Antibodies directed against transducin beta subunits interfere with the regulation of adenylate cyclase activity in brain membranes.

In an attempt to study the mechanisms of action of membrane-bound adenylate cyclase, we have applied to rat brain synaptosomal membranes antibodies raised against purified bovine transducin (T) beta gamma subunits. The antibodies recognized one 36-kDa protein in Western blots of the membranes. Adenylate cyclase activation by GTP non-hydrolyzable analogues was greatly decreased in immune, as compared to preimmune, antibody-treated membranes, whereas the enzyme basal activity was unaffected by both types of antibodies. The inhibition of forskolin-stimulated adenylate cyclase by guanine 5'-(beta, gamma-imino)triphosphate (Gpp-(NH)p) was decreased in membranes preincubated with immune, but not preimmune, antibodies. Anti-T beta antibodies moderately decreased the extent of subsequent adenylate cyclase activation by forskolin, while not affecting activation by Al3+/F-. The enzyme activation by Gpp(NH)p in untreated membranes remained the same upon further incubation in the presence of either type of antibodies. Such results were consistent with the decreased exchange of guanine nucleotides which occurred in membrane treated with immune, but not preimmune antibodies, upon addition of GTP. The blockade of the regulation of adenylate cyclase by Gpp(NH)p observed in membranes pretreated by anti-T beta antibodies thus appears to be caused by the impairment of the guanine nucleotide exchange occurring on Gs alpha subunits. The G beta subunits in the adenylate cyclase complex seem to be instrumental in the guanine nucleotide exchange on G alpha subunits, just as T beta subunits are in the transducin complex.

Structure of brain adenylate cyclase: proteolysis-dependent modifications.

The associations of the components of eucaryotic adenylate cyclase are still poorly characterized. Enzyme activity is, however, thought to depend upon subunit conformations and states of association. Estimates of adenylate cyclase sizes corresponding to given levels of activity may thus give clues as to how the enzyme functions. Studying the rat brain enzyme, we found that samples protected from proteolysis throughout the fractionation procedure yielded, upon Lubrol solubilization, a soluble protein complex of 9.1S sedimentation coefficient and 11.5-nm Stokes radius. These values are much larger than those previously reported. The soluble enzyme specific activity, but not its size, was dependent upon the various effectors preincubated with the membranes. Proteolysis is known to first activate and then decrease adenylate cyclase activity. Proteolysis of the brain samples, whether due to trypsin or to endogeneous proteases, decreased the adenylate cyclase s value, Stokes radius, and specific activity altogether. The magnitude of the shifts depended upon the nature of the enzyme effector preincubated with the membranes. We recently showed that some brain membrane proteins can be ADP-ribosylated by cholera toxin, concomitantly with adenylate cyclase activation [Berthillier, G., d'-Alayer, J., & Monneron, A. (1982) Biochem. Biophys. Res. Commun. 109, 297-304]. Trypsin treatment of such samples led to a quick degradation of the labeled polypeptides and especially of the Mr 47000 protein. This Lubrol soluble protein is likely to be the brain G/F stimulatory subunit.

[Pertussis toxin stimulates brain adenyl cyclase and induces ADP ribosylation of a 40,000 dalton membrane protein]. / La toxine pertussis stimule l'adényl-cyclase cérébrale et provoque l'ADP-ribosylation d'une protéine membranaire de 40 000 dalton.

The adenylate cyclase activity of Rat brain synaptosomal membranes is strongly activated by forskolin. This study demonstrates: 1. That GTP or its analogs, when added to forskolin, decrease the enzyme stimulation by a factor of 40 to 60%; 2. That Pertussis toxin (TP), in the presence of NAD, ADP-ribosylates a single membrane protein of 40,000 dalton apparent molecular weight in a strictly concomitant manner. This Lubrol-soluble protein has a sedimentation coefficient of 3.6 and a Stokes radius of 6 nm. An immune serum against TP blocks the NAD-glycohydrolase activity of the toxin.

[Recurrent paroxysmal myoglobinuria with acute renal insufficiency caused by muscular carnitine (palmitoyltransferase deficiency. A case]. / Myoglobinurie paroxystique récidivante avec insuffisance rénale aiguë secondaire à un déficit en carnitine palmityl transférase musculaire. Une observation.

A 45-year-old man was hospitalized on 3 occasions for recurrent myalgias with paroxysmal myoglobinuria resulting in two episodes of acute renal failure. The second episode was fatal: the patient died of shock and hyperkaliaemia during haemodialysis. The predominant signs and symptoms were muscle pain with functional deficit, signs of renal failure and a rise in serum enzymes. All examinations were negative between attacks. Muscle biopsies showed a major degree of myolysis, and biochemical tests demonstrated severe muscle palmityl transferase deficiency associated with partial deficiency of muscle carnitine. The diagnostic features and physiopathology of the disease are reviewed.

