Effect of vitamin D status on cyclic AMP-dependent protein kinase activity and its heat-stable inhibitor in chick kidney

Cyclic AMP-dependent protein kinase activity in supernatants of homogenates of kidneys from vitamin D-deficient chicks is decreased to 70% of the level measured in kidneys from normal chicks. Activity was restored to normal by oral administration of vitamin D or 1,25-dihydroxyvitamin D3 for 1 or 2 weeks. Both isozymes of cAMP-dependent protein kinase were reduced to the same extent by vitamin D deficiency. The decreased enzyme activity could not be accounted for by a shift to the particulate fraction nor by an increased requirement for cyclic AMP. A heat stable, trichloroacetic acid-precipitable, trypsin-labile inhibitor of protein kinase activity was identified and quantitated in kidneys from vitamin D-deficient chicks (16 to 26 units/mg of protein) and from those given vitamin D (2 to 6 units/mg of protein). The measured difference in inhibitor levels could not be attributed to differential stability in kidney homogenates from vitamin D-deficient or -repleted chicks. The observed increase in inhibitor level with vitamin D deficiency is not sufficient to account for the decrease in cyclic AMP-dependent protein kinase activity, suggesting that the total amount of this enzyme activity is reduced in vitamin D deficiency.     

Stimulation of creatine kinase BB activity by 1 alpha,25-dihydroxycholecalciferol and 24R,25-dihydroxycholecalciferol in rat tissues.

Vitamin D metabolites stimulate creatine kinase BB activity in organs of vitamin D-deficient rats. In epiphyses of long bones, creatine kinase BB activity increases 2.6-fold 24 h after injection of 24R,25-dihydroxycholecalciferol but not of 1 alpha,25-dihydroxycholecalciferol. Contrariwise, 1 alpha,25-dihydroxycholecalciferol, but not 24R,25-dihydroxycholecalciferol, increases creatine kinase BB activity in diaphyses and in kidney. Neither metabolite affects creatine kinase activity in duodenal mucosa.     

Kinetic characteristics of creatine kinase and hexokinase from rat skeletal muscles during dietary deficit of vitamin K and administration of pelentane]

Kinetic parameters of rat creatine kinase isozymes at different vitamin K supply and treatment with antivitamin K--pelentan have been determined. MM-isozyme (skeletal muscle) has selective sensitivity to the vitamin K deficit, while BB and MB-isozymes (brain, kidney and heart) have not. The value KM for ATP of MM-isozymes increases, while maximal activity decreases. Pelentan treatment does not lead to the change of MM-creatine kinase affinity to ATP. Soluble hexokinase of skeletal muscle in rats with vitamin K deficiency and treated with pelentan has higher affinity to glucose as compared to normal rat enzyme. It has been supposed that skeletal muscle hexokinase exists in a particular molecular form under vitamin K deficiency.     

Acute stimulation of creatine kinase activity by vitamin D metabolites in the developing cerebellum

There is increasing evidence that vitamin D metabolites have a developmental function. We have investigated the influence of the vitamin D status on the activity of creatine kinase in the brain. Normally fed rats show an increase in the specific activity of cerebral and cerebellar creatine kinase during postnatal development. Vitamin-D-depleted rats failed to show this normal increase. Developing cerebellum, but not cerebrum, in both vitamin D-depleted rats and in normally fed animals, responded sequentially to a single injection of a vitamin D metabolite by displaying increased creatine kinase specific activity. In 5-25-day-old rats, 24R,25-dihydroxyvitamin D-3 significantly increased creatine kinase specific activity 24 h after injection. In contrast, 1,25-dihydroxyvitamin D-3 stimulated cerebellar creatine kinase activity from 20 days after birth. A similar pattern of sequential responsiveness to vitamin D metabolites, but at an earlier age, was shown in the cerebellum of the rabbit, which is a 'perinatal brain developer' compared to the rat, a 'postnatal brain developer'. Because of the difficulty in obtaining vitamin D-depleted rabbits, studies were carried out in normally fed animals. In these rabbits, 24R,25-dihydroxyvitamin D-3 stimulated cerebellar creatine kinase activity between 6 days before birth and 9 days after birth, while 1,25-dihydroxyvitamin D-3 caused an increase in cerebellar creatine kinase specific activity from 8 days after birth. These developmental differences found in creatine kinase basal activity and responsiveness are correlated with differences in cellular growth rates, both in the rabbit and in the rat, suggesting that vitamin D metabolites may be required for optimal cerebellar development.     

