of enzyme, suggesting the involvement of distinct classes of SH groups in the catalytic function and in the binding of ADP to myosin. Rapid kinetic measurements in a stopped-flow apparatus indicate that small concentrations of N-ethylmaleimide (2–5 moles per mole of enzyme) which activates the Mg2+ or Ca2+-moderated ATPase activity at 25 °, and inhibits at 6 °, decrease the rate of appearance and the magnitude of the ATP- or ADP-induced difference spectrum of H-meromyosin at 288 mμ. Assuming that the rate of appearance of the difference spectrum is a measure of the rate of interaction of ADP with H-meromyosin, SH group modification decreases both the formation (k1) and dissociation (k2) rate constants of the H-meromyosin-ADP complex. The decrease in k2 is accompanied by the inhibition of Mg-moderated ATPase activity by N-ethylmaleimide at 6 °. The ultraviolet difference spectrum at 288 mμ, which accompanies the binding of ATP or ADP to H-meromyosin is abolished by N-ethylmaleimide without major interference with the substrate binding.

The effect of steroids on the active Ca2+ transport of rabbit skeletal muscle microsomes was studied. The inhibitory effect of steroids appears connected with the cis conformation of rings A and B and with the presence of at least two hydrophilic groups in the molecule. Steroids which inhibit the Ca2+ transport of skeletal muscle microsomes activate the microsomal ATPase activity and promote the hemolysis of human red blood cells in similar concentrations.

The number and reactivity of actin SH groups have been studied by reacting actin with 14 C- N -ethylmaleimide (NEM), iodoacetate, iodoacetamide, or N -(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and measuring the distribution of radioactivity among the actin peptides following tryptic hydrolysis and separation by chromatography or electrophoresis. Seven to nine SH peptides were counted in guanidine-treated actin preparations, only one of which is reactive with the above reagents in native G- or F-actin. The number of SH peptides is similar to the number of analytically determined SH groups. The actin SH peptides containing the fast reacting SH group were isolated. Their amino acid composition is characterized by a high content of aspartic and glutamic acid. Limited photoxoidation of actin inhibits polymerization but leaves the ATP binding and myosin ATPase activation unchanged. The reaction of SH groups with 14 C-NEM remains unaffected. Treatment of actin with N -bromosuccinimide inhibits polymerization and myosin ATPase activation but leaves the ATP binding unaffected. The reactivity of one SH group with 14 C-NEM is abolished, presumably due to oxidation to cysteic acid.

The effect of N -ethylmaleimide on the ATPase activity and ADP binding of tryptic H-meromyosin was studied at 6 and 23 °C temperatures. The affinity constant of H-meromyosin for ADP with Mg as activator was increased by small concentrations of N -ethylmaleimide (2.25 moles per mole of enzyme) at both temperatures, accompanied by activation of ATP hydrolysis at 25 °C and inhibition at 6 °C. With higher N -ethylmaleimide concentrations, the ATPase activity was inhibited at both temperatures, without comparable inhibition of ADP binding. Rapid kinetic analysis of the rate of development of difference spectrum after the addition of ATP or ADP to H-meromyosin indicates, that blocking of the S 1 and S 2 SH groups of H-meromyosin decreases both the formation ( k 1 ) and the dissociation ( k 2 ) rate constants of H-meromyosin substrate complex. At 6 °C, in the presence of Mg, the value of k 2 for ADP is similar to the turnover number of ATP hydrolysis, suggesting that dissociation of ADP from the active site may be the rate-limiting step of ATP hydrolysis. At 23 °C, the turnover number of Mg-moderated ATP hydrolysis is much smaller than k 2 , indicating that the rate limitation shifted so another, so far unidentified, step.