Phosphorylase Enzyme Activity Research Articles

Expression of glycogen phosphorylase isozymes in developing rat lung.

Glycogen accumulates to significant levels in epithelial cells of the developing respiratory tract. Mobilization of glycogen stores is regulated differentially along the respiratory epithelium such that glycogenolysis in the alveolar epithelium (the site of surfactant synthesis) precedes that in the bronchial and bronchiolar epithelium. The initial step in glycogen degradation is catalyzed by glycogen phosphorylase, which exists as three genetically distinct isozymes referred to as muscle, liver, and brain isoforms. The goal of this study was to characterize the temporal and spatial expression of each of the glycogen phosphorylase isozymes in developing lung to determine which isoform(s) was associated with glycogen mobilization in the fetal type II epithelial cell. RNA levels encoding glycogen phosphorylase were assessed by ribonuclease protection assay using isoform-specific antisense probes. RNAs encoding the brain and liver isozymes were detected in isolated day 20 fetal type II epithelial cells and at lower levels in adult type II cells. The muscle isoform RNA was barely detectable in fetal type II cells and was undetectable in adult type II cells. Expression of brain and liver isoform RNAs was higher in whole fetal lung than in fetal type II cells. Consistent with this result, in situ hybridization studies demonstrated widespread expression of the brain and liver isoforms in developing lung tissues; in contrast, expression of the muscle isoform was restricted to the pulmonary vein. Glycogen phosphorylase enzyme activity corresponding to the brain isoform was clearly detected in isolated fetal type II cells; however, the majority of enzyme activity migrated as two bands with distinct electrophoretic mobilities that may have been the result of isoform heterodimerization. Collectively, these results suggest that the brain and liver isoforms of glycogen phosphorylase may be involved in mobilization of type II cell glycogen during late fetal lung development.

Construction of a 2.8-megabase yeast artificial chromosome contig and cloning of the human methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p21.

Many human malignant cells lack methylthioadenosine phosphorylase (MTAP) enzyme activity. The gene (MTAP) encoding this enzyme was previously mapped to the short arm of chromosome 9, band p21-22, a region that is frequently deleted in multiple tumor types. To clone candidate tumor suppressor genes from the deleted region on 9p21-22, we have constructed a long-range physical map of 2.8 megabases for 9p21 by using overlapping yeast artificial chromosome and cosmid clones. This map includes the type IIFN gene cluster, the recently identified candidate tumor suppressor genes CDKN2 (p16INK4A) and CDKN2B (p15INK4B), and several CpG islands. In addition, we have identified other transcription units within the yeast artificial chromosome contig. Sequence analysis of a 2.5-kb cDNA clone isolated from a CpG island that maps between the IFN genes and CDKN2 reveals a predicted open reading frame of 283 amino acids followed by 1302 nucleotides of 3' untranslated sequence. This gene is evolutionarily conserved and shows significant amino acid homologies to mouse and human purine nucleoside phosphorylases and to a hypothetical 25.8-kDa protein in the pet gene (coding for cytochrome bc1 complex) region of Rhodospirillum rubrum. The location, expression pattern, and nucleotide sequence of this gene suggest that it codes for the MTAP enzyme.

Purification and characterization of starch phosphorylase from Cuscuta reflexa filaments

Starch phosphorylase (EC.2.4.1.1.) from the filaments of Cuscuta reflexa has been purified using (NH 4) 2SO 4 fractionation, ion-exchange chromatography using DEAE cellulose and gel filtration through Sephadex G-200. The enzyme preparation is estimated to be about 85–90% pure as revealed by native polyacrylamide gel electrophoresis. The native M r of the enzyme as determined by gel filtration through Sephadex G-200 is 400 000 ± 5000. Subunit M r of the enzyme as determined by SDS polyacrylamide gel electrophoresis is 100 000 ± 3000. Unlike other starch phosphorylases, the present enzyme is not inhibited by aromatic amino acids. The kinetic data are characteristic of a sequential reaction mechanism. The optimum temperature for starch phosphorylase enzyme activity is 45°, the energy of activation being 12.2 kcal (°mol) −1 and energy of activation for denaturation is 32.94 kcal (°mol) −1. Maximum activity occurred at pH 5.7 with half-maximum activity at pH 5.2 and 7.8.