Cyclic AMP dependent protein kinase and its endogenous inhibitor protein: tissue distribution and effect of vitamin D status in the chick

1. Vitamin D deficiency in the chick leads to decreased (to 55% of normal) cyclic AMP-dependent protein kinase activity in the kidney but does not alter calcium-dependent phospholipid-sensitive protein kinase activity. 2. Decreased cyclic AMP-dependent protein kinase activity in response to vitamin D deficiency was not observed in other tissues including pancreas, brain, liver, intestinal mucosa, or heart. 3. Vitamin D deficiency leads to elevated levels of the endogenous inhibitor protein of cyclic AMP-dependent protein kinase in kidney, but not heart, muscle, pancreas, or brain.     

Effect of ascorbic acid on 25-hydroxyvitamin D3 metabolism in the kidneys and 1,25-dihydroxyvitamin D3 reception in the small intestine mucosa of guinea pigs

Ascorbic acid deficiency in vitamin D-supplied guinea pigs caused a moderate decrease of Ca in the blood and osseous tissue, a 1.5-fold decrease of 2.5-hydroxyvitamin D (25-OH D) in blood serum, a 2-fold decrease of the 25-OH D 1-hydroxylase activity in kidneys and a 1.6-fold increase of the 24-hydroxylase activity. The concentration of 1.25-dihydroxyvitamin D3 (1.25-(OH)2D3) nuclear receptors in small intestinal mucosa diminished by 20-30%; in this case the percentage of occupied hormone receptors reduced from 11.8 to 8.6%. The affinity of receptors for 1.25-(OH)2D3 did not change thereby (Kd = 0.25-0.26 nM; Kd2 = 0.06-0.10 nM). At the same time the value of cooperativity coefficient showed a decrease-from 1.7 to 1.4, which was accompanied by a reduction of the maximum capacity of receptors (1.2-1.5-fold). Vitamin C depletion augmented the manifestation of vitamin D deficiency in guinea pigs and impeded their correction after administration of cholecalciferol. This markedly retarded the restoration of the 25-OH D level in the blood as well as the number of occupied and unoccupied nuclear receptors for 1.25-(OH)2D3. The experimental results illustrate the effects of ascorbic acid on the vitamin D hormonal system function, which is manifested both at the level of 1.25-(OH)2D3 synthesis in the kidneys and of its receptor binding in target tissues.     

Glycogen phosphorylase and its converter enzymes in haemolysates of normal human subjects and of patients with type VI glycogen-storage disease. A study of phosphorylase kinase deficiency.

1. The properties of phosphorylase a, phosphorylase b, phosphorylase kinase and phosphorylase phosphatase present in a human haemolysate were investigated. The two forms of phosphorylase have the same affinity for glucose 1-phosphate but greatly differ in Vmax. Phosphorylase b is only partially stimulated by AMP, since, in the presence of the nucleotide, it is about tenfold less active than phosphorylase a. In a fresh human haemolysate phosphorylase is mostly in the b form; it is converted into phosphorylase a by incubation at 20degreesC, and this reaction is stimulated by glycogen and cyclic AMP. Once activated, the enzyme can be inactivated after filtration of the haemolysate on Sephadex G-25. This inactivation is stimulated by caffeine and glucose and inhibited by AMP and fluoride. The phosphorylase kinase present in the haemolysate can also be measured by the rate of activation of added muscle phosphorylase b, on addition of ATP and Mg2+. 2. The activity of phosphorylase kinase was measured in haemolysates obtained from a series of patients who had been classified as suffering from type VI glycogenosis. In nine patients, all boys, an almost complete deficiency of phosphorylase kinase was observed in the haemolysate and, when it could be assayed, in the liver. A residual activity, about 20% of normal, was found in the leucocyte fraction, whereas the enzyme activity was normal in the muscle. These patients suffer from the sex-linked phosphorylase kinase deficiency previously described by others. Two pairs of siblings, each time brother and sister, displayed a partial deficiency of phosphorylase kinase in the haemolysate and leucocytes and an almost complete deficiency in the liver. This is considered as being the autosomal form of phosphorylase kinase deficiency. Other patients were characterized by a low activity of total (a+b) phosphorylase and a normal or high activity of phosphorylase kinase in their haemolysate.     

