Identification of a third form of protein phosphatase 1 in rabbit skeletal muscle that is associated with myosin

A third form of protein phosphatase 1 has been identified in skeletal muscle which is distinct from the species composed of the catalytic subunit complexed to the glycogen-binding subunit (protein phosphatase 1G) or inhibitor-2 (protein phosphatase 1I). The third form has an apparent molecular mass of 110 kDa, is not immunoprecipitated by antibody prepared against the glycogen-binding subunit, does not interact with glycogen and is devoid of inhibitor-2. It is tightly bound to myosin and is therefore termed protein phosphatase 1M.     

Purification and characterization of rat skeletal muscle fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Fructose-6-phosphate,2-kinase:fructose-2,6-bis-phosphatase from rat skeletal muscle has been purified to homogeneity, and its structure and kinetic properties have been determined. The Mr of the native enzyme was 100,000 and the subunit Mr was 54,000. The apparent Km values of fructose-6-P,2-kinase for Fru-6-P and ATP were 56 and 48 microM, respectively. The apparent Km value for Fru-2,6-P2 of fructose-2,6-bis-phosphatase was 0.4 microM, and the Ki for Fru-6-P was 12.5 microM. The enzyme was bifunctional, and the phosphatase activity was 2.5 times higher than the kinase activity. The enzyme was not phosphorylated by cAMP-dependent protein kinase. The amino acid composition of the skeletal muscle enzyme was similar to that of the rat liver enzyme, and the carboxyl terminus sequence (His-Tyr) was the same as that of the liver enzyme. The tryptic peptides generated from the liver and skeletal muscle enzymes were identical except for two peptides. A peptide corresponding to nucleotides 14-28 of the rat liver enzyme was not detected in the skeletal muscle enzyme. A peptide whose amino acid sequence was Thr-Ala-Ser-Ile-Pro-Gln-Phe-Thr-Asn-Ser-Pro-Thr-Met-Val-Ile-Met-Val-Gly-Leu-Pro - Ala-Arg was also isolated. This peptide was the same as that of rat liver enzyme (nucleotides 31-52) containing the phosphorylation site except in the muscle enzyme two amino terminus amino acids, Gly-Ser(P), have been altered to Thr-Ala. Thus, the rat skeletal muscle enzyme is very similar in structure to the rat liver enzyme except for the lack of possibly one peptide and the lack of a phosphorylation site by the substitution of the target Ser with Ala.     

Human red blood cell insulin-degrading enzyme and rat skeletal muscle insulin protease share antigenic sites and generate identical products from insulin.

The mechanisms of cellular insulin degradation remain uncertain. Considerable evidence now exists that the primary cellular insulin-degrading activity is a metallothiol proteinase. Two similar degrading activities have been purified and characterized. Insulin protease has been purified from rat skeletal muscle and insulin-degrading enzyme from human red blood cells. Whereas the two degrading activities share a number of similar properties, significant differences have also been reported; and it is not at all established that they are the same enzyme. To examine this, we have compared antigenic and catalytic properties of the two enzymatic activities. Monoclonal antibodies against the red blood cell enzyme adsorb the skeletal muscle enzyme; and on Western blots, the antibodies react with an identical 110-kDa protein. Immunoaffinity-purified enzymes from both red blood cells and skeletal muscle degrade [125I]iodo(B26)insulin to the same products as seen with purified insulin protease and with intact liver and kidney. Chelator-treated muscle and red blood cell enzymes can be reactivated with either Mn2+ or Ca2+. Thus, insulin-degrading enzyme and insulin protease have similar properties. These results support the hypothesis that these activities reside in the same enzyme.     

Processivity and subcellular localization of glycogen synthase depend on a non-catalytic high affinity glycogen-binding site

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS.     

Isolation and sequence analysis of a cDNA clone encoding the entire catalytic subunit of a type-2A protein phosphatase

A 2.5 kb clone containing the full-length coding sequence of a type-2A protein phosphatase catalytic subunit has been isolated from a rabbit skeletal muscle cDNA library constructed in lambda gt10. The sequence of the protein deduced from the cDNA contains 309 residues (35.58 kDa). A major mRNA species at 2.0 kb and a minor component at 2.8 kb were visualized by Northern blotting in both skeletal muscle and liver. The type-2A enzyme showed weak homology with mammalian alkaline phosphatases between residues 55 and 95. The protein sequence of the type-2A phosphatase from rabbit skeletal muscle differs from that reported for the bovine adrenal enzyme in three regions.     

