Clinico-biochemical and molecular studies of purine nucleoside phosphorylase deficiency.
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Blotting, Northern↗
Biomedical subjects
A Akatsuka
Publications and source records attributed to A Akatsuka.
Restoration of myelin formation by a single type of myelin basic protein in transgenic shiverer mice.
A minigene containing mouse cDNA coding for the smallest type of myelin basic protein and including the native promoter was constructed and used to produce transgenic shiverer mice. The hypomyelinating mouse, the shiverer, has a deletion in its myelin basic protein gene, lacks all four types of myelin basic protein in its myelin, and shows abnormal behavior such as violent tremors. Five of twenty-one transgenic shiverer mice showed recovered protein synthesis, compact myelin formation, and normal behavior. These results suggest that a single type of myelin basic protein restores myelin formation and returns the shivering phenotype to normal in the transgenic shiverer mouse.
Animals↗
A newly modified isolation method of single muscle fibers--especially useful in histological, histochemical and electron microscopic studies on branched fibers.
The nitric acid muscle fiber digestion method has generally been used to determine total numbers of muscle fibers and branched fibers. However, it is not suitable for further studies because nitric acid causes protein denaturation known as the xanthoproteic reaction which make it difficult to examine the fiber types and morphology of muscle fibers. Therefore, we attempted to modify this method to preserve the morphology of muscle fibers as much as possible. Our modifications were as follows: (1) samples were immersed in ion exchanged water before nitroc acid treatment; (2) mammalian relaxing solution was used as the post- incubation solution; (3) the temperature of nitric acid was maintained at 4 degrees C; and (4) the nitric acid concentration was reduced to 10% from 15%. The samples obtained by this method were stained with phosphotungstic acid-hematoxylin and their striations were examined by a differential interference contrast method using light microscopy. The cell organella i.e. actin filaments, myosin filaments, T-tubules and mitochondria, were also examined electron microscopy. Actin and myosin filaments in these samples were also stained immunohistochemically to clarify the preservation of antigenicity. As a result, this modified method made it possible to examine actin and myosin filaments of a single muscle fiber light-microscopically and immunohistochemically and also to examine cell organella of a single muscle by electron microscopy. These results indicate that our method is useful for studies on branched muscle fibers such as stereological analysis and innervation of a single branched muscle fiber, in addition to obtaining muscle fiber numbers.
Actins↗
Subcellular localization of acid sphingomyelinase and lipid in Niemann-Pick mice.
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Animals↗
The outer membrane of Pseudomonas aeruginosa is a barrier against the penetration of disaccharides.
The outer membrane of Pseudomonas aeruginosa acted as a barrier against the penetration of di- (Mr, 342), tri- (Mr, 504) and tetrasaccharides (Mr, 666), whereas the membrane allowed the penetration of pentose (Mr, 150) and methylhexoses (Mr, 194) into the periplasm. When the intact cells of P. aeruginosa were treated with 600 mosM saccharides of various sizes and observed under an electron microscope, saccharides of Mr larger than 342 caused the extensive shrinking of the outer membrane. Whereas the cells treated with the saccharides of Mr less than 194 or with sucrose in the presence of EDTA showed plasmolysis. Determination of the extent of saccharide penetration into the periplasm of the cells treated with 600 mosM sodium chloride or with 600 mosM saccharides of various sizes showed that only pentose and hexoses, so far examined, were penetrable but di-, tri- and tetrasaccharides were impenetrable.
Disaccharides↗
Biochemical and morphological studies of human diploid and fluoride-resistant fibroblasts in vitro.
Fluoride-resistant (FR 30) lines were produced from human diploid fibroblast (NDU-1) cells by progressively increasing the concentration of fluoride (F) in the growth medium up to 1.58 mM. This concentration completely inhibited the growth of the original cells. The resistant cells had decreased incorporation of [14C]-leucine and an increased rate of the cell division. The activities of acid phosphatase, alkaline phosphatase, lactate dehydrogenase, leucine aminopeptidase, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and alpha-hydroxybutyrate dehydrogenase in the FR 30 cells were lower than in the NDU-1 cells. The FR 30 cells had irregular shapes and sizes; the amount of undeveloped rough endoplasmic reticulum and the number of lysosomes were increased. These biochemical and morphological changes in FR 30 cells suggest that their metabolic activities were depressed by F, although they have some degree of F resistance.
Cell Division↗
Glucagon-stimulated phosphorylation of rat liver glycogen synthase in isolated hepatocytes.