Glycogen phosphorylase: developmental expression in rat liver.

The developing fetus is vulnerable to hypoglycemia if its transplacental substrate supply is compromised. Consequently, we examined hepatic glycogen phosphorylase gene expression in the developing rat liver by comparing the relative activities of the hepatic phosphorylase enzyme, concentrations of cellular phosphorylase mRNA, and rates of transcription in isolated liver nuclei of fetal rats at 19 days of gestation, 21 days of gestation, neonatal rats, and suckling rats. Cellular phosphorylase mRNA and enzyme activity (total and phosphorylase a) increased until term. Reciprocally, the rate of phosphorylase mRNA transcription was rapid in the rats at 19 days of gestation, and declined progressively until term. These data indicate that glycogen phosphorylase gene expression is regulated post-transcriptionally in late gestation, perhaps by an increase in phosphorylase mRNA stability towards term. This results in increased phosphorylase mRNA and enzyme expression.

Elevated Thymidine Phosphorylase Activity in Psoriatic Lesions Accounts for the Apparent Presence of an Epidermal “Growth Inhibitor,” But Is Not in Itself Growth Inhibitory

An apparent tissue-specific growth inhibitor, or chalone, obtained from psoriatic lesions was tentatively identified in the 100-kDa fraction based upon inhibition of DNA synthesis, as measured by [3H]-thymidine uptake by a squamous cell carcinoma cell line, SCC 38. This fraction, however, failed to inhibit SCC 38 cell growth when assessed directly in a neutral red uptake assay. Characterization of the inhibitor of [3H]-thymidine uptake revealed it to have biochemical properties identical to thymidine phosphorylase: 1) molecular weight close to 100 kDa, 2) isoelectric point of 4.2, and 3) thymidine phosphorylase enzyme activity. Thus, we conclude that its ability to inhibit [3H]-thymidine uptake was due to thymidine catabolism rather than inhibition of DNA synthesis or growth inhibition. Examination of thymidine phosphorylase activity in keratome biopsies from psoriatic and normal skin demonstrated a twentyfold increase in activity in psoriatic lesions relative to non-lesional or normal skin. This increase in metabolism of thymidine was due to thymidine phosphorylase rather than uridine phosphorylase activity. The correlation between increased thymidine phosphorylase activity and increased keratinocyte proliferation in vitro (cultured) and in vivo (psoriasis), suggests that this enzyme may play a critical role in providing the thymidine necessary for keratinocyte proliferation.

Glyconeogenic and glycogenic enzymes in chronically active and normal skeletal muscle

The chronically active (pseudomyotonic) gastrocnemius muscle in the C57B16J dy2J/dy2J mouse contains both elevated lactate and glycogen as well as fibers that have high amounts of glycogen and enhanced glyconeogenic activity. In the present study we analyze the activities of some key glyconeogenic enzymes to assess the causes of elevated muscle glycogen and to determine the pathway for glycogen synthesis from lactate. Glycogen synthase, malate dehydrogenase, phosphoenolpyruvate carboxykinase, and malic enzyme were all elevated in homogenates of the chronically active muscle. Activities of glycogen phosphorylase and fructose 1,6-bisphosphatase were decreased in whole muscle homogenates. Histochemistry demonstrated that the high-glycogen fibers were typically fast-twitch glycolytic fibers that had high glycogen synthase, glycogen phosphorylase, and malic enzyme activities. Malate dehydrogenase activity followed succinate dehydrogenase activity and did not correlate to high-glycogen fibers. Thus the high-glycogen fibers have an elevated enzymatic capacity for glycogen synthesis from lactate, and the pathway may involve use of the pyruvate kinase bypass enzymes.

Skeletal muscle biochemistry and histology in ambulatory patients with long-term heart failure.

