Adenosine-triphosphatase activity and nicotinamide nucleotide coenzymes in the parotid gland of the young lamb and adult sheep

1. The activity of a Mg(2+)-dependent Na(+)-plus-K(+)-activated adenosine triphosphatase and the concentrations of nicotinamide nucleotide coenzymes have been measured in the immature parotid glands of young lambs and in the actively secreting glands of adult sheep. 2. The activity of the adenosine triphosphatase increased during development and attained relatively high levels in the mature secreting gland. 3. A high ([NAD]+[NADH(2)])/([NADP]+[NADPH(2)]) ratio (approx. 10:1) was observed in the parotid glands of lambs and sheep. 4. The high concentrations of NAD and the very low concentrations of NADPH(2) have been discussed in relation to metabolic activity, the activity of the Na(+)-plus-K(+)-activated adenosine triphosphatase and the secretion of saliva by the parotid gland.     

The adenosine diphosphate–adenosine triphosphate-exchange reaction of cerebral microsomes and its relation to the sodium ion-stimulated adenosine-triphosphatase reaction

Microsomes from guinea-pig cerebral cortex contain a system capable of exchanging ADP with ATP at rates of about 20mumoles/mg. of protein/hr. The ADP-ATP-exchange reaction requires Mg(2+) for activity. The reaction is not stimulated by Na(+) or K(+) and is not inhibited by ouabain, in contrast with the Na(+)-plus-K(+)-stimulated adenosine triphosphatase. The pH optimum also differs from that of the adenosine triphosphatase. The ADP-ATP-exchange reaction is stimulated two- to three-fold by non-ionic, anionic and cationic detergents, even when these agents are inhibiting the adenosine-triphosphatase reaction. This reaction may represent a component of the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction but is more likely to be due to other enzyme systems present in microsomal subfractions.     

Separation of adenosine diphosphate-adenosine triphosphate–exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase

1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated.     

The influence of external sodium ions on the sodium pump in erythrocytes

1. A study has been made of the interaction between Na(+) and K(+) on the adenosine triphosphatase activity of erythrocyte ;ghosts', and on the K(+) influx and Na(+) efflux of intact erythrocytes. The adenosine triphosphatase activity and the ion movements were greater at a low external K(+) concentration in the absence of Na(+) than they were in the presence of 150mm-Na(+). The inhibition by external Na(+) of K(+) influx had an inhibitory constant of 5-10mm. 2. Activation by K(+) of kidney microsomal adenosine triphosphatase was retarded by Na(+), and activation by Na(+) was retarded by K(+). Fragmented erythrocyte membranes behaved similarly. 3. These observations suggest that there is competition between Na(+) and K(+) at the K(+)-sensitive site of the membrane.     

The phosphatidylinositol kinase of rat brain

1. The presence of a phosphatidylinositol kinase in homogenates of adult rat brain was shown by using labelled ATP or labelled phosphatidylinositol. 2. The kinase was activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+), Cu(2+), K(+), Na(+) and F(-). 3. The detergents sodium deoxycholate, Cutscum and Triton X-100 markedly stimulated the reaction; sodium taurocholate, Tween-20 and cetyltrimethyl-ammonium bromide were less effective. 4. The activity of the enzyme was dependent on SH groups. 5. The subcellular distribution of the kinase in brain resembled that of Na(+)-plus-K(+)-stimulated adenosine triphosphatase and 5'-nucleotidase.     

The cerebral sodium-plus-potassium ion-stimulated adenosine triphosphatase of bovine brain and its microsomal matrix

