Osmotic effectors in kidneys of xeric and mesic rodents: corticomedullary distributions and changes with water availability

Summary Urea, sodium, the methylamines glycine betaine and glycerophosphorylcholine (GPC), and the polyols sorbitol and myo-inositol are reported to be the major osmolytes in kidneys of laboratory mammals. These were measured (millimoles per kilogram wet weight) in kidney regions and urines of three species of wild rodents with different dehydration tolerances: the pocket mousePerognathus parvus (xeric), voleMicrotus montanus (mesic), and deer mousePeromyscus m. gambeli (intermediate). In animals kept without water for 4–6 days, sodium, urea, betaine and GPC+choline were found in gradients increasing from cortex to outer to inner medulla in all species, withPerognathus having the highest levels. Sorbitol was high in the inner medulla but low in the cortex and outer medulla; inositol was highest in the outer medulla. Totals of methylamines and methylamines plus polyols in the medulla showed high linear correlations (positive) with urea and with sodium values.Whole medullae were analyzed at several time points inMicrotus andPeromyscus subject to water diuresis followed by antidiuresis. In 102 h diuresis inMicrotus, all osmolytes decreased except inositol; however, only urea, sodium and sorbitol reached new steady states within 24 h. Urea returned to initial values in 18 h antidiuresis, while other osmolytes required up to 90 h. InPeromyscus, all osmolytes except the polyols declined in diuresis (max. 78 h test period). During antidiuresis, urea and GPC+choline rose to initial values in 18 h, with sodium and betaine requiring more time. In plots of both species combined, total methylamines+polyols correlated linearly (positive) with sodium, and GPC+choline with urea.Estimates of tissue concentrations suggest that total methylamines+polyols can account for intracellular osmotic balance in all species in antidiuresis and that sufficient concentrations of methylamines may be present to counteract perturbing effects of urea on proteins.Abbrevations GPC Glycero-3-phosphorylcholine - TCA trichloroacetic acid - M+P methylamines plus polyols     

Effect of crude extract of Stevia rebaudiana on renal water and electrolytes excretion.

To evaluate the effect of crude extract of Stevia rebaudiana on renal water, Na+ and K+ excretion, male Wistar rats (250-350 g each) under antidiuresis or water diuresis conditions, were evaluated. During intravenous infusion of the extract (0.05 mg/min/100 g) no significant differences were detected in mean arterial pressure or renal hemodynamics parameters. In contrast, fractional water and sodium excretion and solute clearance increased significantly, in both groups of animals. In antidiuresis rats the extract significantly increased reabsorption of water by the collecting duct and in water diuresis animals the extract significantly increased free water clearance. The data suggest preferential action of the extract in the proximal tubular cells involved with salt transport mechanism.     

Renal mechanisms involved in stress-induced antinatriuresis and antidiuresis in rats

The present study was conducted to investigate if changes in sodium and water excretion in stressed animals were due to modifications in the glomerular filtration rate (GFR) and to determine the participation of angiotensin II (Ang II) and alpha and beta-adrenoceptors on sodium and water renal excretion in rats subjected to immobilization stress (IMO). Male Wistar rats (250-300 g) were randomly separated into five different groups and vehicle (0.9% NaCl) via intraperitoneal (i.p.) or propanolol (3 mg/kg i.p.) or captopril (6 mg/kg i.p.) or yohimbine (3 mg/kg i.p.) or prazosin (1 mg/kg i.p.) were injected respectively. During experimental measurements, the animals were kept in metabolic cages for 6 h and sodium, potassium and water renal excretion and saline (1.5% NaCl) and water intake were determined at day 1 (drug effect) and day 7 (drug + IMO effects). GFR was measured by creatinine clearance in control and IMO rats. A stress-induced antinatriuresis and antidiuresis was reversed by alpha 1 and alpha 2-adrenoceptor antagonists, while captopril inhibited only the antidiuresis and propranolol had no effect on either parameter. No differences were observed in creatinine clearance in the studied groups. Since yohimbine blocks alpha 2-adrenoceptors and prazosin blocks alpha 1-adrenoceptors and alpha 2B-adrenoceptors, the stress-induced renal sodium reabsorption mainly could be attributed to alpha 2B-adrenoceptors. The present results indicate that beta-adrenoceptors do not participate in this response and, Ang II only reverses the antidiuresis and shows a slight participation in antinatriuresis. The increment in sodium and water reabsorption caused by IMO occurred without changes in the glomerular filtration rate.     

Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte.

1. Six rat liver plasma-membrane subfractions of different density and morphological, enzymic and chemical properties were prepared from homogenates by a combination of differential, rate-zonal and density-gradient centrifugation. They consisted of three vesicular 'light' subfractions of density 1.12-1.13 and three 'heavy' subfractions of density 1.16-1.18 containing membrane strips and intercellular junctions. 2. All six subfractions contained a basal adenylate cyclase activity. One of the 'light' subfractions that showed the highest glucagon-stimulated adenylate cyclase activity was identified as deriving form the blood-sinusoidal face of the hepatocyte. This subfraction, unlike the others, was contaminated by Golgi components, as indicated by its morphological properties and the presence of galactosyl- and sialyl-transferase activities. 3. All the six subfractions showed high activities of the following plasma-membrane marker enzymes: 5'-nucleotidase, alkaline phosphodiesterase (nucleotide pyrophosphatase), alkaline phosphatase, leucine naphthylamidase and Mg2+-activated adenosine triphosphatase. A 'light' subfraction that showed the highest specific activities of all the above marker enzymes, but lacked a glucagon-stimulated adenylate cyclase activity, was identified as deriving from the bile-canalicular face of the hepatocyte. 4. The 'heavy' subfractions, which showed generally the lowest activities of the above plasma-membrane enzyme markers, and were characterized by the presence of desmosomes and gap junctions, were taken to originate from the contiguous faces of the hepatocyte. 5. The protein composition of the six subfractions was generally similar, as shown by polyacrylamide-gel electrophoresis. Differences in the amounts of various protein and glycoprotein bands among the subfractions correlated with their morphology, enzymic composition and sialic acid content. 6. Hormonal and histochemical evidence supporting the identification of a bile-canalicular subfraction, a blood-sinusoidal subfraction and contiguous-face subfractions is discussed.     

UDP-galactose-ceramide galactosyltransferase in rat brain myelin subfractions during development.

The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70--75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2--5% of the protein was recovered in the light-myelin fraction, and about 7--12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3--4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.     

Kinetic studies of D-glucose transport in renal brush-border membrane vesicles of streptozotocin-induced diabetic rats

Renal brush-border membrane vesicles prepared from streptozotocin-induced 4-day-diabetic rats possessed a Na+-dependent D-glucose transport system that exhibited apparent Kt and Vmax values about 2-fold greater than normal. Apparently, hyperglycemia and probably other stimuli cause the induction and membrane incorporation of a low-affinity transporter in these membranes; this increased sugar-transport capacity is retained for at least 4 weeks so long as the animals maintained or increased their body weight. Membranes prepared from 28-day-diabetic, severely ill ketoacidotic animals lose this enhanced transport ability and the decrease in Vmax was found to correlate directly with the weight loss. Furthermore, the transporter in brush-border membranes prepared from these cachectic animals had an even lower affinity for glucose than those from the acute hyperglycemic animals. That these changes in the diabetic animals represent major alterations in renal brush-border membrane construction is further supported by our observation that the specific activity of the marker enzymes, alkaline phosphatase and neutral alpha-glucosidase, are profoundly increased and decreased, respectively, in this condition.     

Comparison of cholinergic systems in Ep neocortical regions of cats with high and low cognitive ability

Cats were behaviorally tested for the ability to solve the abstraction and generalization tasks. Fractions of light (C) and heavy (D) synaptosomes of the associative temporal (Ep) areas were prepared, and subfractions of synaptic membranes and synaptoplasm were isolated. Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity and the content of protein and protein sulphydric (SH-) groups were measured in synaptic subfractions. All the studied characteristics were lower in subfractions C of cats with higher cognitive abilities. In subfractions D, the ChAT activity was correlated neither with ChAT activity in the respective C fraction, nor with cognitive abilities of cats. It is suggested that cholinergic terminals originating from neurons of the basal magnocellular nuclei are concentrated in the C fractions, and those from the cortical cholinergic neurons are concentrated in the D fractions. Physiological significance of the "deficiency" of cholinergic inputs of the Ep areas from the basal magnocellular nuclei in animals with higher cognitive abilities is discussed.     