Changes in kinetic properties of pyridoxal-dependent enzymes during dietary vitamin B6 deficiency in rats

The erythrocyte aspartate aminotransferase and renal and intestinal glycogen phosphorylase activities in rats are determined as dependent on their provision with vitamin B6. It has been shown that the aspartate aminotransferase activity decreases and the shape of the aspartate concentration-activity curve changes in the vitamin B6-deficient animals. The B6 insufficiency does not affect the intestinal mucosa glycogen phosphorylase. However the renal phosphorylase activity decreases by 30 percent in the vitamin B6 deficient rats. It occurs due to changes in the affinity of phosphorylase A and B to glucose-1-phosphate but not to AMP. The activation of these investigated enzymes by exogenous pyridoxal phosphate reveals no essential differences between the vitamin B6-deficient and normal rats. The possible causes of the observed changes in the aspartate aminotransferase and phosphorylase activity are discussed.     

The biochemical and structural maturation of human skeletal muscle cells in culture: The effect of the serum substitute Ultroser G

On the basis of the percentage creatine kinase-MM, human skeletal muscle cells cultured on growth and differentiation media containing the serum substitute Ultroser G reach a significantly higher maturation grade after 7 days of differentiation than cells cultured on serum-containing media. They also remain viable for longer periods. The myotubes are much longer, their nuclei are often localized in rows on the periphery, and they show cross-striation more frequently. The activities of creatine kinase, citrate synthase, cytochrome c oxidase, AMP deaminase, and phosphorylase are significantly higher. Extending the differentiation period to 3 weeks increases the maturation grade of the cultures and the activities of all the enzymes mentioned before, except phosphorylase. A correlation exists between the enzyme activities and the maturation grade of the muscle cells. The content of fatty acid-binding protein also increases significantly with the maturation grade in contrast to the palmitate oxidation rate. The AMP deaminase and creatine kinase activity and the percentage MM-type remain lower in cultured cells than in adult muscle and the hexokinase activity remains higher, but the other enzyme activities become comparable after 20 days of differentiation. The myotubes, derived from Ultroser G-containing culture media, show spontaneous contractions after 12 days and cross-striation after 20 days when immunostained for the M-subunit of creatine kinase. These cells possess clusters of acetylcholine receptors, but aggregation of desmin at the site of the clusters was never detectable. The possibility of cultivating muscle cells with a predictable maturation grade allows the study of muscle development and muscular diseases caused by differentiation defects or by deficiency of a maturation-dependent (iso)enzyme.     

Effect of fluorine on enzyme activity in the small intestine mucosa during absorption of sodium, potassium, monosaccharides and amino acids

Effect of NaF on ATP-ase, creatine kinase, acid and basic phosphatases activity of small intestine mucosa in white rats during enteral administration of NaCl, KCl solutions has been studied under conditions of acute experiment. Inhibition of the Mg2+-ATP-ase activity by 13%, general ATP-ase activity by 25%, creatine kinase by 22% and a 2.2-3 fold inhibition of Na, K+-ATP-ase activity is observed. Acid and basic phosphatase activity does not change, swelling of mucosa is observed. The acid phosphatase activity in the intestinal contents increases 1.5 times, basic phosphatase activity--1.7 times, creatine amount--1.7 times, Pi amount 1.8 times. In experiments in vitro F- produces 20% activation of the basic phosphatase of mucosa and a 2.6-fold inhibition of the acid phosphatase. Rate of fructose absorption falls by 34%, that of methionine--by 29%, glucose--by 24%, glutaminic acid--by 10%. Activity of general ATP-ase in this case decreases by 22, 15, 27, 20% respectively. It is supposed that the F- effect results in destabilization of the membrane structures of the intestine mucosa.     

The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system.