Recent studies in patients with long-term heart failure have suggested that intrinsic abnormalities in skeletal muscle can contribute to the development of early lactic acidosis and fatigue during exercise. The present study provides an analysis of substrate and enzyme content, fiber typing, and capillarization in skeletal muscle biopsy samples obtained at rest from the vastus lateralis in 11 patients with long-term heart failure (left ventricular ejection fraction, 21 +/- 8%) and nine normal subjects. Patients demonstrated a reduced peak exercise oxygen consumption (13.0 +/- 3.3 ml/kg/min) when compared with normals (30.2 +/- 8.6 ml/kg/min, p less than 0.001) and had an accelerated rise in blood lactate levels during exercise. In mixed fiber skeletal muscle, total phosphorylase and glycolytic enzyme activities were not different in the two groups, whereas mitochondrial enzymes involved in terminal oxidation were decreased in patients as compared with normal subjects as indicated by reductions in succinate dehydrogenase (51 +/- 15 vs. 81 +/- 17 microM/g protein/min, p less than 0.001) and citrate synthetase (26 +/- 7 vs. 43 +/- 20 microM/g protein/min, p less than 0.05). 3-Hydroxyacyl-CoA-dehydrogenase, an important enzyme mediating beta-oxidation of fatty acids, was also reduced in patients as compared with normals (18 +/- 7 vs. 27 +/- 10 microM/g protein/min, p less than 0.05). There was no difference in high-energy phosphagens or lactate concentration of mixed muscle in the two groups, whereas glycogen content was decreased in patients (262 +/- 29 vs. 298 +/- 35 microM glucosyl units/kg dry wt, p = 0.01). Patients demonstrated a reduced percentage of slow twitch type I fibers (36 +/- 7% vs. 52 +/- 22%, p less than 0.05) and had a higher percentage of type IIb fast twitch fibers (24 +/- 9% vs. 11 +/- 12%, p = 0.02), which were smaller than the type IIb fibers seen in normal subjects (p less than 0.05). In patients, the number of capillaries per fiber was decreased for type I and type IIa fibers (both, p less than 0.03), but the ratio of capillaries to cross-sectional fiber area was not different for the two groups. These data demonstrate major alterations in skeletal muscle histology and biochemistry in patients with long-term heart failure, including fiber atrophy, a decrease in percentage of composition of type I fibers, and an increase in type IIb fibers accompanied by a decrease in oxidative enzyme capacity.(ABSTRACT TRUNCATED AT 250 WORDS)

Depressed activities of purine enzymes in lymphocytes of patients infected with human immunodeficiency virus.

Enzyme activities were studied in peripheral blood lymphocytes from patients infected with, or at risk for, infection with human immunodeficiency virus (HIV). No significant differences were observed in the HIV-infected and HIV-seronegative high-risk patients with regard to enzyme activities of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and purine nucleoside phosphorylase (EC 2.4.2.1) in peripheral blood. Adenosine deaminase (EC 3.5.4.4) was significantly (P less than 0.02) depressed in asymptomatic HIV-seropositive patients and HIV-seronegative patients at high risk of HIV infection as compared with a healthy HIV-seronegative population. Adenosine kinase (AK, EC 2.7.1.20) was significantly increased in the asymptomatic seropositive (P less than 0.02) and also in the HIV-seronegative high-risk groups (P = 0.01) compared with the normal controls. AK activity was significantly lower in subjects with AIDS than in the asymptomatic (P less than 0.002) and high-risk groups (P less than 0.01). Taken together, these results indicate that adenosine deaminase and AK activities are influenced by the health of the patient, and that measurement of AK activity may prove useful in monitoring the clinical progress of patients with HIV infection.

Long term regulation of glycogen metabolizing enzymes by insulin in H4 hepatoma cells.