Carnitine metabolism in early stages of Duchenne muscular dystrophy.

Muscle carnitine deficiency was found in 12 children affected with Duchenne muscular dystrophy (DMD), the diagnosis being made at a preclinical stage or at the beginning of the clinical symptoms. Enzymatic activities related to fatty acid transport and carnitine metabolism were studied in these patients and normal subjects: palmitoyl carnitine transferase was increased, palmitoyl carnitine hydrolase was not found in the muscle, palmitoyl coenzyme A synthetase was normal and palmitoyl coenzyme A hydrolase was increased.

Evidence of the mannosylation of a non-histone protein in monkey liver chromatin.

Mannose is incorporated in monkey liver chromatin by the means of a nuclear membrane mannosyltransferase. 14C-labelled chromatin is dissociated either by sulfuric acid or 6 M urea and 0.4 M GuCl. The fractions then enriched in non-histone 14C-labelled proteins are excluded from Ultro-gel AcA 202, their analysis in SDS-polyacrylamide gel electrophoresis shows that radioactivity fits with one major protein band, confirming the presence of at least a non-histone protein labelled with mannose in monkey liver chromatin, with an apparent molecular weight of 13,000.

[The presence of glycosyltransferases on chromatin acceptors in monkey liver nuclear membranes (author's transl)]. / Présence de glycosyltransferases à accepteurs chromatiniens dans les membranes nucléaires d'hépatocytes de singe.

Nuclei were prepared from monkey hepatocytes by centrifugation of the homogenate on a cushion of 2.3 M sucrose, during 45 min at 100000 X g. The yield was 2.2 x 10(7) nuclei per g of liver, and 70% of te homogenate DNA was recovered in these nuclei. An electron microscopic study as well as a biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. A mannosyltransferase and an N-acetylglucosaminyltransferase, working on endogenous glycoproteic acceptors, are present in the nuclei for 1.4 and 6.5% of the homogenate activities, respectively. The nuclei are hydrolysed by DNAse I. The suspension, adjusted in 1.9 M sucrose, was centrifuged for 2 h at 100000 X g, under buffer layer. Purified nuclear membranes were collected at the interface. These membranes did not contain any more endoplasmic reticulum enzyme activities, but the mannosyl and N-acetylglucosaminyltransferase activities were still present. They essentially work on an exogenous chromatin acceptor, prepared by lysis of the nuclei. The eventual role of these glycosyltransferases in the glycosylation of non-histone proteins is discussed.

Carnitine levels in normal children and adults and in patients with diseases muscle.

An assay for evaluating carnitine levels in normal children and adults is described. After a 12-hour period of fasting, individual variations in 24-hour urinary excretion of carnitine were observed in adults. In children, there was a significant decrease in excretion from the 10th month to the third year, and then an increase until the 10th year. There was no significant difference between children and adults in the serum and skeletal muscle levels. Muscle carnitine levels were also studied in 12 cases of lipid-storage myopathy and in cases of other muscle diseases, including Duchenne muscular dystrophy in children.

[Primary cardiomyopathy in children with lipid infiltration of the myocardium and skeletal muscles and demonstration of a palmityl-carnitine transferase deficiency. Apropos of 4 cases]. / Myocardiopathie primitive de l'enfant avec surcharge lipidique des fibres myocardiques et musculaires et mise en évidence d'un déficit en enzyme palmityl-carnitine-transférase. A propos de 4 observations.

The authors report four cases of metabolic cardiomyopathy with lipid infiltration diagnosed by skeletal muscle and myocardial biopsy in children with no clinical signs of muscular dystrophy. Normal or increased serum and urinary carnitine levels excluded a primary carnitine deficiency. A deficiency in muscular-palmityl-carnitine-transferase was demonstrated. This pathogenic mechanism may be an indication for treatment with carnitine, but the results are less spectacular than in primary carnitine deficiency states.

[Presence of UTP:D-GLUCOSE-1-phosphate uridylyltransferase in liver microsomes of the eel (Conger vulgaris)]. / Présence d'une UTP:D-glucose-1-phosphate uridylyltransférase dans les microsomes du foie de congre (Conger vulgaris).

An activity UTP : D-glucose-1-phosphate uridylyltransferase is located in the microsomal membranes of conger liver. The properties of this enzyme are studied and compared to the soluble activity. The microsomal activity is partially liberated from the membrane by freezing and thawing and by the means of a neutral detergent, Triton X-100. The enzyme is latent in the membranes and totally inhibited by phospholipase A2. This microsomal enzyme could be the last of a membranous biosynthetic pathway for UDP-glucose, as conger liver microsomes contain also a membranous glucokinase and a membranous phosphoglucomutase.