L-type glycogen synthase. Tissue distribution and electrophoretic mobility

We previously reported (Kaslow, H.R., and Lesikar, D.D.FEBS Lett. (1984) 172, 294-298) the generation of antisera against rat skeletal muscle glycogen synthase. Using immunoblot analysis, the antisera recognized the enzyme in crude extracts from rat skeletal muscle, heart, fat, kidney, and brain, but not liver. These results suggested that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type, referred to as "M-type" glycogen synthase. We have now used an antiserum specific for the enzyme from liver, termed "L-type" glycogen synthase, to study its distribution and electrophoretic mobility. Immunoblot analysis using this antiserum indicates that L-type glycogen synthase is found in liver, but not skeletal muscle, heart, fat, kidney, or brain. In sodium dodecyl sulfate-polyacrylamide gels of crude liver extracts prepared with protease inhibitors, rat L-type synthase was detected with electrophoretic mobility Mapp = 85,000. In contrast, the M-type enzyme in crude skeletal muscle extracts with protease inhibitors was detected with Mapp = 86,000 and 89,000. During purification of L-type synthase, apparent proteolysis can generate forms with increased electrophoretic mobility (Mapp = 75,000), still recognized by the antiserum. These M-type and L-type antisera did not recognize a protein with Mapp greater than phosphorylase. The anti-rat L-type antisera recognized glycogen synthase in blots of crude extracts of rabbit liver, but with Mapp = 88,000, a value 3,000 greater than that found for the rat liver enzyme. The anti-rat M-type antisera failed to recognize the enzyme in blots of crude extracts of rabbit muscle. Thus, in both muscle and liver, the corresponding rat and rabbit enzymes are structurally different. Because the differences described above persist after resolving these proteins by denaturing sodium dodecyl sulfate electrophoresis, these differences reside in the structure of the proteins themselves, not in some factor bound to the protein in crude extracts.     

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.     

Identification of a putative collagen-binding protein from chicken skeletal muscle as glycogen phosphorylase.

We have purified and generated antisera to a 95 kDa skeletal muscle protein that constitutes the largest mass fraction of gelatin-agarose binding proteins in skeletal muscle. Preliminary results indicated that this 95 kDa chicken skeletal muscle protein bound strongly to gelatin-agarose and type IV collagen-agarose, suggesting a possible function in muscle cell adhesion to collagen. However, N-terminal sequencing of proteolytic fragments of the 95 kDa protein indicates that it is the chicken skeletal muscle form of glycogen phosphorylase, the binding of which to gelatin-agarose is unlikely to be biologically relevant. Further characterization showed that the skeletal muscle form of glycogen phosphorylase is immunologically distinct from the liver and brain forms in the chicken, and suggests that, unlike mammalian skeletal muscle, chicken skeletal muscle may have two phosphorylase isoforms. Furthermore, immunolocalization data and solubility characteristics of glycogen phosphorylase in muscle extraction experiments suggest the enzyme may interact strongly with an unidentified component of the muscle cytoskeleton. Thus, this study yields a novel purification technique for skeletal muscle glycogen phosphorylase, provides new information on the distribution and isoforms of glycogen phosphorylase, and provides a caveat for using gelatin affinity chromatography as a primary step in purifying collagen-binding proteins from skeletal muscle.     

Muscle and liver protein metabolism in rats fed raw or heat-treated pea seeds

Raw or extruded pea (Pisum sativum, cv. Ballet) diets with or without supplementary amino acids were fed for 15 days to young growing rats and the effects on tissue weights, liver and muscle protein metabolism and hormone levels monitored. Body weight gain, liver and gastrocnemius muscle weights and protein contents were reduced and some key hormones altered when rats were fed unsupplemented raw pea diets. This appeared to be a result of amino acid deficiencies in the diet, the action of antinutritional factors and the refractory nature of the reserve proteins and other seed components. However, this did not in itself improve the nutritional performance of the rats due to the overriding effects of the amino acid deficiencies in the pea diets. After supplementation, extruded peas supported much higher rates of growth and skeletal muscle deposition than did supplemented raw peas. Despite this, the weight gains remained less than achieved on a high quality control diet. Protein synthesis and degradation rates in skeletal muscles and total protein contents were similar to control values. The lower growth rate did not appear to be due to impaired deposition of skeletal muscle. Deposition of other body components, possibly lipids, may have been lowered by supplemented extruded pea diets. Liver protein levels were reduced in rats fed supplemented raw peas and blood corticosterone was elevated. In conclusion, extrusion treatment of peas in combination with amino acid supplementation appeared to abolish the negative effects of peas on skeletal muscle deposition.     