Addition of glucagon (20 nM) to the isolated hepatocytes from 24-h starved male rats results in an inactivation of glycogen synthase. The A0.5 for glucose-6-P is increased 2-fold over the control but the S0.5 for UDP-glucose is not significantly affected. The glucagon-stimulated inactivation of glycogen synthase is also accompanied by a 60-120% increase in the phosphorylation of the synthase. Glycogen synthase labeled with 32P by incubation of the hepatocytes with [32P] PO4(3-) was recovered by immunoprecipitation and the resulting immunoprecipitate was subjected to tryptic digestion. Analysis of the 32P-labeled peptides reveals that the sites corresponding to those phosphorylated by cAMP-dependent protein kinase and glycogen synthase (casein) kinase-1 (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057) are rapidly phosphorylated in response to glucagon. These results demonstrate that glucagon not only triggers the activation of cAMP-dependent protein kinase through an increase in the intracellular level of cAMP but also, by an unknown mechanism, activates a Ca2+- and cAMP-independent protein kinase.
Animals↗
Phosphorylation of rat liver glycogen synthase bound to the glycogen particle.
Rat liver glycogen synthase bound to the glycogen particle was partially purified by repeated high-speed centrifugation. This synthase preparation was labeled with 32P by incubations with cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1 in the presence of [gamma-32P]ATP. The phosphorylated synthase was separated from other proteins in the glycogen pellet by immunoprecipitation with rabbit anti-rat liver glycogen synthase serum. Analysis of the immunoprecipitates by sodium dodecyl sulfate-gel electrophoresis showed that synthase subunits of Mr 85,000 and 80,000 were present in varying proportions. The 32P-labeled synthase in the immunoprecipitate was digested with trypsin, and the resulting peptides were analyzed by isoelectric focusing. Synthase bound to the glycogen particle was phosphorylated by cAMP-dependent protein kinase at more sites and by cAMP-independent synthase (casein) kinase-1 at less sites than when the homogeneous synthase was incubated with these kinases. Phosphorylation of synthase in the glycogen pellet by either cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1 did not cause a significant inactivation as has been observed when the synthase was incubated with these kinases. Inactivation of synthase in the glycogen pellet, however, can be achieved by the combination of both kinases. This inactivation appears to result from the phosphorylation of a new site by cAMP-independent synthase (casein) kinase-1 neighboring a site previously phosphorylated by cAMP-dependent protein kinase.
Animals↗
Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1.
We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.
Animals↗
Phosphorylation of rat liver glycogen synthase by phosphorylase kinase.
Phosphorylation of rat liver glycogen synthase by rabbit skeletal muscle phosphorylase kinase results in the incorporation of approximately 0.8-1.2 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and thin layer chromatography reveal the presence of two major 32P-labeled peptides. Similar results were obtained when the synthase was phosphorylated by rat liver phosphorylase kinase. This extent of phosphorylation does not result in a significant change in the synthase activity ratio. In contrast, rabbit muscle glycogen synthase is readily inactivated by rabbit muscle phosphorylase kinase; this inactivation is further augmented by the addition of rabbit muscle cAMP-dependent protein kinase or cAMP-independent synthase (casein) kinase-1. Addition of cAMP-dependent protein kinase after initial phosphorylation of liver synthase with phosphorylase kinase, however, does not result in an inactivation or additional phosphorylation. The lack of additive phosphorylation under this condition appears to result from the phosphorylation of a common site by these two kinases. Partial inactivation of liver synthase can be achieved by sequential phosphorylation with phosphorylase kinase followed by synthase (casein) kinase-1. Under this assay condition, the phosphate incorporation into the synthase is additively increased and the synthase activity ratio (-glucose-6-P/+glucose-6-P) is reduced from 0.95 to 0.6. Nevertheless, if the order of the addition of these two kinases is reversed, neither additive phosphorylation nor inactivation of the synthase is observed. Prior phosphorylation of the synthase by phosphorylase kinase transforms the synthase such that it becomes a better substrate for synthase (casein) kinase-1 as evidenced by a 2- to 4-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase appears to result from the rapid phosphorylation of a site neighboring that previously phosphorylated by phosphorylase kinase.
Animals↗
Morphology and function of human neuroblastoma xenotransplanted in nude mice.