1. Adenosine triphosphatase activities of dispersions prepared from bovine cerebral cortex that had been frozen, were greater than those of dispersions prepared from fresh tissue. The subcellular distribution of components of the dispersion was not altered by freezing the tissue and a microsomal fraction enriched in Na(+)+K(+)-stimulated adenosine triphosphatase activity was prepared. 2. The bovine cerebral microsomes were further treated with a 2m-sodium iodide reagent to obtain a particulate preparation with minimal Na(+)+K(+)-independent adenosine triphosphatase activity. Na(+)+K(+)-stimulated activity was increased by the sodium iodide treatment and this preparation was shown to be enriched in lipid constituents. 3. Density-gradient centrifugation of the sodium iodide treated preparation gave three main subfractions each containing approximately equal amounts of phospholipid and protein. Further exposure of the sodium iodide-treated preparation to the 2m-sodium iodide reagent altered the distribution of protein and phospholipid among the fractions obtained by density-gradient centrifugation. Dissociation of phospholipids from protein in the sodium iodide-treated preparation was brought about also by high concentrations of arginine. Concentrated solutions of arginine and sodium thiocyanate brought about dissociation of phospholipids from protein of the microsomal preparation. 4. Many amino acids were found to inhibit Na(+)+K(+)-stimulated adenosine triphosphatase activity when present in high concentrations. The inhibition was complex but resulted, in part at least, from diminished affinity for ATP and Na(+) in the presence of the amino acids. 5. A non-ionic detergent, Lubrol W, solubilized up to 40% of the enzyme activity of the sodium iodide-treated preparation together with 30% of the protein and phospholipid in the preparation. Protein was released from the sodium iodide-treated preparation by pancreatic elastase but Na(+)+K(+)-stimulated adenosine triphosphatase activity of the residue was diminished. Ultrasonic treatment of the sodium iodide-treated preparation failed to release a significant proportion of Na(+)+K(+)-stimulated adenosine triphosphatase activity into a form not deposited by ultracentrifugation.     

Inhibition of Mg2+ and Na(+)-K+ ATPases in selected tissues of fish, Cyprinus carpio under fenvalerate toxicity.

The changes in the magnesium adenosine triphosphatase (Mg2+ ATPase) and sodium-potassium adenosine triphosphatase (Na(+)-K+ ATPase) in gill, brain, liver and muscle tissues of freshwater fish, Cyprinus carpio at 6, 12, 24 and 48 hr exposure periods were studied after subjecting to sublethal concentration (10 micrograms/lit) of fenvalerate. Mg2+ ATPase and Na(+)-K+ ATPase activities were inhibited in all the tissues of fenvalerate exposed fish. The per cent inhibition increased with increase in the period of exposure and the possible reasons for the inhibition patterns are discussed.     

Use of Antibody to Membrane Adenosine Triphosphatase in the Study of Bacterial Relationships

An antiserum to Ca(2+)-activated adenosine triphosphatase from membranes of Micrococcus lysodeikticus cross-reacted in agar gels with membrane adenosine triphosphatases from other pigmented micrococci and related species. Species of Micrococcus and Sarcina showed different levels of inhibition of adenosine triphosphatase activities in heterologous reactions with antiserum. Inter- and intraspecific relationships based on the inhibition reaction were compared with an independent parameter, namely the quantitative and qualitative composition of the bacterial membrane phospholipids and fatty acids. The guanine plus cytosine contents in the deoxyribonucleic acid of the species studied correlated well with the serological cross-reactivity of adenosine triphosphatases from their membranes. The types of cross-bridges found in the peptidoglycans of these cocci were also compared with the other properties. The results suggest that an antiserum specific for a major membrane protein may be a reliable and most useful adjunct in studying bacterial serotaxonomy.     

The action of thiol reagents on the adenosine-triphosphatase activities of heavy meromyosin and L-myosin

1. The Ca²⁺-activated adenosine triphosphatase of heavy meromyosin is maximally stimulated by lower relative molar concentrations of phenylmercuric acetate than are required with myosin. 2. Stimulation of the Ca²⁺-activated adenosine triphosphatase of both heavy meromyosin and myosin by thiol reagents is markedly affected by ionic strength, the effects being greater with the former than with the latter. In particular, N-ethylmaleimide strongly inhibits the Ca²⁺-activated adenosine triphosphatase of heavy meromyosin at ionic strength below about 0·2. 3. The precise behaviour of the thiol reagents at low ionic strength is slightly modified by the age of the heavy meromyosin and myosin preparations. 4. Stimulation of the Mg²⁺-activated adenosine triphosphatase of heavy meromyosin by thiol reagents is relatively insensitive to ionic strength. 5. The adenosine triphosphatases of heavy meromyosin and myosin activated by potassium chloride in the absence of bivalent activators are inhibited by thiol reagents over the range of ionic strength at which stimulation occurs in the presence of calcium chloride as activator. 6. The modifying effects of potassium chloride and sodium chloride are qualitatively different when heavy-meromyosin adenosine triphosphatase is stimulated with phenylmercuric acetate. No such difference is observed when the enzyme is stimulated with N-ethylmaleimide.     