The effects in kidney of streptozotocin-induced diabetes and of insulin supplementation to diabetic animals on glycogen-metabolizing enzymes were determined. Kidney glycogen levels were approximately 30-fold higher in diabetic animals than in control or insulintreated diabetic animals. The activities of glycogenolytic enzymes i.e., phosphorylase (both a and b), phosphorylase kinase, and protein kinase were not significantly altered in the diabetic animals. Glycogen synthase (I form) activity decreased in the diabetic animals whereas total glycogen synthase (I + D) activity significantly increased in these animals. The activities were restored to control values after insulin therapy. Diabetic animals also showed a 3-fold increase in glucose 6-phosphate levels. These data suggest that higher accumulation of glycogen in kidneys of diabetic animals is due to increased amounts of total glycogen synthase and its activator glucose 6-phosphate.     

The role of vitamin E in metabolism and reception of vitamin D

It was found that calcium exchange disturbances under vitamin E deficiency is due to changes in the metabolism of vitamin D. In vitamin E-deficient rats the serum blood levels of hydroxyvitamin D (25-OHD) showed no significant changes, whereas the concentration of the hormonal form of 1.25-hydroxyvitamin D [1.25(OH)2D], decreased by 40%. In vitro studies showed that the 25-hydroxylase D3 activity in the livers of rats with E-avitaminosis had a tendency to decrease (by 22%), whereas that of 24-hydroxylase dropped drastically (by 52%). The serum blood levels of the parathyroid hormone (PTH) and kidney levels of cAMP under E-avitaminosis were significantly lowered. Preincubation of kidney slices with the adenylate cyclase activator, forskolin, increased the activity of 1-OHase in about the same degree as that in vitamin E-rich rats. The free radical scavenger, BHT, added to kidney slices suppressed the activity of the both enzymes; this finding testifies to the low O2-binding affinity of these monooxygenases. The content of 1.25(OH)2D3 receptors occupied in vivo in the kidneys of vitamin E-deficient rats decreased 2.5-fold; however, the binding of 1.25(OH)2D3-receptor complexes to heterologous DNA was unaffected thereby. The vitamin deficiency in vivo results in the inhibition of vitamin D metabolism in the liver and kidney concomitant with the formation of active metabolites and decreases the concentration of hormone-receptor complexes in target tissues.     

Hormonal and ionic control of the glycogenolytic cascade in rat liver.

1. A parallel dose-dependent activation of histone kinase, phosphorylase kinase and phosphorylase was observed in isolated hepatocytes incubated in the presence of glucagon; the effect of suboptimal concentrations of glucagon was antagonized by insulin. 2. An activation of phosphorylase which was not accompanied by a stable change in the activity of phosphorylase kinase was observed in hepatocytes incubated with phenylephrine, isoproterenol or vasopressin as well as on decapitation of unanesthetized animals. A dissociation of the two enzymic activities was also observed in hepatocytes incubated in the presence of a high concentration of glucose, in which phosphorylase was strongly inactivated with no change in the activity of phosphorylase kinase. 3. The activation of phosphorylase by phenylephrine in isolated hepatocytes was counteracted by insulin, greatly decreased by the absence of Ca2+ from the incubation medium, and completely suppressed by the replacement of Na+ by K+. 4. In a liver extract, phosphorylase kinase could also be activated by trypsin. Control, glucagon-activated or trypsin-activated phosphorylase kinase was inhibited by about 70% by EGTA and the activity was restored by the addition of Ca2+. 5. The mechanisms that control the activity of phosphorylase kinase and of phosphorylase are discussed.     

Differentiation in cultures derived from embryonic chicken muscle: II. Phosphorylase histochemistry and fluorescent antibody staining for creatine kinase and aldolase