Insulin alone at concentrations of less than 1 to 5 uU/ml increased the enzyme activities of glycogen synthase, synthase phosphatase, phosphorylase, and phosphorylase phosphatase in hepatoma H4 cells in culture in the presence and absence of serum. Increase in total and active forms of glycogen synthase and phosphorylase were observed. Cycloheximide blocked the action of insulin on glycogen synthase, glycogen synthase phosphatase and phosphorylase phosphatase. The enzymes with the exception of glycogen synthase phosphatase were expressed with greater hormonal sensitivity in the absence as compared to the presence of serum in terms of hormone concentration required and or time of onset. These results demonstrate that these glycogen metabolizing enzymes are under long term control by insulin, with glycogen synthase being the most sensitive of the enzymes studied to the action of the hormone.

Enzymes of the purine metabolism in rat brain microsomes.

Rat brain microsomes, when they are suspended in moderate ionic strength medium, released enzyme activities of lactate dehydrogenase (LDH, E.C.1.1.1.27), malate dehydrogenase (MDH, E.C.1.1.1.37), adenosine deaminase (ADA, E.C.3.5.4.4), guanine deaminase (GAH, E.C.3.5.4.3), and purine nucleoside phosphorylase (PNP, E.C.2.1.2.4). The activities released decreased when the saline concentration of the medium was increased and the opposite occurred when 50 mM, pH 7.4 sodium phosphate medium was used. Rat brain microsomes that had been extracted previously by moderate ionic strength solutions still had activities of all the enzymes tested, and released these activities upon sonication or deoxycholate (DOC) treatment. The proportion of the activity released was similar for all the enzymes. DOC treatment released higher enzymic activities and a smaller amount of protein than sonication did. The proportion of activities released was similar to that found in the 105,000 g supernatant. The suspension of microsomes still retained activities of the above-mentioned enzymes after consecutive extractions with increasing concentrations of detergent solutions (DOC and Triton X-100). The amount of enzymic activities released from the microsomes by sonication or DOC treatment did not depend on the protein composition of the homogenization medium. Thus, on increasing the enzyme concentration in the homogenization medium, the activities released did not increase in parallel. The set of results obtained showed that the microsomal fraction is as useful as the cytosolic one for studying purine catabolism in rat brain. Furthermore, the conditions in which purine enzymes are attached to the microsomal fraction are probably closer to "in vivo" conditions than those in which these enzymes are found in the soluble fraction.

Regulation of neonatal heart glycogen metabolism by thyroxine.

We investigated the effect of hyperthyroidism and hypothyroidism on the myocardial glycogen metabolism by quantitating 125I-insulin binding, glycogen content, glycogen synthase and phosphorylase enzyme activities in the newborn rabbit. Although an increase in 125I-insulin binding was observed in response to hyperthyroidism (p less than 0.01), a decrease in myocardial glycogen (p less than 0.01) along with no change in the synthase and phosphorylase enzyme activity was demonstrated. On the other hand, propylthiouracil-induced hypothyroxinemia did not affect the 125I-insulin binding, glycogen content or the two enzyme systems. We conclude that the depletion of myocardial glycogen secondary to hyperthyroidism is independent of an increase in 125I-insulin binding or any change in the major glycogen enzyme activities (synthase and phosphorylase). We speculate that this decline in neonatal heart glycogen may be secondary to thyroxine-induced altered glucose uptake or modified postinsulin receptor events.

The loss of enzyme activities involved in early steps of energy metabolism being a parameter for the reduction of physical performances during adult life-span of Phormia terrae novae females

1. 1. The enzyme activities of phosphorylase a and b, trehalase, arginine phosphokinase, α-glycerophosphate (α-GDH) dehydrogenase were measured during the life-span in mated and unmated Phormia terrae novae females, fed with and without protein. 2. 2. The activity profiles of these enzymes were set into correlation to physical activities of the single groups. 3. 3. Except the profile of α-GDH, all other enzymes show a direct correlation to the maximum of physical performance expressed in flight activity and reproduction rate. 4. 4. Although these activities depending on rearing conditions of the single populations took place at different points in time, the same relationships between physical and enzyme activities could establish in each group. 5. 5. In each population the loss of enzyme activities is to be seen before the reduction of physical performances took place. 6. 6. It is suggested that the metabolic regulation of life-span and life-processes is ruled out by the early steps of energy releasing metabolism. The hormonal control of this metabolism is under investigation.

Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells

Mammalian cells have enzymes that deaminate adenosine to inosine, which can readily be phosphorolysed to hypoxanthine. They do not, however, possess enzymes to form adenine by the cleavage of adenosine. For this reason, the release of adenine from adenosine by mammalian cell cultures has usually been interpreted as indicating the presence of mycoplasma, a frequent microbial contaminant that contains high levels of adenosine phosphorylase. We found that some human lymphoblast cultures free of mycoplasma showed high levels of adenosine cleavage and that this activity resulted from adenosine phosphorylase in the bovine serum used as the culture growth supplement. A survey of 13 serum supplements disclosed that fetal bovine serum (six lots) contains the highest adenosine phosphorylase activity, ranging from 9 to 648 nmol adenine produced per hour per ml serum; newborn calf serum (four lots) has much less activity, ranging from 0 to 5 nmol adenine produced per hour per ml serum; and donor horse serum (three lots) contains no detectable activity. These results suggest that mycoplasma tests dependent on the presence of adenosine phosphorylase or other enzyme activities may give false-positives with cultures containing fetal bovine serum supplements.

Production, purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

The production of dextransucrase from Leuconostoc mesenteroides NRRL B-512F was stimulated 2-fold by the addition of 0.005% of calcium chloride to the medium; levansucrase levels were unaffected. Dextransucrase was purified by concentration and dialysis of the culture supernatant with a Bio-Fiber 80 miniplant, and by treatment with dextranase followed by chromatography on Bio-Gel A-5m. A 240-fold purification, with a specific activity of 53 U/mg, was obtained. Contaminating enzyme activities of levansucrase, invertase, dextranase, glucosidase, and sucrose phosphorylase were decreased to non-detectable levels. Poly(acrylamide)-gel electrophoresis of the purified enzyme showed only two protein bands, both of which had dextransucrase activity. These bands also gave a carbohydrate stain, indicating that the dextransucrase could be a glycoprotein. Acid hydrolysis, followed by paper chromatography, of the purified enzyme showed that the major carbohydrate was mannose. ConcanavaIin A completely removed dextransucrase activity from solution, confirming the mannoglycoprotein character of the enzyme. Dextransucrase activity was not altered by the addition of 0.008−4 mg/ml of dextran, but its storage stability was increased by the addition of 4 mg/ml of dextran. As previously shown by others, the activity of dextransucrase was decreased by EDTA, and was restored by the addition of calcium ions. Zinc, cadmium, lead, mercury, and copper ions were inhibitory to various degrees.

Metabolism of RNA-ribose by Bdellovibrio bacteriovorus during intraperiplasmic growth on Escherichia coli

