Molecular mechanisms of McArdle's disease (muscle glycogen phosphorylase deficiency). RNA and DNA analysis.

Lack of muscle glycogen phosphorylase activity leads to McArdle's disease, a rare metabolic myopathy. To investigate its molecular basis at the nucleic acid level, we isolated muscle phosphorylase cDNA clones from a human cDNA library in Escherichia coli plasmid pBR 322. Subcloning of one insertion of M13 bacteriophage permitted its definite identification by sequencing. Northern blot experiments revealed one specific messenger RNA of 3.4 kilobases found uniquely in tissues expressing muscle phosphorylase. We show that McArdle's disease exhibits a molecular heterogeneity at the messenger RNA level. In eight unrelated cases of McArdle's disease in which no inactive proteins had been detected, we assayed muscle biopsies for phosphorylase mRNA by Northern blotting. In five cases, no muscle phosphorylase mRNA could be detected, while in three other cases, normal length mRNA was present in lower amounts. Moreover, Southern blot analysis of DNA isolated from white blood cells in four McArdle patients revealed no major deletion or rearrangements of the phosphorylase gene as compared with controls.

Dietary and hormonal regulation of aldolase B gene expression.

Aldolase B is an enzyme of the glycolytic pathway whose activity and mRNA levels in the liver fluctuate according to dietary status. Both the enzyme activity and the mRNA concentration decline during fasting and increase four- to eightfold upon refeeding of a carbohydrate-rich diet. The mechanism, however, of the mRNA induction remains unknown. To elucidate the mechanisms that regulate this induction responsive to dietary stimuli, we have studied the roles of hormones and glycolytic substrates on aldolase B gene expression in three tissues that synthesize the enzyme. Using a cDNA probe complementary to rat aldolase B mRNA, we determined the amount of cytoplasmic RNAs in the liver, kidney, and small intestine of normal, adrenalectomized, thyroidectomized, diabetic, and glucagon- or cAMP-treated animals refed either a fructose-rich or a maltose-rich diet. The in vivo hormonal control of gene expression was found to be very different in the three organs tested. In the liver, cortisone and thyroid hormones were required for the induction of the specific mRNA by carbohydrates, while in the kidney none of the hormonal modifications tested altered the level of mRNA induction. In the liver, but not in the kidney, diabetes and glucagon administration abolished the induction of aldolase B mRNAs in animals refed the maltose-rich diets. In the small intestine, only diabetes and thyroidectomy affected the gene expression. Finally, no induction occurred when normal fasted rats were given any of the hormones. Thus, the in vivo hormonal control of liver aldolase B gene expression differs significantly from that of kidney and small intestine. In the liver, the mRNA induction requires the presence of dietary carbohydrates, of permissive hormones, and the cessation of glucagon release, while in the kidney, the induction of the mRNAs by fructose occurs regardless of the hormonal status of the animals. The hormonal control of aldolase B mRNA levels in the small intestine is intermediate.

Alpha-mannosidase in human red cells.

1. A search for lysosomal hydrolases and related enzymes has been made in hemolysates from human and rabbit red cells. Apart from acid phosphatases, significant activities were found only for alpha-mannosidase, neutral alpha-glucosidase and beta-hexosaminidase. 2. alpha-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) activity per cell in human red blood cells was 200-times lower than in white cells. The optimal pH was 5.5--6.0. Electrophoresis on cellulose acetate showed three bands. Hemolysates from four patients with mannosidosis were not deficient in alpha-mannosidase. pH activity curves and elctrophoretic pattern were similar to those of controls. From its biochemical and genetic properties, it is concluded that red cell mannosidase differs from the lysosomal acid mannosidase.

Hexosaminidase C in Tay-Sachs and Sandhoff disease.