Nine human neuroblastomas were serially transplanted in nude mice. All transplanted tumors were taken from patients older than the age of 1 year 10 months. In contrast, the authors were unable to transplant tumors from patients younger than 1 year 7 months of age, in spite of many attempts. All transplanted tumors produced varying amounts of cathecolamines as revealed by the liquid chromatography and formalin-induced fluorescence techniques. The content of the cathecolamines was closely correlated with the number and size of secretory granules. In high cathecolamine-producing tumors, cell clusters showing strong yellow-orange fluorescence plus a positive argyrophil reaction suggesting pheochromoblastic differentiation were demonstrated by our combined cathecolamine fluorescence and argyrophil reaction method. One of the transplantable tumor lines was demonstrated by unique host responses and biochemical data, to produce adrenocorticotropic hormone and increased cathecolamines. Our observations indicate that neuroblastoma derived from neural crest cells seem to have a nearly complete hormone production function as compared with their normal counterparts.
Adrenal Gland Neoplasms↗
Cyclic nucleotide- and Ca2+-independent phosphorylation of tubulin and microtubule-associated protein-2 by glycogen synthase (casein) kinase-1.
MAP-2 and tubulin are both shown to be substrates for glycogen synthase (casein) kinase-1 (CK-1). Greater than 40 mol 32P is incorporated into MAP-2 by CK-1 compared to only 14 mol 32P observed when cyclic AMP-dependent protein kinase (A-kinase) is the catalyst. Peptide mapping shows that CK-1 and A-kinase recognize a few common sites; the majority of the sites phosphorylated on MAP-2 by CK-1 are quite distinct. Up to 4 mol 32P can be incorporated into the tubulin dimer by CK-1 compared to only 0.9 mol 32P by A-kinase. The preferred substrate for both kinases is beta-tubulin.
Animals↗
Phosphorylation and inactivation of rabbit skeletal muscle glycogen synthase: distinction between kinase Fa-, phosphorylase kinase-, and glycogen synthase (casein) kinase-1-catalyzed reactions.
Rabbit skeletal muscle glycogen synthase was phosphorylated by kinase Fa, phosphorylase kinase, and cAMP-independent synthase (casein) kinase-1 to determine the differences among these kinase-catalyzed reactions. The stoichiometry of phosphate incorporation, the extent of inactivation, and the sites of phosphorylation were compared. Synthase (casein) kinase-1 catalyzes the highest level of synthase phosphorylation (4 mol/subunit) and inactivation (reduction of the activity ratio to below 0.05). The sites, defined by characteristic tryptic peptides, phosphorylated by synthase (casein) kinase-1 are distinguishable from those by kinase Fa and phosphorylase kinase. In addition, synthase (casein) kinase-1, unlike kinase Fa, does not activate ATP X Mg2+-dependent protein phosphatase. These results demonstrate that synthase (casein) kinase-1 is a distinct glycogen synthase kinase.
Animals↗
Phosphorylation of smooth muscle myosin light chain by five different kinases.
Phosphorylation of the 20 kDa myosin light chain from smooth muscle by five different kinases was investigated. Three of the kinases (myosin light chain kinase, phosphorylase kinase, and cAMP-dependent protein kinase) phosphorylate serine residues, the fourth (casein-kinase-2) mainly threonine, and the fifth (glycogen synthase (casein) kinase-1) both serine and threonine. Isoelectric focusing analyses of 32P-labelled chymotryptic peptides indicate that phosphorylase kinase and cAMP-dependent protein kinase phosphorylate the same site as myosin light chain kinase. However, both casein kinase-2 and glycogen synthase (casein) kinase-1 phosphorylate different sites.
Animals↗
Phosphorylation and inactivation of rat liver glycogen synthase by cAMP-dependent protein kinase and cAMP-independent synthase (casein) kinase-1.
Phosphorylation of liver glycogen synthase by cAMP-dependent protein kinase (A-kinase) results in the incorporation of approximately 0.7 to 1.0 mol of PO4/subunit. Analyses of the tryptic peptides by isoelectric focusing and peptide mapping reveal the presence of a single 32P-labeled peptide. This extent of phosphorylation does not result in a significant reduction of the synthase activity ratio. Phosphorylation of the liver synthase by cAMP-independent synthase (casein) kinase-1 results in the incorporation of 1.6 to 2.2 mol of PO4/subunit. Although at least 4 tryptic peptides have been found to be labeled with 32P, no significant reduction of the synthase activity ratio was observed. Under the same assay conditions, the muscle synthase is effectively inactivated by either kinase alone or in combination. Inactivation of liver synthase can be achieved after phosphorylation by A-kinase and followed by synthase (casein) kinase-1. However, the inactivation becomes less effective if the order of the addition of these two kinases is reversed. Under the latter assay condition, the phosphate incorporation is less than additive in the presence of both kinases. Prior phosphorylation of the synthase by A-kinase transforms the synthase to become a better substrate for synthase (casein) kinase-1 as evidenced by a 3- to 5-fold increase in the rate of phosphorylation. This increased rate of phosphorylation of the synthase by synthase (casein) kinase-1 results from the rapid phosphorylation of a site neighboring to that previously phosphorylated by A-kinase.