During intraperiplasmic growth of Bdellovibrio bacteriovorus 109J on Escherichia coli some 30 to 60% of the initial E. coli RNA-ribose disappeared as cell-associated orcinol-positive material. The levels of RNA-ribose in the suspending buffer after growth together with the RNA-ribose used for bdellovibrio DNA synthesis accounted for 50% or less of the missing RNA-ribose. With intraperiplasmic growth in the presence of added U-14C-labeled CMP, GMP, or UMP, radioactivity was found both in the respired CO2 and incorporated into the bdellovibrio cell components. The addition of exogenous unlabeled ribonucleotides markedly reduced the amounts of both the 14CO2 and 14C incorporated into the progeny bdellovibrios. During intraperiplasmic growth of B. bacteriovorus on [U-14C]ribose-labeled E. coli BJ565, ca. 74% and ca. 19% of the initial 14C was incorporated into the progeny bdellovibrios and respired CO2, respectively. Under similar growth conditions, the addition of glutamate substantially reduced only the 14CO2; however, added ribonucleotides reduced both the 14CO2 and the 14C incorporated into the progeny bdellovibrios. No similar effects were found with added ribose-5-phosphate. The distribution of 14C in the major cell components was similar in progeny bdellovibrios whether obtained from growth on [U-14C]ribose-labeled E. coli BJ565 or from E. coli plus added U-14C-labeled ribonucleotides. After intraperiplasmic growth of B. bacteriovorus on [5,6-3H-]uracil-[U-14C]ribose-labeled E. coli BJ565 (normal or heat treated), the whole-cell 14C/3H ratio of the progeny bdellovibrios was some 50% greater and reflected the higher 14C/3H ratios found in the cell fractions. B. bacteriovorus and E. coli cell extracts both contained 5'-nucleotidase, uridine phosphorylase, purine phosphorylase, deoxyribose-5-phosphate aldolase, transketolase, thymidine phosphorylase, phosphodeoxyribomutase, and transaldolase enzyme activities. The latter three enzyme activities were either absent or very low in cell extracts prepared from heat-treated E. coli cells. It is concluded that during intraperiplasmic growth B. bacteriovorus degrades some 20 to 40% of the ribonucleotides derived from the initial E. coli RNA into the base and ribose-1-phosphate moieties. The ribose-1-phosphate is further metabolized by B. bacteriovorus both for energy production and for biosynthesis, of non-nucleic acid cell material. In addition, the data indicate that during intraperiplasmic growth B. bacteriovorus can metabolize ribose only if this compound is available to it as the ribonucleoside monophosphate.

Sensitivity of scanning electron microscopy for the detection of mycoplasma contamination

Mycoplasma contamination of cell cultures causes a serious problem in the interpretation of experimental data. These organisms interfere with cell metabolism, induce heritable chromosome abnormalities, alter the antigenicity and function of the cell membrane, contaminate virus preparations and make the interpretation of cell surface structures difficult. Mycoplasmas can be isolated by direct culture. However certain strains will not grow in cell-free media. Indirect methods based on the staining of cultures and the assay of uridine phosphorylase enzyme activity require large numbers of organisms to yield positive results. With SEM, individual particles or clusters on only a few cells in the culture can be visualized.The actual sensitivity of SEM as a detection method is difficult to evaluate since many mycoplasmas grow in aggregates and reproduce by budding or the breaking apart of filaments as in Fig. 1.

Studies on the effect of oral hypoglycaemic agents on hepatic glycogenolysis

The effect of three sulphonamide derivatives, tolbutamide, acetohexamide and glycodiazine and two of the biguanide derivatives, phenformin and metformin on hepatic glycogenolysis was investigated in the rat. The in vitro studies in both normal and alloxan-diabetic rats reveal that tolbutamide, acetohexamide and glycodiazine inhibited hepatic glucose-6-phosphatase enzyme activity while phenformin and metformin produced no effect. Hepatic phosphorylase enzyme activity was inhibited by tolbutamide and glycodiazine, while acetohexamide, phenformin and metformin failed to produce any effect. The in vivo studies showed that an acute dose of any of the sulphonamide derivatives studied did not affect the two enzyme activities in both normal and alloxan-diabetic rats. However, the two biguanides, phenformin and metformin although not affecting the enzyme activities in normal animals, they produced significant decrease in glucose-6-phosphatase enzyme activity in livers of diabetic rats. The conclusion is reached that sulphonamide derivatives affect the glycogenolytic enzymes directly only in vitro , while a single, moderately big, hypoglycaemic dose produced no effect in vivo . On the other hand, the two biguanides affect glucose-6-phosphatase enzyme activity in diabetic rats by increasing glucose utilization, an effect not related to insulin.

Glycogen synthesis in rat diaphragm in vivo: a biphasic effect of insulin on glycogen synthetase enzyme.

