oligomycins and Vitamin-D-Deficiency

The basolateral segment of the rat renal tubular plasma membrane possesses Ca2+-dependent ATPase activity which was independent of Mg2+. Two kinetic forms were found: one, was a high affinity (apparent Km for free Ca2+ of 172 nM) low capacity (Vmax of 144 nmol of Pi X min-1 mg-1 protein) type; the other, had low affinity (apparent Km of 25 microM) and high capacity (896 nmol of Pi X min-1 X mg-1 protein). Mg2+ inhibited both Ca2+-ATPases. The high affinity enzyme exhibited positive cooperativity with respect to ATP, with a n value of 1.6. Ca2+-ATPase activity was not affected by calmodulin and was not inhibited by vanadate. On the other hand, both high and low affinity Ca2+-ATPase activities were increased when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient rats. Kinetically, the enhanced activities were due to an increase in the Vmax values; the apparent affinities for free Ca2+ were not changed. The physiological function of the vitamin D-sensitive, Mg+-independent, Ca2+-ATPase activities remains to be established.

Topics: Adenosine Triphosphate; Animals; Basement Membrane; Calcitriol; Calcium-Transporting ATPases; Hydrolysis; Kidney Cortex; Kinetics; Magnesium; Male; Oligomycins; Ouabain; Rats; Rats, Inbred Strains; Ruthenium Red; Theophylline; Vanadates; Vanadium; Vitamin D Deficiency

Isolated kidney mitochondria prepared from Vitamin D-deficient chicks catalyze the conversion of 25-hydroxyvitamin D3 to 1,25 dihydroxyvitamin D3. It wasfound that changes in the concentrations of Ca-2plus, HPO4-2minus, and Hplus altered synthesis in an interrelated fashion. Increasing the Ca-2plus concentration from 10-6 to 10-5 M caused a four- to fivefold increase in 1 alpha-hydroxylase activity when the medium pH was between 6.5 and 7.0. increasing the [Ca2+] to 10-4 M caused to furhter stimulation. At higher pH values, Ca-2plus had little effect upon 1 alpha-hydroxylase activity. In the absence of calcium [Ca2+] less than or equal to 10-7 M), a change in pH from 6.5 to 7.1 had no effect upon 1 alpha-hydroxylase activity in the presence of 10-5 M calcium, increasing the medium pH had a biphasic effect. An increase in pH from 6.5 to 6.9 caused a 1.5-fold increase in 1 alpha-hydroxylase activity, but a further increase of the pH to 7.1 caused a profound decrease in rate of hydroxylation to approximately 20% of the peak value. Neither 10-5 M LaC13 nor 10 mug/ml of oligomycin altered the effects of Ca2+ upon hydroxylate activity. However, the effect of calcium was blocked by 2.5 times 10-5 M ruthenium red, 0.83 mug/ml of antimycin A, and 500 muM dinitrophenol. The clcium ionophore, A23187, decreased but did not prevent the stimulatory effect of calcium. These data are consistent with the concept that the [Ca2+ in the mitochondrial matrix space is of importance in regulating the 1 alpha-hydroxylase. Phosphate exerted a biphasic effect on 1,25(OH)2D3 production with maximal stimulation (approximately twofold) at 1-3 mM. Calcium enhanced the stimulation by phosphate at all concentrations studied. The presence of potassium modified the interrelated effects of calcium and phosphate in two ways: 10-3 M calcium blocked the stimulation by phosphate; and in the presence of phosphate, 10-3 M calcium resulted in less 1,25(OH)2D3 production by production by isolated mitochondria are qualitatively similar to the effects of these ions on 1,25(OH)2D3 production yb isolated renal tubules.

Topics: Animals; Antimycin A; Calcium; Chickens; Diet; Dihydroxycholecalciferols; Dinitrophenols; Hydrogen-Ion Concentration; Hydroxycholecalciferols; In Vitro Techniques; Kidney; Mitochondria; Mixed Function Oxygenases; Oligomycins; Phosphates; Ruthenium; Vitamin D Deficiency

Topics: Adenosine Triphosphatases; Animals; Benzoates; Biological Transport, Active; Calcium; Chickens; Cholecalciferol; Duodenum; Hydrogen-Ion Concentration; Injections, Intravenous; Intestinal Mucosa; Intestine, Small; Magnesium; Nucleotides; Oligomycins; Organomercury Compounds; Ouabain; Phosphoric Monoester Hydrolases; Potassium; Protein Biosynthesis; Sodium; Time Factors; Vitamin D Deficiency

Topics: Adenosine Triphosphate; Animals; Calcium; Carbon Dioxide; Carbon Isotopes; Citric Acid Cycle; Cortisone; Depression, Chemical; Dinitrophenols; Gluconeogenesis; Kidney; Ligases; Magnesium; Male; Mitochondria; Mitochondria, Liver; Oligomycins; Potassium; Pyruvates; Rats; Stimulation, Chemical; Vitamin D Deficiency