Animals↗
Comparison of the liver glycogen synthase from normal and streptozotocin-induced diabetic rats.
Glycogen synthase in the liver extracts of short-term (3 days) streptozotocin-induced diabetic rats is poorly activated by the endogenous synthase phosphatase as well as phosphatase(s) from the liver extracts of normal animals. However, synthase in the liver extracts of diabetic rats is readily activated by the 35,000 Mr rabbit liver protein phosphatase (H. Brandt, F.L. Capulong, and E. Y. C. Lee (J. Biol. Chem. 250, 8038-8044 (1975)). The purified synthases from normal and diabetic animals respond differently after incubations with three different phosphatases. Both normal and diabetic synthase are activated by the 35,000 Mr protein phosphatase; however, the total activity of diabetic, but not the normal, synthase is significantly increased. Normal, but not the diabetic, synthase is activated by a synthase phosphatase from normal rats; this activation is accompanied by an increase in total synthase activity. Incubation of the diabetic synthase with calf intestinal alkaline phosphatase results in a reduction of the total synthase activity, whereas synthase activity of the normal is not significantly affected. The reduction in total activity of the diabetic synthase by treatment with alkaline phosphatase was prevented by prior dephosphorylation with 35,000 Mr rabbit liver protein phosphatase. In addition to their differences in responses to different phosphatases, the normal and diabetic synthases are also different in their molecular weights as determined by sucrose density gradient centrifugation (154,000 +/- 17,000 (n = 6) for the normal and 185,000 +/- 15,000 (n = 8) for the diabetic synthase) and their kinetic properties.
Animals↗
Phosphorylation of troponin and myosin light chain by cAMP-independent casein kinase-2 from rabbit skeletal muscle.
Casein kinase-2 from rabbit skeletal muscle was found to phosphorylate, in addition to glycogen synthase, troponin from skeletal muscle, and myosin light chain from smooth muscle. Troponin T and the 20,000 Mr myosin light chain are phosphorylated by casein kinase-2 at much greater rates than glycogen synthase. The V values for the phosphorylation of troponin and myosin light chain are nearly an order of magnitude greater than that of glycogen synthase; however, the Km values for these two substrates are greater than that for glycogen synthase. The kinase activities with the various protein substrates are stimulated approximately three- and fivefold by 5 mM spermidine and 3 mM spermine, respectively. Heparin is a potent inhibitor of the kinase when casein, glycogen synthase, or myosin light chain is the substrate. However, with troponin as substrate the kinase is relatively insensitive to inhibition by heparin. The amount of heparin required for 50% inhibition with troponin as substrate is at least 10 times greater than with casein as substrate. The phosphorylation of troponin by casein kinase-2 results in the incorporation of phosphate into two major tryptic peptides, which are different from those phosphorylated by casein kinase-1. The site in myosin light chain phosphorylated by casein kinase-2 is different from that phosphorylated by myosin light chain kinase.
Animals↗
Phosphorylation and activation of rabbit skeletal muscle phosphorylase kinase by a cyclic nucleotide- and Ca2+-independent protein kinase.
Phosphorylase kinase from rabbit skeletal muscle can be phosphorylated and activated by a cyclic nucleotide- and Ca2+-independent protein kinase previously identified as a glycogen synthase kinase (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This independent kinase phosphorylates the beta subunit of phosphorylase kinase approximately 15 times faster than it does the alpha subunit. The cAMP-dependent and -independent kinases separately catalyze the incorporation of 1 mol of phosphate into the beta subunit. Analyses of the tryptic peptides from the beta subunit phosphorylated with either kinase by isoelectric focusing and peptide mapping indicate that both kinases phosphorylate the same site on the beta subunit. Activation of phosphorylase kinase catalyzed by the independent kinase is only 60% of that observed with cAMP-dependent kinase. If phosphorylase kinase is first incubated with the independent kinase to phosphorylate the beta subunit, subsequent addition of cAMP-dependent kinase results in a predominant phosphorylation of the alpha subunit. This additional phosphorylation of the alpha subunit is accompanied by a further activation of the alpha subunit is accompanied by a further activation of phosphorylase kinase to the same extent as that achieved by cAMP-dependent kinase alone. Hence, the phosphorylation of the alpha subunit is clearly required for full activation of phosphorylase kinase, even at low [Mg2+].
Animals↗