50 Hz magnetic fields activate mussel immunocyte p38 MAP kinase and induce HSP70 and 90

We studied the effects of a sublethal concentration of pyrethroid insecticide fenvalerate on metabolic enzymes, RNA and protein of brain, liver and skeletal muscle of the freshwater catfish, Clarias batrachus. Exposure to fenvalerate gradually decreased the activity of citrate synthase (CS), glucose 6-phosphate dehydrogenase (G6-PDH) and lactate dehydrogenase (LDH) in brain, liver and skeletal muscle up to 21 days. The maximum decrease in enzyme activity was 23-47%. Withdrawal of fenvalerate from the medium for 21 days restored enzyme activity to their control level in all three tissues. RNA and protein content in brain, liver and skeletal muscle decreased significantly with exposure of fenvalerate up to 21 days. The maximum decrease in RNA and protein was 22-32%. Withdrawal of fenvalerate from the medium for 21 days restored the RNA and protein contents to control levels. The present study suggests that fenvalerate impairs cellular metabolism and its biochemical effects are reversible after withdrawal of fenvalerate.     

Differential response of AMP deaminase isozymes to changes in the adenylate energy charge

AMP deaminase has been prepared from white skeletal muscle fibers, red skeletal muscle fibers, cardiac muscle and liver. The isozymes from skeletal muscle, cardiac muscle and liver can be readily distinguished from one another by the shape of the adenylate energy charge response curve. However, the enzyme prepared from different skeletal muscles which consists of predominately red or white fibers are indistinguishable from one another by this criterion.     

Changes of the NADP-linked malic enzyme in the developing rat skeletal muscle

The activities of NADP-linked malic enzyme, hexose monophosphate shunt dehydrogenases and NADP-linked isocitrate dehydrogenase were studied during development of skeletal muscle and compared with those in the liver. The variation patterns of malic enzyme activity in the liver and in the skeletal muscle were very similar, however the amplitude of the changes was different. The enzyme activity increased approx 16-fold in the liver and about 2-fold in skeletal muscle at the same stage of development. In skeletal muscle the increase of the malic enzyme activity was only slightly higher than of lactic dehydrogenase and citrate synthase. Studies on the intracellular distribution of malic enzyme in skeletal muscle showed that both mitochondrial and extramitochondrial enzymes increased between 20th and 37th day of life, the increase of the extramitochondrial enzyme being more pronounced. The hexose monophosphate shunt dehydrogenases activity showed an increase in the liver but no change was observed in the skeletal muscle at the weaning time. Changes in the activity of the liver and skeletal muscle isocitrate dehydrogenase were not significant between 10th and 80th day of life. The results suggest that the malic enzyme in the liver is playing a different physiological role than in the skeletal muscle.     

Developmental regulation of the activation of signaling components leading to translation initiation in skeletal muscle of neonatal pigs

The rapid growth of neonates is driven by high rates of skeletal muscle protein synthesis. This high rate of protein synthesis, which is induced by feeding, declines with development. Overnight-fasted 7- and 26-day-old pigs either remained fasted or were refed, and the abundance and phosphorylation of growth factor- and nutrient-induced signaling components that regulate mRNA translation initiation were measured in skeletal muscle and liver. In muscle, but not liver, the activation of inhibitors of protein synthesis, phosphatase and tensin homolog deleted on chromosome 10, protein phosphatase 2A, and tuberous sclerosis complex 1/2 increased with age. Serine/threonine phosphorylation of the insulin receptor and insulin receptor substrate-1, which downregulates insulin signaling, and the activation of AMP-activated protein kinase, an inhibitor of protein synthesis, were unaffected by age and feeding in muscle and liver. Activation of positive regulators of protein synthesis, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), and eIF4E-binding protein-1 (4E-BP1) decreased with age in muscle but not liver. Feeding enhanced mTOR, S6K1, and 4E-BP1 activation in muscle, and this response decreased with age. In liver, activation of S6K1 and 4E-BP1, but not mTOR, was increased by feeding but was unaffected by age. Raptor abundance and the association between raptor and mTOR were greater in 7- than in 26-day-old pigs. The results suggest that the developmental decline in skeletal muscle protein synthesis is due in part to developmental regulation of the activation of growth factor and nutrient-signaling components.     

