Anthony Martonosi

Sarcoplasmic reticulum: VIII. Use of 8-anilino-1-naphthalene sulfonate as conformational probe on biological membranes

Abstract 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.

Sarcoplasmic reticulum. V. The structure of sarcoplasmic reticulum membranes.

Electron microscope analysis of skeletal muscle microsomes was carried out by negative staining with potassium phosphotungstate. The size and shape of microsomes are influenced by species differences, the conditions of preparation and storage. The outer surface of the microsomal membrane is covered by subunits of 40 A diameter, with center to center separation of 90–120 A. Chemical modification of microsomal membrane by treatment with phospholipase C, digitonin, or trypsin produced characteristic changes in the surface structure, shape or size of microsomes, parallel with the loss of Ca2+ transport function. The surface structure of microsomes does not indicate temperature-dependent structural changes of the kind observed on liver cell surface membranes, and the Arrhenius plot of ATPase activity is linear.

Biochemical and Clinical Aspects of Sarcoplasmic Reticulum Function

Publisher Summary This chapter discusses a critical appraisal of information about the possible involvement of sarcoplasmic reticulum in various muscle diseases such as cardiac failure, muscular dystrophy, myotonia, and denervation atrophy. Because of the singular concentration of attention on the role of sarcoplasmic reticulum in the regulation of sarcoplasmic Ca 2+ concentration, conspicuous gaps developed in the knowledge regarding other possible functions of sarcoplasmic reticulum such as its involvement in the energy metabolism of muscle cells. Recent evidence indicates that the sarcoplasmic reticulum may represent a compartment for hexokinase, glyceraldehydephosphate dehydrogenase, and phosphofructokinase activities. Other metabolic functions of sarcoplasmic reticulum include the synthesis of phospholipids and proteins, as suggested by their turnover in Sarcoplasmic reticulum membrane. Finally, a detailed biochemical analysis of the function of transverse tubular system and the triad may soon become possible. The distribution of sarcoplasmic reticulum in the muscle cell and its relationship to the myofibrils, surface membranes, and the T-system tubules bear considerable importance in relation to its physiological function.

Sarcoplasmic reticulum: XI. The mode of involvement of phospholipids in the hydrolysis of ATP by sarcoplasmic reticulum membranes☆

Abstract 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 protein composition of sarcoplasmic reticulum membranes.

Abstract By the use of various polyacrylamide electrophoresis methods the proteins of sarcoplasmic reticulum membranes were resolved into a large number of distinct bands. A major band was tentatively identified with the ATPase enzyme implicated in the active Ca transport.

Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membranes.

Abstract The proteins of Sarcoplasmic reticulum membranes were resolved by polyacrylamide gel electrophoresis into several fractions ranging in mol wt from 300,000 to about 30,000. The ATPase enzyme involved in Ca 2+ transport is associated with a major protein fraction and its molecular weight based on its electrophoretic mobility on polyacrylamide gels in the presence of sodium dodecylsulfate is about 106,000. Reducing agents (β-mercaptoethanol or dithiothreitol) cause the dissociation of membrane proteins into subunits of 20,000–60,000 mol wt, which can be separated by electrophoresis or Sephadex G-150 chromatography. Methods were developed for the isolation of a 32 P-labeled phosphopeptide from peptic hydrolysates of microsomal membranes. The presence of glycolipids was demonstrated in sarcoplasmic reticulum membranes with periodate-Schiff reaction.

Sarcoplasmic reticulum. XVI: The permeability of phosphatidyl choline vesicles for calcium

Abstract 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.

Elementary processes in the hydrolysis of ATP by sarcoplasmic reticulum membranes.

The energy required for the active transport of calcium by sarcoplasmic reticulum is derived from the hydrolysis of ATP 1 l or carbamyl phosphate through a transport ATPase that is absolutely dependent upon membrane phospholipids for activity Tr and constitutes about 70% of the protein content of microsomal membranes.f1, The hydrolysis of ATP 8-10 or acetylphosphate occurs with the formation and decomposition of a phosphoprotein intermediate that has several characteristics of an acylphosphate.9 A possible mechanism that relates ATP hydrolysis and Ca transport to the phosphoprotein intermediate may be written as follows :

Sarcoplasmic reticulum: XII. The interaction of 8-anilino-1-naphthalene sulfonate with skeletal muscle microsomes

Abstract The relationship between fluorescence intensity and the binding of 8-anilino-1-naphthalene sulfonate (ANS) to fragmented sarcoplasmic reticulum membranes was studied. Under various conditions the fluorescence intensity increased proportionally with the concentration of ANS and divalent metal ions bound to microsomal membranes. The dependence of ANS binding on free ANS concentration is complex, which may be attributed to heterogeneous binding sites, or negative interactions between similar sites. The polarization of ANS fluorescence in microsomal membranes is greater than in micellar dispersions of phospholipids and is largely independent of medium viscosity in both systems. The low polarization of fluorescence at physiological temperature indicates considerable mobility of the probe in the phase of the membrane. Local anesthetics and polymyxin B increase the intensity of fluorescence of ANS—microsome systems and decrease its polarization. The relationship of these effects to the inhibition of Ca 2+ transport remains to be established. The marked effect of ions on the intensity and polarization of fluorescence should be considered before attributing the fluorescence response to conformational change.

The binding of arsenazo III to cell components

The Ca2+ indicator, arsenazo III, binds to subcellular fractions of rabbit skeletal muscle with sufficient affinity that in living muscle containing 1--2 mM arsenazo III, the estimated free arsenazo III concentration is only 50--200 microM; 80--90% of the bound arsenazo III is associated with soluble proteins. The binding of arsenazo III to soluble proteins decreases the optical response of the dye to Ca2+; this is due to a decrease in the affinity of the protein-bound dye for Ca2+. Approximately half of the bound arsenazo III is released from the particulate fraction and soluble proteins upon addition of 5 mM Ca2+, suggesting that the Ca-arsenazo complex has lower affinity for the protein binding sites than the free dye. The Ca2+ binding to the soluble protein fraction of rabbit skeletal muscle is attributable largely to its parvalbumin content.