[Presence of enzymes catalyzing UDP-glucose biosynthesis in a low density Golgi fractions of cat hepatocytes]. / Présence des enzymes catalysant la biosynthèse d'UDP glucose dans une fraction légère de l'appareil de Golgi des hépatocytes de chat.

A Golgi-rich fraction is prepared from cat hepatocytes by the means of a four-step sucrose density gradient. The material applied to this gradient is composed either of smooth microsomes prepared from healthy animals, or of total microsomes prepared from cat treated by 50 per cent ethanol (0.6 g/100 g body weight, administered by stomach tube). A light fraction (d : 1.10) is obtained by the two procedures. It does not show any glucose-6-phosphatase activity, but is enriched in sialyltransferase, known as a marker enzyme for Golgi apparatus. It also contains the three enzymes implicated in the biosynthetic pathway for UDP-glucose (glucokinase, phosphoglucomutase and UTP : glucose-1-phosphate uridylyltransferase). UDP-glucose being the ultimate substrate in membranous glucosylation reactions, these results could support the hypothesis that sugar-nucleotides necessary for the glycoprotein biosynthesis are produced in the Golgi vesicles directly.

[Glycogen synthase activity in parodontal disease (author's transl)]. / Activite glycogene-synthase dans la maladie paradontale.

A biochemical study of an enzyme participating in the synthesis of glycogen is presented, with particular regard to the fluctuations in the amounts of this polysaccharide in human gingival epithelium, during inflammation. The increase in the activity of UDPglucose : glycogen glucosyltransferase can be related to the accumulation of glycogen. Some kinetic parameters of this enzyme are described.

[Presence of a pathway for biosynthesis of UDP-glucose in the Golgi apparatus of rat hepatocytes]. / Présence d'une voie de biosynthèse de l'UDP-glucose dans l'appareil de Golgi des hépatocytes de rat

Glucokinase, phosphoglucomutase and glucose-1-phosphate uridylyltransferase are the three enzymes involved in a microsomic pathway for the synthesis of UDP glucose. Evidence is given, in this paper, for the localization of these three enzymes in a Golgi-rich fraction of rat liver. This fraction is prepared, from smooth microsomes, by the means of a discontinuous four-step sucrose gradient. Three of the lighter fractions (d = 1.08-1.13) are enriched in the Golgi markers (galactosyltransferase, sialytransferase and thiamin pyrophosphatase), especially the one with density 1.13. The three enzymes we are interested in are enriched in the two upper hands (d 1.08-1.11), which display an activity for the biosynthesis of UDP-glucose from glucose equivalent to the one obtained in a crude microsomic preparation, and which are not contaminated by other subcellular components.

The topographical location and unique nature of a glucokinase associated with the Golgi apparatus of rat liver.

A particulate glucokinase was recovered in the Golgi-rich fraction of rat liver prepared by the method of Morré [Methods Enzymol. (1971) 22, 130-148], thus extending the demonstration by Berthillier et al. [Biochim. Biophys. Acta (1973), 293, 370-378] of particulate glucokinase activity in a microsomal subfraction that showed enrichment in Golgi characteristics. The purity of this fraction was examined and it was then subjected to several treatments, the action of Triton X-100, freezing and thawing, and sonication to establish the topographical location of the glucokinase activity thus solubilized. The evidence suggests that the glucokinase activity is either soluble in the lumen of the Golgi apparatus or loosely associated with the inside of the Golgi membranes.

[Perparation and comparative analytical study of cat liver microsomes]. / Préparation et étude analytique comparée des microsomes de foie de chat

Cat liver homogenates have been fractionated by differential centrifugation. Four particulate fractions (1 000 X g, 10 000 X g, and 145 000 X g) and a supernatant have been obtained. The biochemical composition of these fractions has been established from the assay and distribution pattern of 22 enzymatic and chemical constituents including marker enzymes for mitochondria, lysosomes, peroxisomes, plasma membranes, endoplasmic reticulum, Golgi apparatus and cell sap. The microsomal fraction was characterized by a moderate contamination with large cytoplasmic granules and by a low yield in protein and cholesterol. It contained 50 per cent of Golgi complex and about 40 per cent of plasma membranes. Morphological analysis of subcellular fractions was performed and confirmed biochemical results.

[Molecular weight of a membrane receptor of UDP-glucose]. / Poids moléculaire d'un récepteur membranaire d'udp-glucose

Rat liver microsomal membranes have been shown to contain an UDP-glucose binding protein. Its mol. wt was estimated to be 120 000 by gel filtration and by ultracentrifugation in a sucrose gradient. The receptor activity was purified by gel filtration on Sephadex G200 and analysed by gel-electrofocusing.