1. Hexosaminidase C has been purified from human placenta. Complete separation from hexosaminidases A and B was achieved. 2. The following properties of hexosaminidase C differ from those of the A and B isozymes. Presence in the supernatant rather than the lysosomes, neutral pH optimum, higher molecular weight, lack of activity on beta-N-acetylgalactosamine derivatives, and lack of immunological relationship. 3. Hexosaminidase C is active in patients deficient in hexosaminidases A and B and can be recognized by its characteristic electrophoretic mobility. It is concluded that the genetic origin of hexosaminidase C is probably different from that of hexosaminidases A and B.

Molecular heterogeneity of rabbit heart phosphorylase kinase.

Phosphorylase kinase (ATP: phosphorylase-b phosphotransferase, EC 2.7.1.38) from rabbit heart, when submitted to electrophoresis on Pevikon, separates into two discrete peaks A and B. The two peaks have been analyzed using reelectrophoresis, chromatography on DEAE-cellulose, thermal stability, inactivation by EGTA (ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) and reaction with an anti-muscle phosphorylase kinase antiserum. It can be concluded that rabbit heart extracts contain two isozymes of phosphorylase kinase. The more negatively charged isozyme seems to be identical with the muscle enzyme. The other isozyme resembles the liver enzyme but differs from the major fraction of the latter by its charge. It is likely that there exist at least three molecular types of phosphorylase kinase.

Studies on the nature of different molecular forms of glucose-6-phosphate dehydrogenase purified from human leukocytes.

Several molecular forms of human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) corresponding to different stages of post-synthetic modifications have been purified from human leukocytes. The various enzyme forms were different in their specific activity, their kinetic properties and their isoelectrofusing pattern. The molecular weight of the subunits of the different forms was not modified. The changes in the electrofocusing pattern were not due to modifications of the N-terminal ends, the oxidation of thiol groups or the non-covalent fixation of an acid molecule upon the enzyme. Carboxypeptidase B cleaved a C-terminal lysine from the different enzyme forms and shifted the isoelectric point of the different enzyme active bands towards the acid pH. The different enzyme forms studied here seemed to result from the action upon 'native glucose-6-phosphate dehydrogenase' of 'modifying factors' especially abundant in some leukemic granulocytes. The modifying factors did not seem to be consumed during the 'modification' of glucose-6-phosphate dehydrogenase. Moreover, the storage for one year of unmodified enzyme resulted in changes in its electrofocusing pattern similar to those quickly induced by the 'modifying factors'. Consequently it appears that the modifying factors are catalysts of the modification of special residues of glucose-6-phosphate dehydrogenase. The hypothesis that this modification involves the deamination of asparagine or glutamine residues is put forward.

Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.

Calmodulin ligands. The interaction of muscle phosphorylase kinase with phosphodiesterase. Comparison of calmodulin ligands in muscle extracts from normal and phosphorylase kinase-deficient mice.

Interactions between phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38) and calmodulin were studied with pure preparations of muscle phosphorylase kinase, and with crude extracts from muscles of control (C57 Black) and deficient (ICR/IAn) mice, which lack muscle phosphorylase kinase activity. Calmodulin was determined by its ability to stimulate a calmodulin-dependent phosphodiesterase. The amount of calmodulin bound to phosphorylase kinase in muscle extract was estimated to a maximum of 30% of the total amount of calmodulin. In the muscle of the deficient strain a decrease of 35% in the total amount of calmodulin was observed. This correlates with the absence of the calmodulin fraction specifically bound to phosphorylase kinase. From sucrose gradient studies we demonstrated that in the presence of Ca2+ the amount of calmodulin bound to phosphorylase kinase was enhanced, compared to the control in the presence of EGTA. This observation was made both in crude extracts and in pure phosphorylase kinase preparations. Sucrose gradient also showed that muscle phosphorylase kinase can be dissociated to low molecular species when extracts are made in the presence of Ca+; this dissociation was found to be related to a Ca2+-dependent proteolytic effect.

Two different species of messenger RNAs specify synthesis of M1 and M2 pyruvate kinase subunits.