The presence or absence of five proteins (glycogen phosphorylase, aldolase A, aldolase C, creatine kinase M, creatine kinase B) in the various classes of cells found in primary cultures derived from embryonic chick breast muscle was investigated using cytological staining methods. Histochemical staining for phosphorylase and indirect fluorescent antibody staining for aldolase A and C as well as for creatine kinases M and B showed the following: All five proteins were found in the many myotubes present in standard medium cultures and in the very few myotubes found in cultures containing 5-bromodeoxyuridine (10^?5M). The elongated bipolar cells prevented from fusing in medium containing EGTA also contain all five proteins. The flattened myogenic cells that predominate in the 5-bromodeoxyuridine-treated cultures contain no phosphorylase or creatine kinase M, though many of them contain creatine kinase B and aldolases A and C. These results are interpreted as indicating that: (1) phosphorylase and creatine kinase M, but not aldolase A, are suitable all-or-none markers for terminal muscle differentiation; (2) the small amounts of creatine kinase M detected in electrophoreses of 5-bromodeoxyruridine-treated cultures can be accounted for by the few myotubes present and are not due to “protodifferentiation” of large numbers of cells; (3) proteins typical of differentiated muscle are produced only in cells that have passed through the last step in myogenesis that is susceptible to 5-bromodeoxyuridine inhibition, and (4) if fusion is blocked by reducing the concentration of calcium ions, accumulation of characteristic muscle proteins can continue in those cells that have initiated terminal differentiation.     

The phosphorylase kinase activity of hearts from phosphorylase kinase-deficient mice

In an assay measuring radioactive incorporation from gamma--P32P]ATP into phosphorylase b, cardiac muscle extracts from mice with the phosphorylase kinase deficiency mutation showed significant, calcium-dependent phosphorylase kinase activity that was 10 to 15% of that of Swiss mice, the control strain. Isoproterenol stimulated significant phosphorylase a accumulation in both isolated atria and right ventricular strips of phosphorylase kinase-deficient mice, and the drug-stimulated increases in phosphorylase a activity the the contractile responses of right ventricular strips were similar in Swiss and phosphorylase kinase/deficient mice.     

The role of vitamin K in the interaction of 1,25-dihydroxyvitamin D3 receptors with DNA

Vitamin K deficiency in rats caused a rise of in vivo occupied 1,25(OH)2D3 receptor level in chromatin of the intestinal mucosa and a marked (2-2.5-fold) increase of intestinal cytosolic 1,25(OH)2D3-receptor complex binding with heterologous DNA, whereas maximum binding capacity and equilibrium dissociation constant of cytosolic 1,25 (OH)2D3 receptors did not change. Preincubation of renal and intestinal cytosol of vitamin K-deficient rats with microsomal vitamin K-dependent gamma-carboxylating system reduced sharply 1,25(OH)2D3-receptor complex binding with DNA. In rats treated by vitamin K antagonist along with a low calcium diet, no dramatic decrease of occupied 1,25(OH)2D3 receptors occurred after the animals were maintained with a high calcium diet. No such effect was observed in vitamin K-replete rats. The data demonstrate vitamin K-dependent Ca-sensitive qualitative modification of 1,25(OH)2D3 receptor dropping its binding performance to DNA.     

Regularities of organ-specific expression of enzyme systems in cattle]

The organ specificity of creatine kinase, esterase, isocitrate dehydrogenase lactate dehydrogenase, nucleoside phosphorylase, adenylate kinase, hexokinase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase of black-white cattle has been studied. Esterases, creatine kinase, adenylate kinase, hexokinase and glucose-6-phosphate dehydrogenase have a very wide spectrum of the organ variabilities. Liver and heart have the largest specificity of enzymes activity. Some peculiarities of isozyme spectrum are found in ovaries and spleen.     

Purification and localization of brain-type creatine kinase in sodium chloride transporting epithelia of the spiny dogfish, Squalus acanthias.