Effects of sodium and potassium ions on oxidative phosphorylation in relation to respiratory control by a cell-membrane adenosine triphosphatase

1. A study has been made of the oxygen consumption of kidney homogenates in relation to the ADP concentration as regulated by the cell-membrane adenosine triphosphatase. Stimulation of this enzymic activity by Na(+) and K(+) caused parallel increases in oxygen consumption and ADP concentration. Similarly, inhibition with ouabain caused a parallel fall. The membrane adenosine triphosphatase concerned in active transport therefore appears to regulate respiration through its control of ADP concentration. 2. The respiration of homogenates and mitochondria was also stimulated by K(+) in a way independent of adenosine-triphosphatase activity. It was shown that K(+) facilitates oxidative phosphorylation and the respiratory response to ADP. A K(+) concentration of 25-50mm was needed for maximum oxidative phosphorylation in the presence of physiological concentration of Na(+). Na(+) counteracted K(+) in the effects on mitochondria. It is concluded that K(+) regulates cellular respiration at two structures, one directly in mitochondria, and the second indirectly through control of ADP production at the cell membrane.     

Fatty acid utilization during development of the rat

The effects of dimethyl sulphoxide and glycerol on ox brain microsomal Na(+)+K(+)-stimulated adenosine triphosphatase (EC 3.6.1.3), K(+)-stimulated p-nitrophenyl phosphatase and K(+)-dependent muscle pyruvate kinase (EC 2.7.1.40) were studied. Dimethyl sulphoxide at concentrations below 20% (v/v) was found to stimulate the p-nitrophenyl phosphatase and pyruvate kinase by increasing their affinity for K(+) but to inhibit the Na(+)+K(+)-stimulated adenosine triphosphatase. The latter enzyme activity was also inhibited by glycerol, which like dimethyl sulphoxide, stimulated the K(+)-activated p-nitrophenyl phosphatase at a wide range of concentrations. The solvent effects were promptly reversed by dilution. Similarity was found between glycerol and dimethyl sulphoxide, on one hand, and ATP, on the other, in their stimulatory effect and their ability to increase the ouabain- and oligomycin-sensitivity of the K(+)-stimulated p-nitrophenyl phosphatase. However, only the solvents, not the ATP, increased the binding of K(+) by the microsomes. From the above findings it is suggested that solvents may act on K(+)-dependent enzymes by altering the state of solvation of the activating cation as well as by changing the enzyme structure.     

A protein factor inhibiting the magnesium-activated adenosine triphosphatase of desensitized-actomyosin

1. The preparation and properties of a myofibrillar protein factor which inhibits the Mg(2+)-activated adenosine triphosphatase of desensitized actomyosin is described. 2. This factor had negligible effect on the Mg(2+)-activated adenosine triphosphatase of natural actomyosin and on the Ca(2+)-activated adenosine triphosphatases of desensitized actomyosin and myosin. 3. The Mg(2+)-activated inosine triphosphatase activity of desensitized actomyosin was not affected by the factor. 4. The inhibitory effect was sensitive to ionic strength. In addition to their ionic effects Mg(2+) and Ca(2+) appeared to have a specific action in reducing the effect of the inhibitor. 5. F-actin reduced the inhibition whereas Bailey-type tropo-myosin had little effect. 6. As far as can be judged from the reported experiments this factor is different from any of the previously described myofibrillar components.     