A study was performed to determine whether M1 and M2 pyruvate kinases were synthesized under the direction of one or two messenger RNAs. We compared M1 and M2 pyruvate kinases purified from fresh tissues with those neosynthesized under the direction of messenger RNAs from tissues synthesizing either M1 or M2. RNA was isolated from rat muscle, lung, spleen and kidney by ethanol precipitation in 7 M guanidium chloride, translated in rabbit reticulocyte system and newly-synthesized pyruvate kinase subunits were purified by microimmunoaffinity chromatography. Pyruvate kinase from fresh muscle and spleen was purified in one step by a similar process. Muscle and spleen RNA directed the synthesis of M subunits with molecular weights of approx. 61000 and 62000, respectively, the same as those of the corresponding fresh tissue monomers. In addition, peptide maps obtained by partial digestion of neosynthesized M1 and M2 with V8 protease from Staphylococcus aureus confirmed that these polypeptides were clearly different.

Transient transcriptional inhibition of the transferrin gene by cyclic AMP.

Transferrin mRNA content and gene transcription rate were measured in the liver of rats submitted to iron overload or depletion, castration, treatment with sexual steroid hormones, glucagon and cyclic AMP. The influence of puberty in males and females and of pregnancy was also analysed. Glucagon and cyclic AMP reduced mRNA level by about 50% at the 12th hour of treatment and transferrin gene transcription by as much as 95% at the 30th minute of drug infusion, with a secondary increase of the transcription rate for a protracted treatment. None of the other hormones tested had any detectable effect on transferrin gene expression, the same being true for iron overload or depletion.

Muscle-type phosphorylase activity present in muscle cells cultured from three patients with myophosphorylase deficiency.

Skeletal muscle fibers cultured from three patients whose mature fibers are deficient in glycogen myophosphorylase (EC 2.4.1.1) were shown to become rather mature, to have no excessive glycogen accumulation, and to develop signifcant myophosphorylase activity. That activity was characterized electrophoretically and immunologically and shown to be muscle phosphorylase rather than a genetically different type, thereby demonstrating true "rejuvenation" in culture of an enzyme genetically programmed ultimately to be deficient.

Purification of human liver uridylyl transferase and comparison with the erythrocyte enzyme.

Uridylyl transferase (UDP glucose: alpha-D-galactose 1 phosphate uridylyl transferase, EC 2.7.7.12) has been purified 1350-fold from human liver to complete homogeneity. The purification procedure involved ammonium sulfate fractionation, batch treatment, chromatography on DEAE-cellulose, hexylagarose and hydroxylapatite. The specific activity of the homogeneous enzyme was 27 units/mg protein. The liver enzyme was compared to the red cell enzyme previously purified by us. The liver enzyme was similar to the red cell enzyme with respect to subunit molecular weight, kinetic studies and immunological properties. Differences in electrophoretic behaviour were found: the liver transferase has a more basic pI (between 5.5 and 5.8) than that of the erythrocyte enzyme (pI between 5.0 and 5.45). It is very likely that the liver uridylyl transferase and the red blood cell transferase are the same enzymes with post-translational modifications.

Modifications of aldolase during in vivo aging of rabbit red cells.

Crude hemolysates, partially purified aldolase and aldolase purified to homogeneity from reticulocytes and mature erythrocytes, were incubated with a specific antiserum raised against crystalline rabbit muscle aldolase. We show that the same aldolasic activity corresponds to a greater amount of antigen in older than in younger cells, in crude hemolysates as well as in the above mentioned preparations; that is to say, old-cell aldolase contains cross-reacting material (CRM). Properties of purified enzyme from reticulocytes and mature erythrocytes were compared to those of muscle crystalline aldolase: -- the molecular specific activity of purified aldolase from erythrocytes is lower than with crystalline muscle aldolase, i.e. CRM is maintained throughout the purification steps. -- the specific activity of red cell aldolase towards both substrates (FDP and F1P) is lower than that of crystalline muscle aldolase. However, the ratio of activity towards the two substrates FDP/F1P is decreased in erythrocytes and reticulocytes. -- no other difference was found: Michaelis constant towards FDP, thermodenaturation constant and C terminal extremities are identical as are the molecular weights.

Electrophoretic modifications of three enzymes in extracts of human and bovine lens. Posttranslational "aging" of lens enzymes.

