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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.

Using a procedure of ethanol precipitation in concentrated guanidine . HCl solutions followed by chloroform/isoamylic alcohol extraction and washing in 3 M sodium acetate, we isolated high-molecular-weight cellular RNA from human fetal and adult skeletal muscle. About 500 micrograms RNA were obtained/g of fetal muscle and 50 micrograms RNA/g of adult muscle. Both RNA preparations were efficiently translated in a cell-free reticulocyte lysate system and directed synthesis of various polypeptides, one of them of Mr 200,000 probably corresponding to myosin heavy chains. Dodecylsulphate/polyacrylamide gel electrophoretic pattern of polypeptides neosynthesized using either fetal or adult RNA exhibited several differences. Three neosynthesized cytosolic muscle enzymes were purified from the translation mixtures using a micro-method of immunoaffinity chromatography; specificity of the neosynthesized polypeptides purified according to this procedure was checked by immunological competition with the corresponding unlabeled pure muscle enzymes. Successful cell-free translation of RNA from adult skeletal muscle and purification of neosynthesized human enzymes are reported for the first time. These methods, indispensable for further studies on human adult muscle gene expression, could also shed light on the mechanism of some inherited molecular diseases and tumoral or dystrophic processes.

Using a cloned rat transferrin cDNA probe, we looked for transferrin mRNA in the various rat tissues during development. In all the cases the mRNA detected seemed to be the same and to be product of a single gene. The transferrin gene is early expressed at a high level during liver differentiation. In the muscle and other non-hepatic and non-nervous tissues, the gene expression is maximal just before birth (19-20th day of gestational age), then markedly decreases during the postnatal development, the mRNA level being very low in the adult tissues. In brain, by contrast, transferrin mRNA level is very low before birth, then gradually increases during the postnatal development and reaches a plateau in the adult. Maximal mRNA concentration in fetal muscle (2 days before birth) and adult brain is about 1:7 to 1:10 of that obtained in adult liver. These results are analyzed in the light of the evidence that transferrin is not only an iron-binding protein, but also a factor involved in cell proliferation and differentiation, and particularly in nerve control of muscle differentiation.

(1) Two unrelated cases of McArdle's disease (glycogen storage disease type V, deficiency in muscle-type glycogen phosphorylase, EC.2.4.1.1) with no detectable inactive protein, and two heterozygous relatives of one patient, have been investigated for the presence or absence of functional messenger RNA. Methods were developed and scaled down to be compatible with clinical-size biopsies. (2) Total muscle RNA was prepared and translated in a rabbit reticulocyte cell-free system. Neosynthesized products were isolated by immunoaffinity microchromatography. Phosphorylase was compared with control enzymes neosynthesized and purified under the same conditions. (3) No functional mRNA for phosphorylase could be detected in the muscles of the two patients. A decreased amount of messenger for phosphorylase, compared with the control enzymes, was found in the muscles of the two heterozygotes. (4) Exploration of functional messenger RNA in clinical enzymopathies should be readily adaptable to a number of enzymatic diseases.

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.

Using specific immunoprecipitation of M-type phosphofructokinase and assay of immunoprecipitate enzyme activity, it was possible to detect some M-type enzyme in normal blood cells and fibroblasts, although this isoenzyme represents a very small part of total phosphofructokinase. White blood cells and cultured fibroblasts from a patient with hereditary muscle phosphofructokinase deficiency showed normal phosphofructokinase activity and electrophoretic pattern; direct immunoneutralization results were also normal. Nevertheless, it was possible to prove the defect in these cells using the immunoprecipitation method: no active immunoprecipitates could be obtained with anti M-type antibody. The patient's red blood cells had a reduced phosphofructokinase activity which was only neutralized by anti L-type antiserum. The purification of partially deficient red cell phosphofructokinase confirmed that this enzyme only consisted of L-type subunits while, under normal conditions, both L- and M-type subunits are observed. The possibility of detecting specific enzyme defects in apparently non-affected cells could be of practical importance, especially in prenatal diagnosis.