The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1. The intrinsic Na(+), K(+), Mg(2+) and Ca(2+) contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na(+) from 90+/-20 to 24+/-12, the bound K(+) from 27+/-3 to 7+/-2, the bound Mg(2+) from 20+/-2 to 3+/-1 and the bound calcium from 8+/-1 to <1nmol/mg of protein. 3. The activities of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase and the Na(+)-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5mum (ATP/protein ratio 12.5pmol/mug). 4. The Na(+)-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5mum-magnesium chloride and 2mum-potassium chloride. Addition of 2.5mum-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na(+)-dependent ATP hydrolysis was partly restored with 2.5mum-magnesium chloride; addition of K(+) in the range 2-10mum-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0 degrees C with 0.5nmol of K(+)/mg of protein so that the final added K(+) in the reaction mixture was 0.1mum restored the Na(+)-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [(42)K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K(+)/mg of protein was linear over a period of 20min and was inhibited by Na(+). Half-maximal inhibition of (42)K(+)-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na(+)-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K(+) and Mg(2+) of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K(+) from a solution of 0.5mum-potassium chloride.     

Cytochemical studies on the localization of alkaline phosphatase and HCO-3-activated adenosine triphosphatase in the brush border membrane of rat duodenal enterocytes

Cytochemical techniques were used to demonstrate, with appropriate controls, alkaline phosphatase and HCO-3-activated adenosine triphosphatase (ATPase) in rat duodenal brush border microvillus membranes. Intense activity of ecto-alkaline phosphatase activity was demonstrated with 2-glycerophosphate as substrate. Although biochemical assays suggested that L-phenylalanine inhibited both alkaline phosphatase and HCO-3-activated ATPase, cytochemical studies indicated that there was marked inhibition of alkaline phosphatase revealing a specific HCO-3-activated ATPase on the inner aspect of the microvillus membrane. While it is tempting to suggest that this HCO-3-activated ATPase is implicated in active bicarbonate secretion by the duodenum, decisive identification is not yet possible.     

Comparison of some minor activities accompanying a preparation of sodium-plus-potassium ion-stimulated adenosine triphosphatase from pig brain

1. An ATPase (adenosine triphosphatase) preparation obtained from pig brain microsomes by treatment with sodium iodide showed four apparently different ouabain-sensitive activities under various conditions. They were (a) ouabain-sensitive Mg(2+)-stimulated ATPase, (b) K(+)-stimulated ATPase, (c) (Na(+),K(+))-stimulated ATPase and (d) Na(+)-stimulated ATPase activities. 2. These activities showed the same substrate specificity, ATP being preferentially hydrolysed and CTP slightly. AMP was not hydrolysed. 3. These activities were inhibited by low concentration of ouabain. The concentration producing 50% inhibition was 0.1mum for ouabain-sensitive Mg(2+)-stimulated ATPase, 0.2mum for K(+)-stimulated ATPase, 0.1mum for (Na(+),K(+))-stimulated ATPase and 0.003mum for Na(+)-stimulated ATPase activity. 4. The ouabain-sensitive ATPase activities were inactivated by N-ethylmaleimide but the insensitive ATPase activity was not. 5. The three ouabain-sensitive ATPase activities were inhibited about 50% by 1mm-Ca(2+), whereas the ouabain-sensitive Mg(2+)-stimulated ATPase activity was activated by the same concentration of Ca(2+). The preparation was treated with ultrasonics at 20kcyc./sec. The 2min. ultrasonic treatment inactivated the ATPase activities by 50%. 7. The temperature coefficient Q(10) was 6.6 for K(+)-stimulated ATPase activity, 3.7 for (Na(+),K(+))-stimulated ATPase and 2.6 for Na(+)-stimulated ATPase. 8. Organic solvents inactivated the ATPase activities, to which treatment the K(+)-stimulated ATPase was the most resistant. 9. The phosphorylation of the enzyme preparation became less dependent on Na(+) with decreasing pH. This Na(+)-independent phosphorylation at low pH was sensitive to K(+) and hydroxylamine as well as the Na(+)-dependent phosphorylation at neutral pH.     