Predominant osmotically active organic solutes in rat and rabbit renal medullas

The mechanism that concentrates the urine to an osmolality several times that of systemic plasma results in high concentrations of solutes (particularly NaCl and urea) in extracellular fluid of renal medulla, but not in the labyrinth of the renal cortex. Intracellular and extracellular osmolality must be equal in animals, but the known intracellular levels of Na and K salts and urea in renal medullas are much too low to balance the high extracellular osmolality. The purpose of these studies was to identify the other intracellular osmolytes that must be present. Cortexes and medullas from rabbit and rat kidneys were analyzed by proton nuclear magnetic resonance, mass spectrometry, and chemical assays to determine the identity and amount of organic solutes. Large amounts of glycerophosphorylcholine, betaine, sorbitol, and inositol were found in both species localized almost exclusively to the inner medulla. In rabbits during antidiuresis glycerophosphorylcholine, betaine, and sorbitol were present in the inner medulla, at concentrations of 21.1, 34.8, and 20.8 mumol/g wet weight, respectively, but were not detected in the cortex. Inositol was present in rabbit inner medulla at 10.7 mumol/g wet weight and was also present in the cortex, but at lower concentration. None of the above metabolites was present in measurable amounts in urine or peripheral plasma. The accumulation in the cells of the inner medulla of relatively large amounts of betaine, sorbitol, glycerophosphorylcholine and inositol during antidiuresis suggests that they may play a significant role in the maintenance of intracellular osmotic balance.     

Antidiuretic action of prolactin in the rat with diabetes insipidus.

Rats with hereditary hypothalamic diabetes insipidus, devoid of endogenous ADH, exhibited a prompt antidiuresis when injected subcutaneously or intraarterially with ovine prolactin. The antidiuresis was accompanied by a decrease in free water clearance and an increase in urine osmolality without a change in osmolal clearance or creatinine excretion. Measurement of PAH and insulin clearances indicated that prolactin had no effect on renal plasma flow or glomerular filtration rate. Prolactin injection caused a transient decrease in urinary sodium excretion, but proximal tubular sodium reabsorption, estimated by lissamine green transit time, was unaffected. The antidiuretic effect of prolactin could not be attributed to ADH contamination of the ovine prolactin preparation. Kidney cyclic AMP content was increased significantly 5 min after injection of prolactin. Thus, prolactin has an antidiuretic effect similar to that which occurs as a result of ADH action on the kidney and does not require either the release or the presence of ADH in order to cause the antidiuresis. Further, the impaired water excretion cannot be attributed to an increase in proximal tubular sodium reabsorption or to alteration of renal hemodynamics. It is suggested that prolactin has a direct ADH-like action on the kidney resulting in antidiuresis.     

Biosynthesis of glycerolipids by hepatoma and liver microsomes. I. Fatty acyl-CoA ligase and acyl-CoA:sn-glycerol-3-phosphate acyltransferase

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on ouabain-Sepharose. By the method used two subfractions were obtained, one eluting freely from the affinity gel (MF1oua) and a second specifically retained by matrix-bound ouabain (MF2oua), with a total recovery of 95 per cent. Fractionation required the binding of matrix-bound ouabain to its plasma membrane receptor, i.e. (Na+ + K+)-ATPase. Increasing the temperature and binding time did not significantly alter the fractionation of plasma membranes into the two subfractions. Both plasma membrane subfractions separated by ouabain-Sepharose were of plasma membrane origin, as revealed by the identical specific activities of several membrane bound enzymes, gamma-glutamyl transpeptidase, alkaline phosphatase and Mg2+-ATPase in unseparated plasma membranes and in both subfractions, and by the identical amounts of the cytoskeletal protein actin in unseparated plasma membranes and subfractions. The plasma membrane subfractions MF1oua and MF2oua showed different structural and functional properties. In SDS-polyacrylamide gel electrophoresis polypeptides of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2oua. The phospholipid fatty acid composition of the plasma membrane subfractions proved to be different, as well. MF2oua contained significantly higher amounts of saturated fatty acids as compared to MF1oua. The specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysolecithin acyltransferase were highly enriched in the adherent fraction MF2oua, as compared to MF1oua. The data suggest that by the means of affinity chromatography on ouabain-Sepharose plasma membrane domains of the lymphocyte plasma membrane can be isolated, most probably implicated in the initiation of lymphocyte activation.     

Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins

HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside G_M1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A–C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein populations associated with detergent-resistant membranes, and their potential interactions in cell signaling.     

Synergistic effects of nitric oxide and prostaglandins on renal escape from vasopressin-induced antidiuresis

Recent results from our laboratories indicate that renal escape from AVP-induced antidiuresis is accompanied by marked downregulation of kidney aquaporin-2 (AQP2) and AVP V2 receptors. The present studies evaluated the effect of nitric oxide (NO) and PG synthesis blockade on escape from antidiuresis. dDAVP-infused rats were water loaded (WL) for 5 days. l-NAME, an NO synthesis inhibitor, or diclofenac, a cyclooxygenase inhibitor, was infused subcutaneously beginning 1 day before WL. As early as 2 days after WL, urine volume increased and urine osmolality decreased, indicating the onset of escape. Endogenous NO synthesis, measured as urinary NO2 + NO3 excretion, was significantly increased in the WL group compared with the non-WL controls during all 5 days of WL. l-NAME (20 mg. kg(-1). day(-1)) markedly decreased urine volume on days 4 and 5 of WL, indicating inhibition of the escape phenomenon. Kidney AQP2 protein was significantly increased by this dose of l-NAME as well. A lower dose of l-NAME (10 mg. kg(-1). day(-1)) or diclofenac (2.5 mg. kg(-1). day(-1)) did not significantly affect the escape phenomenon by itself, but the combination of l-NAME and diclofenac showed a marked inhibitory effect on the escape phenomenon, which was also accompanied by a significant increase in kidney AQP2 expression. These results therefore suggest that renal NO and PG both play important roles in escape from AVP-induced antidiuresis by acting synergistically to downregulate kidney AQP2 expression.     

Rat liver mitochondrial contact sites and carnitine palmitoyltransferase-I

In hepatic mitochondria, the outer membrane enzyme, carnitine palmitoyltransferase-I (CPT-I), appears to colocalize with contact sites. We have prepared contact sites that are essentially devoid of noncontact site membranes. The contact site fraction has a high specific activity for CPT-I and contains a protein at 88 kDa that is recognized by antibodies directed at two different peptide epitopes on CPT-I. Similarly long-chain acyl-CoA synthetase (LCAS) specific activity is high in this fraction; a protein at 79 kDa is recognized by an antibody against LCAS. Although activity of carnitine palmitoyltransferase-II (CPT-II) is present, it is not enriched in the contact site fraction, and a protein of 68 kDa weakly reacted with anti-CPT-II antibody. Likewise, carnitine-acylcarnitine translocase (CACT) protein is present, but at a somewhat reduced level. Using an analytical continuous sucrose gradient, we demonstrate that the activities of CPT-I and LCAS and their associated immunoreactive proteins are present in a constant amount throughout the contact site subfractions. The enzymatic activity of CPT-II and its associated immunoreactive protein, as well as immunoreactive CACT, is absent in the lighter density gradient subfractions and is present in the higher density subfractions only in trace amounts. This heterogeneity of the contact site fraction is due to unvarying amounts of outer membrane and increasing amounts of attached inner membrane with increasing density of the subfractions.     

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose

Purified plasma membranes of mouse EL4 lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including gamma-glutamyl transpeptidase, 5'-nucleotidase and Mg2+-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL4 lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.     