Total conversion of glycogen synthase from the I- to the D-form by a cyclic AMP-independent protein kinase from rabbit skeletal muscle.

A newly discovered cyclic AMP-independent protein kinase, which catalyzes the total conversion of glycogen synthase from the I- to the D-form, has been isolated from rabbit skeletal muscle. This enzyme, designated glycogen synthase kinase, is separable from cyclic AMP-dependent protein kinase by column chromatography on phosphocellulose. Synthase kinase and cyclic AMP-dependent protein kinase are distinct in their specificity for protein substrates, the effects of cyclic AMP and the inhibitor of cyclic AMP-dependent protein kinase on their activities, and the extent to which they phosphorylate I-form glycogen synthase. The phosphorylation of I-form enzyme by synthase kinase results in the incorporation of 4 mol of phosphate/85,000 subunit; however only two of the phosphate sites seem predominantly to determine glucose-6-P dependence. The resulting multiply phosphorylated enzyme, which is highly dependent on glucose-6 P for activity, has a phosphate content comparable to the D-form enzyme isolated from rabbit muscle.     

Hydroxylation of gamma-butyrobetaine by rat and ovine tissues.

Ovine tissues were assayed for the capacity to synthesize carnitine from γ-butyrobetaine. Activity in liver, kidney and muscle was 0.25, 0.10 and 0.08 nmoles per mg protein per min, respectively. Heart was devoid of the enzyme. Of the rat tissues that were assayed only liver contained the hydroxylase (0.39 nmoles per mg per min). Although the specific activity of the enzyme was approximately three fold higher in sheep liver than in sheep skeletal muscle, on the basis of total activity, muscle would constitute the major portion of the total hydroxylase activity present in the body. The synthesis of carnitine in ovine skeletal muscle may in part explain the high level of carnitine found in that tissue and emphasizes the existence of species differences in the localization of carnitine synthesis.     

Glycogenosis Type III in the Dog

Enzyme and glycogen structure studies have been carried out on tissues of a glycogenotic dog, the clinical and pathological characteristics of which are reported in the accompanying paper. Liver glucose-6-phosphatase, leukocyte and liver acid maltase, and liver and skeletal muscle glycogen Phosphorylase all appeared largely unaffected. The activity of the muscle and liver debranching enzyme (amylo-l,6-glucosidase), determined by two independent assay methods, was, however, reduced to between 0 and 7 % of normal activity. Glycogen structure studies with Phosphorylase or iodine spectra revealed that the abnormally large amounts of glycogen found in liver and skeletal muscle had abnormally short branches, as would be expected for a deficiency of debranching enzyme. It is thus clear that the dog had suffered from the equivalent of Cori's disease (limit dextrinosis, type III glycogen storage disease). Preliminary data indicate that it may be possible to identify heterozygotes based on a study of the debranching enzyme of leukocytes.     

Effect of phosphorylation by different protein kinases on the behaviour of glycogen synthase as a substrate for hepatic synthase phosphatases

Glycogen synthase a from skeletal muscle was phosphorylated in vitro and then used as substrate for the two major synthase phosphatases from liver. Synthase phosphorylated by cAMP-dependent protein kinase (1.4-1.7 P/subunit) was preferentially activated by the cytosolic S-component; in contrast, progressive phosphorylation by casein kinase-1 (0.9-6.5 P/subunit) yielded substrates that were always better dephosphorylated and activated by the glycogen-bound G-component. We have previously isolated from dog liver several types of synthase b that differ by their need for the S- and/or G-component for prompt activation. After additional phosphorylation by a mixture of synthase kinases the activation of these enzyme preparations required the presence of both components.     

Three major forms of adenylate kinase from adult and fetal rat tissues.

The major enzymatic forms of adenylate kinase have been purified to homogeneity from fetal liver and adult brain of the rat. The two enzymes differ with respect to isoelectric points, Km (ATP), Km (AMP), and Ka (citrate). Antibody to adult liver adenylate kinase does not inhibit either enzyme, while entibody to adult skeletal muscle enzyme inhibits the brain enzyme but not the fetal liver enzyme. It is therefore probable that there are three major forms of adenylate kinases in fetal and adult rat tissues.