Influence of temperature acclimatization on the ionic activation of goldfish intestinal adenosine triphosphatase

1. An adenosine triphosphatase membrane system, dependent on Mg²⁺ and activated further by Na⁺+K⁺, was prepared from goldfish anterior intestine by differential centrifugation of homogenized intestinal scrapings. 2. The affinity of this preparation for Na⁺ in the presence of K⁺+Mg²⁺, for K⁺ in the presence of Na⁺+Mg²⁺ and for Mg²⁺ alone, measured at 37°, did not depend on the previous environmental temperature of the fish. When Na⁺+K⁺ were added to preparations from 8°-acclimatized fish the affinity for Mg²⁺ increased; this was not seen with preparations from 30°-acclimatized fish. 3. Part of the Mg²⁺-activated adenosine triphosphatase was inhibited by Na⁺ and the amount of inhibition appeared to increase at high acclimatization temperatures. 4. This Na⁺-inhibited adenosine triphosphatase was separated from the (Na⁺+K⁺)-activated enzyme by centrifugation on sucrose density gradients. 5. Preparations from 8°-acclimatized fish contained less Mg²⁺-activated and more (Na⁺+K⁺)-activated adenosine triphosphatase than did similar fractions from 30°-acclimatized fish. 6. Acclimatization to different environmental temperatures might involve one form of adenosine triphosphatase changing to another. The origin of various membranes seen in microsomal fractions must, however, be established before this hypothesis can be tested further.     

Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.     

Differentiation of the changes in alkaline phosphatase from calcium ion-activated adenosine triphosphatase activities associated with increased calcium absorption in chick intestine.

The pattern of response of the intestinal enzymes Ca2+-activated adenosine triphosphatase and alkaline phosphatase in the chick to 1,25-dihydroxycholecalciferol is consistent with a role for the former but not the latter enzyme in the vitamin D-dependent absorption of calcium.     

Chemical modification of a cerebral sodium-plus-potassium ion-stimulated adenosine triphosphatase preparation

1. A particulate Na(+)+K(+)-stimulated adenosine triphosphatase preparation obtained by treatment of bovine cerebral microsomes with a sodium iodide reagent has been further treated with acid anhydrides likely to convert amino groups into acidic derivatives. 2. The extent of acylation of amino groups was determined by reaction of the remaining amino groups with 2,4,6-trinitrobenzenesulphonic acid. The unmodified preparation contains about 1.2 muequiv. of amino groups/mg of protein of which only about 0.5 muequiv. are accounted for by protein amino groups. Kinetics of the trinitrobenzenesulphonic acid reaction with the unmodified preparation are complex and are altered by ATP or ouabain. 3. The compounds examined cause loss of Na(+)+K(+)-stimulated adenosine triphosphatase activity when relatively few amino groups are modified but ATP was found to afford partial protection against inactivation by methylmaleic anhydride. Na(+)+K(+)-stimulated adenosine triphosphatase activity is partly restored to the dimethylmaleylated preparation by hydrolysis of the dimethylmaleyl-amide bonds but not if more than about 20% of the amino groups have been acylated. 4. Supernatants obtained by high-speed centrifugation of the dimethylmaleylated preparation contained up to 45% of the total protein with less than 10% of the total phospholipid. Methylmaleyl and benzenetricarboxylyl derivatives of the enzyme preparation behaved similarly but tetrafluorosuccinylated material was almost entirely deposited by centrifugation.     

Fractionation of liver plasma membranes prepared by zonal centrifugation

1. Plasma membranes were isolated from crude nuclear sediments from mouse and rat liver by a rate-dependent centrifugation through a sucrose density gradient contained in the ;A' type zonal rotor. 2. The membranes were further purified by isopycnic centrifugation, and characterized enzymically, chemically and morphologically. 3. When the plasma-membrane fraction of sucrose density 1.17g/cm(3) was dispersed in a tight-fitting homogenizer, two subfractions of densities 1.12 and 1.18 were obtained by isopycnic centrifugation. 4. The light subfraction contained 5'-nucleotidase, nucleoside diphosphatase, leucine naphthylamidase and Mg(2+)-stimulated adenosine triphosphatase activities at higher specific activities than unfractionated membranes. The heavy subfraction was deficient in the above enzymes but contained higher Na(+)+K(+)-stimulated adenosine triphosphatase activity. 5. The light subfraction contained twice as much phospholipid and cholesterol, and three times as much N-acetylneuraminic acid relative to unit protein weight as the heavy subfraction. Polyacrylamide-gel electrophoresis indicated differences in protein composition. 6. Electron microscopy showed the light subfraction to be vesicular. The heavy subfraction contained membrane strips with junctional complexes in addition to vesicles.