Electrophoretic modifications have been found in extracts from human and bovine lenses for three enzymes: glucose-6-phosphate dehydrogenase, triosephosphate isomerase and nucleoside phosphorylase. Increased anodic mobility is observed in all cases. It is more pronounced than in red cell lysates, also more evident in lenses from adult than from young animals. These results give evidence of posttranslational "aging" of enzyme molecules in lenses.

Human amniotic fluid alpha-glucosidase.

Amniotic fluid in midpregnancy contains significant alpha-glucosidase activity. This enzyme is distinguishable from the lysosomal acid alpha-glucosidase, deficiency of which is associated with Pompe's disease. The two enzymes differ in optimum pH, thermal stability, electrophoretic migration, isoelectric point, molecular mass, and immunological response. Amniotic alpha-glucosidase is also different from the classical neutral form. Immuno-cross reactions suggest that the amniotic fluid enzyme has a double fetal origin: renal and intestinal. It seems that alpha-glucosidase in amniotic fluid is linked to lipids.

Effects of antisera raised against native and denatured human alpha-glucosidase and beta-hexosaminidases on native enzyme activity.

Antisera were raised in rabbits against native and sodium dodecylsulfate denatured forms of human acid alpha-glucosidase and beta-hexosaminidases A and B. Anti-native enzyme antisera were able to precipitate all or nearly all enzyme activity from cell extracts, and to eliminate all stainable activity on electrophoresis. Antisera prepared against denatured enzymes precipitated only a minor part of enzyme activity. Electrophoretic analysis showed that these antisera were able to bind to the enzyme molecule. The result was a slowing down of the anodic migration but not immobilization. The use of variants with hexosaminidase deficiencies helped to clarify the action of the antisera on the various hexosaminidase isozymes.

Isoenzyme pattern of phosphorylase in white blood cells and fibroblasts from patients with liver phosphorylase deficiency.

Isoenzyme patterns of phosphorylase in white blood cells and cultured fibroblasts of a patient affected with liver-type phosphorylase deficiency were studied. Three bands were observed with electrofocusing of white blood cells and liver from controls. In the white blood cells of the patient only two bands were observed. Patient and control fibroblasts showed two bands, probably identical to the two bands observed in the patient's white blood cells. These results indicate that the liver-type phosphorylase is not expressed in the cultured fibroblasts.

Reactivating factors of human alpha-mannosidase mutant.

In a previous study, we compared the alpha-mannosidase from mannosidosis tissues to that from normal one and we characterized the mutant. In this work, we show that the mutant inactivation by dialysis is reversible in different conditions and we investigate the nature of mannosidosis reactivating factors. The results obtained on pathological tissue by dialysis and by addition of dialysis fluid (DF), pronase treated DF, amino acid mixture, bivalent ions: Ca++, Zn++, Mg++, Co++ DF containing EDTA or DF heated to 600 degrees C suggest the reactivating factor includes both peptides and ions.

Testosterone secreting adrenal cortical adenomas.

We report the hormonal and radiological evaluation of two cases of adrenal cortical adenomas that secreted testosterone exclusively. We discuss some of the pitfalls in the diagnosis of this lesion, and summarize the current knowledge of the characteristic hormonal features in the two cases and the 12 cases previously reported.

[Assessment of the myocardial infarct size and kinetic study of the necrosis process by serial determinations of creatine kinase]. / Estimation de la masse de l'infarctus du myocarde et étude cinétique du processus de nécrose par les dosages en série de la créatine kinase.

Assessment of the size of a myocardial infarct is important from a prognostic point of view, given the opportunities for surgical and pharmacological limitation of the process of necrosis. Serial doses of creatine kinase and its isoenzyme MB given every 4 hours for the first 48 hours of the infarct have allowed us to estimate the size of the infarct and to study the kinetics of enzyme liberation during necrosis. Unknown factors limit the sensitivity of this means of assessing the size of an infarct. The kinetic study showed that the enzyme is liberated by differing mechanisms.