Formation of diacyl- and alkylacylphosphatidylcholine by the membranes of human platelets

We have investigated the distribution and fatty acid preference of two acyl-CoA transferase activities in a human platelet mixed membrane fraction and in well-characterised surface and intracellular membrane subfractions prepared from it by high-voltage free-flow electrophoresis. One transferase inserts long-chain unsaturated fatty acids into 1-acyllysophosphatidylcholine (1-acyl-LPC) and the other into lyso-platelet-activating factor (LPAF). Both transferase activities were approx. 4-fold enriched in the intracellular membranes with respect to their specific activities in the mixed membranes. The surface membrane activities were correspondingly depleted. Using 1-acyl-LPC as the acceptor, all the intracellular membrane preparations showed transferase preference for the CoA ester of 8,11,14-eicosatrienoic acid. In contrast when LPAF was the acceptor the CoA esters of linoleic and arachidonic acid were the preferred donors.     

Diuretic and natriuretic effect of a brain soluble fraction that inhibits neuronal Na+,K(+)-ATPase

The separation by Sephadex G-50 of two subfractions, peak I and II, from the brain soluble fraction has been previously described. These fractions were able to stimulate and inhibit synaptosomal membrane Na+,K(+)-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gómez de Lima, Neurochem. Res. 11, 933-948, 1986). Experimental evidence indicates that the alteration of Na+,K(+)-ATPase activity may result in changes of renal and cardiovascular parameters. In the present study, we have analyzed the effect of peak I and II fractions prepared from rat cerebral cortex on water and sodium excretion and on heart rate and arterial pressure in normotensive anesthetized rats. It was observed that water and sodium excretion were not modified by the administration of peak I fraction but that they were increased by peak II fraction. The cardiovascular parameters were not significantly modified by either of the fractions. The results indicate that brain soluble factor (s) which is (are) present in peak II fraction may modify some aspects of renal physiology after systemic administration.     

Preparation of plasma-membrane subfractions from isolated rat hepatocytes.

1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two 'light' vesicular and one 'heavy' junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed.     

Restoration by T-activin and its subfractions of splenocyte membrane structures in thymectomized animals

It was shown that thymectomy induces the injury of plasma membranes of mouse splenocytes. This membrane disorders completely inhibit T-cell response on Concanavalin. A. The incubation of splenocytes with Tactivin or injection of Tactivin or its subfractions to the animals restore the membrane structure and T-cell response.     

LOCALIZATION OF THE ENZYMES OF KETOGENESIS IN RAT LIVER MITOCHONDRIA

The localization of the enzymes of ketogenesis in isolated rat liver mitochondria has been investigated. Mitochondrial subfractions were isolated after disruption of this subcellular organelle by (a) hypotonic lysis in water, which permitted the ultracentrifugal separation of the soluble and membranous compartments of the mitochondrion, or by (b) a procedure involving swelling, contraction, and ultrasonic treatment, which permitted the isolation from discontinuous sucrose gradients of subfractions rich in intermembrane space protein, outer membrane, and inner membrane-matrix particles. Two membrane subfractions were invariably present as distinct bands at the lower interface of the discontinuous gradient. The upper of these two bands was found to be a highly purified preparation of outer mitochondrial membrane. Subfractions rich in matrix and in inner membrane were isolated from inner membrane-matrix particles after hypotonic treatment. The content of the various mitochondrial compartments in all subfractions was assessed from their enzymic and electron microscopic characteristics. The ketogenic activity of each subfraction was determined by measuring its capacity to form ketone bodies from acetyl CoA. The activity of this process was markedly enhanced by dithiothreitol. These measurements of ketone body formation, together with assays of individual enzymes of the ketogenic pathway, show that thiolase, HMGCoA synthase, and HMGCoA cleavage enzyme are localized in the matrix of the inner membrane-matrix particles. The rates of ketone body formation indicate that the HMGCoA synthase is the rate-limiting enzyme of the pathway in subfractions of high matrix content. Studies with sodium chloride indicate that a large portion of the HMGCoA synthase, which remains present in membrane subfractions derived from water-treated mitochondria, is bound by ionic interaction to component(s) of the membrane.