The targeting of creatine kinase isoenzymes to specific sites within muscle cells provides a system for the regeneration of ATP in situ from ADP and creatine phosphate. We have recently reported the colocalization of brain-type (B) creatine kinase and the nonsarcomeric mitochondrial creatine kinase isoenzymes in the thick ascending limb of the loop of Henle in the rat kidney, suggesting that creatine kinase may regenerate ATP for sodium transport (Friedman, D.L., and Perryman, M.B. (1991) J. Biol. Chem. 266, 22404-22410). In order to test the hypothesis regarding the association of B creatine kinase with sodium transport, we examined the creatine kinase enzymes in the rectal (salt-secreting) gland of the dogfish shark which contains high levels of the Na+/K(+)-ATPase. The creatine kinase isoform composition was determined by non-denaturing electrophoresis, immunoblotting, protein purification, and amino acid sequence analysis. The results demonstrate both B creatine kinase and mitochondrial creatine kinase proteins are present in the rectal gland, an isoform composition which is the same as in the mammalian kidney. By using a combination of chromatographic techniques, shark B creatine kinase was purified to homogeneity and partial sequence data was obtained from two cyanogen bromide peptide fragments. One of these fragments contains the active site and is identical at all sequenced residues with the corresponding region from the echinoderm sperm flagellar creatine kinase, and is 96% homologous with both chicken and rat B creatine kinase subunits. The other fragment corresponds to a region near the N-terminal of mammalian creatine kinases and is 89% homologous with B creatine kinase from chicken. The localization of these isoforms was examined by immunocytochemistry using subunit specific antisera. Mitochondrial creatine kinase and B creatine kinase immunoreactivity are detected in all tubules, and is restricted to the basal region of the cells, which is the site of the Na+/K(+)-ATPase. The conservation of creatine kinase isoform expression in excretory tissue, and the localization of creatine kinase immunoreactivity in the basal region of the tubule cells, demonstrate that subcellular compartmentation of B creatine kinase may underly the functional coupling of creatine kinase activity with sodium transport.     

Identification of glycogen phosphorylase and creatine kinase as calpain substrates in skeletal muscle

The goal of this study was to identify calpain substrates in muscle cells. Our hypothesis was that the yeast two-hybrid method could be used to identify novel calpain substrates. To accomplish this, native mu- and m-calpains, as well as a variety of calpain DNA fragments, were expressed in yeast cells and used to screen for binding proteins in a human skeletal muscle cDNA library. Calpain constructs that were used in the screening process included native mu- and m-calpains, a dominant negative (DN) m-calpain (i.e. active site modified), N-terminal truncated DN m-calpain (i.e. autolyzed DN-m-calpain) and, finally, an N- and C-terminal truncated m-calpain (i.e. autolyzed DN-m-calpain lacking a calcium-binding domain). Yeast cells were transformed using yeast two-hybrid expression vectors containing the different calpain constructs as "baits". Beta-galactosidase activity was assayed as an index of interaction between calpain and its potential target proteins. From this analysis, four clones (Ca2+-ATPase, novel nebulin-related protein (N-RAP), creatine kinase and glycogen phosphorylase) were recovered. Two of these, creatine kinase and glycogen phosphorylase, were selected for further study. In in-vitro assays, calpain was able to partially digest both proteins, suggesting that both creatine kinase and glycogen phosphorylase are natural calpain substrates.     

Autosomal recessive phosphorylase kinase deficiency in liver, caused by mutations in the gene encoding the beta subunit (PHKB).

The association of autosomal recessive phosphorylase kinase deficiency in liver of a 3 1/2-year-old female child with mutations in the gene encoding the common part of the beta subunit of phosphorylase kinase is reported. The proband had a severe deficiency of phosphorylase kinase in liver, while the phosphorylase kinase activity in erythrocytes was only slightly diminished. She had no symptoms of muscle involvement. The complete coding sequences of the liver gamma subunit and of the beta subunit of phosphorylase kinase of the proband were analyzed for the presence of mutations, by either reverse-transcribed PCR or SSCP analysis. Three deviations from the normal sequence were found in the region encoding the common part of the beta subunit of phosphorylase kinase-namely, a 1827G-->A (W609X) transition, a 2309A-->G (Y770C) transition, and a deletion of nucleotides 2896-2911-whereas no mutations were detected in the sequence encoding the liver gamma subunit of phosphorylase kinase. The 1827G-->A mutation and the deletion both result in the formation of early stop codons. Investigation of DNA showed that the deletion is caused by a splice-acceptor site mutation (IVS30(-1),g-->t). Family analysis revealed that the 1827G-->A and IVS30(-1),g-->t substitutions are located on different parental chromosomes and that compound heterozygosity for these mutations segregates with the disease. The 2309A-->G mutation was detected in 2%-3% of the normal population. Thus, it is concluded that the deficiency of phosphorylase kinase in this proband is caused by compound heterozygosity for the 1827G-->A and the IVS30(-1),g-->t mutations and that the 2309A-->G mutation is a polymorphism. This implies that a defect in the sequence encoding the common part of the beta subunit of phosphorylase kinase may present as liver phosphorylase kinase deficiency.