AbstractDuring enhanced glycogen synthesis an increase in the activity of the enzyme rate‐limiting for glycogen synthesis is expected. This enzyme, glycogen synthetase (UDP‐glucose: α‐1,4‐glucan, α‐4‐glucosyltransferase; EC 2.4.1.11), can be activated by transformation of its D‐form to I‐form. Insulin is known to stimulate glycogen synthesis in skeletal muscle. However, Søvik did not find any change in I‐form in the rat diaphragm 30–240 min after an intraperitoneal (i.p.) injection of insulin, although glycogen synthesis was highly stimulated (O. Søvik, Acta physiol. scand. 1966. 68. 246–254). In the present study glycogen content and synthetase enzyme activity were measured in rat diaphragms 10–120 min after i.p. injection of insulin. Phosphorylase enzyme activity was also measured. Insulin administration produced a biphasic change in the synthetase enzyme; compared to controls there was an increase of the % I‐form after 10 and 20 min and a decrease after 50, 80 and 120 min. Phosphorylase enzyme activity was unaffected. Glycogen increased continuously up to 120 min. Thus, the increase in I‐form was shown to be a transient effect of insulin. In Søvik's experiment this increase had probably subsided at the time of the first measurement. To account for the increase in glycogen when the % I‐form was decreased, an activation of the synthetase enzyme through an increase of its activator, glucose‐6‐phosphate, may be considered.

Effect of insulin on glycogen synthesis and synthetase enzyme activity in the perfused rat heart

Insulin stimulates glycogen synthesis in the heart in vivo and in vitro. During insulin stimulation an activation of the glycogen synthetase enzyme (UDP-glucose: α-1, 4-glucan α-4-glucosyltransferase, EC 2.4.1.11) is expected since this enzyme is rate limiting for the synthesis of glycogen. However, in vitro such an activation was not found by Huijing et al., who perfused isolated rat hearts with insulin for 30 min (F. Huijing, F.O. Nuttall, C. Villar-Palasi and J. Larner, Biochim. Biophys. Acta, 177 (1969) 204). In the present study isolated rat hearts were perfused for 2–60 min with and without insulin. With insulin in the perfusate an activation of the synthetase enzyme by means of an increase in its active I-form was observed after 5 and 10 min of perfusion but not after 30 and 60 min. Glycogen was rapidly accumulated and after only 2 min of perfusion with insulin there was a significant effect. After 30 min no further increase in glycogen was obtained with insulin. The I-form of the synthetase enzyme was inversely related to the glycogen level. Insulin changed this relationship so that for a given glycogen level there was a higher level of I-form. Glucose 6-phosphate, considered to be an activator of the synthetase enzyme, was increased by insulin. Incorporation of labelled glucose was also increased by insulin. Phosphorylase enzyme activity was unaffected. Thus, in stimulating cardiac glycogen synthesis in vitro insulin activates the synthetase enzyme by increasing the I-form. An additional activation may be exerted by the increased level of glucose 6-phosphate found in presence of insulin.

Effect of foreign innervation on the androgen-sensitive levator ani muscle of the rat.

Cross-union of the tibial with the pudendal nerve innervating the androgen-sensitive levator ani (LA) muscle of male rats, results in reversal of the histochemical muscle fibre pattern concerning myofibrillar ATPase, succinate dehydrogenase and phosphorylase enzyme activities. The homogeneous muscle fibre pattern of the LA muscle is changed to a mosaic pattern of muscles normally innervated by the tibial nerve. The success of the hetero-reinnervation is shown by practically full recovery of muscle weight and of isometric twitch-contraction properties of the LA muscle. Castration of 2-months duration, i. e. lack of the male sex hormone, leads to marked atrophy but no change in histochemical muscle fibre pattern. Hetero-reinnervation of the LA muscle results in change of histochemical enzyme pattern even if the cross-union of nerves is performed after long periods of castration leading to very marked decrease of muscle fibre size. However, testosterone application alone after castration increases markedly muscle fibre size but does not lead to reversal of muscle fibre pattern. The myotropic hormonal influence on the target (LA) muscle is therefore primarily of myogenic origin and specificity of hormonal action is maintained even with a foreign nerve innervating the muscle. The experiments, thus, provide evidence for the differentiation of specific neural influences affecting muscle fibre pattern and hormonal influences in respect to the myotropic action of the sex hormone on the androgen-sensitive LA muscle.