Calcium accumulation by chick intestinal mitochondria: Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3

We have evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca²⁺ accumulation by chick intestinal mitochondria. Ca²⁺ accumulation appears to occur in two phases: an early, transient accumulation into an Na⁺-labile pool followed by an ATP-dependent accumulation into an Na⁺-resistant pool. Ca²⁺ accumulation is extensive at free Ca²⁺ concentrations greater than 3 · 10^?6 M in the presence of ATP. Ruthenium red and dinitrophenol block Ca²⁺ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)₂D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca²⁺, or the rate and extent of ATP-supported Ca²⁺ accumulation. Intestinal cytosol stimulated Ca²⁺ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca²⁺-binding protein. 30 ng/ml 1,25(OH)₂D-3 stimulated Ca²⁺ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 > 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca²⁺ concentration from 3 · 10^?6 to 1 · 10^?5 M increased Ca²⁺ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)₂D-3 in vitro increased Ca²⁺ accumulation less than 2-fold, we conclude that 1,25(OH)₂D-3 influences mitochondrial accumulation of Ca²⁺ in vivo primarily by altering cytosol concentrations of free Ca²⁺.     

Thin-layer isoelectric focusing of polyphenoloxidase of Sephadex and its detection by the print technique.

Separation of polyphenoloxidase isoenzymes based on their charge properties was achieved by isoelectric focusing on Sephadex G-75 thin layers containing a mixture of ampholytes in the pH ranges 4–6 and 3–10. The separated isoenzymes can be detected as colored zones by a print technique in which a dried filter paper, previously buffered with 0.1 m phosphate buffer, pH 7,0, and impregnated with 1% substrate in methanol, is placed onto the gel layer. d-Catechin and tyramine were the best substrates for detecting the diphenolase and monophenolase activities, respectively. Using this technique, two commercial preparations of mushroom tyrosinase were found to consist of 7 and 15 isoenzymes, while enzyme preparations from two potato varleties showed 11 to 15 isoenzymes. The isoenzymes of potato and mushroom polyphenoloxidase showed marked differences in their pI values.     

Interaction between bacteriophage PM2 protein IV and DNA.

The interaction between DNA and the structural protein IV of bacteriophage PM2 was studied by co-sedimentation, filter binding and electron microscopy. The co-sedimentation data and the sigmoid-shaped filter binding curve were interpreted in terms of co-operative binding. At a given DNA/protein input ratio, some DNA molecules were associated with a large amount of protein IV while others had no detectable protein bound to them. Electron microscopic examination of DNA-protein IV mixtures showed highly condensed DNA molecules alongside uncomplexed native DNA. Dissociation experiments revealed the presence of two types of complexes. Type I dissociated rapidly while type II had a long half-life. Dissociation of complexes obtained with increasing protein/DNA ratios suggested that the type I complex was a precursor of type II complex. Protein IV binds equally well to superhelical, relaxed or linear DNA as well as to single-stranded DNA. These observations lead to a model for the interaction and for the consequent alterations in the DNA structure.     

Renal parathyroid hormone-dependent adenylate cyclase activity after repletion of vitamin D-deficient rats with vitamin D-2

Rats fed a diet deficient in both vitamin D and Ca²⁺ exhibited a greater depression of the renal parathyroid hormone (PTH)-dependent adenylate cyclase than was observed in rats fed diets deficient in either vitamin D or calcium. Total serum Ca²⁺ was decreased from a control level of 11.2 mg/dl to 8.5 mg/dl in rats fed the diet deficient in calcium alone, and to 5.4 mg/dl in rats fed the diet deficient in vitamin D. Serum calcium was decreased further to 4.3 mg/dl in rats fed the diet deficient in both vitamin D and Ca²⁺. Serum immunoreactive PTH was significantly elevated over control levels when rats were fed the test diets; however, there were no significant differences between the elevated levels in the three experimental groups. Repletion of rats deficient in vitamin D only with a single oral dose of 3200 I.U. vitamin D-2 resulted in restoration of serum calcium to normal levels, a return of serum PTH to the control state, and an associated increase in PTH-dependent adenylate cyclase activity to the control level by 72 h. Repletion of rats deficient in both vitamin D and Ca²⁺ with the same dose of vitamin D-2 raised serum Ca²⁺ to 7.2 mg/dl by 72 h, but did not cause a reduction in circulating PTH, nor did it result in any significant improvement in the responsiveness of the membrane adenylate cyclase to PTH. These results suggest that elevated PTH is a factor in the down regulation of the PTH-dependent adenylate cyclase, but do not rule out a role for calcium as a regulatory factor.     

Enzyme-activated inhibition of bacterial D-amino acid transaminase by beta-cyano-D-alanine

beta-Cyano-D-alanine is an efficient suicide substrate (Ki = 10 microM) of D-amino acid transaminase. This apparent inactivation is temperature dependent: it is irreversible at 10 degrees C or below and becomes progressively reversible at higher temperatures. Since at higher temperatures the apparent reactivation process predominates over the inactivation reaction, the reactivation process is considered to be endothermic. The nature of this reversibility suggests the formation of a heat labile bond between the inhibitor molecule and a nucleophilic group on the enzyme.     

The occurrence of γ-carboxyglutamate in a protein isolated from ox liver mitochondria

A calcium-binding protein has been isolated from ox liver mitochondria. The purification procedure includes ammonium sulfate fractionation and chromatographies on Sephadex G-100 and DEAE-Sephadex, respectively. The isolated protein contains γ-carboxyglutamate, has a molecular weight of 59,000, and an isoelectric point of about 3.8. By submitochondrial fractionation it was shown that the calcium-binding protein is located between the inner and outer mitochondrial membranes.     

Relationship between bone resorption and lysosomal enzyme release as demonstrated by the effect of 1α-hydroxy-vitamin D-3 in an organ culture system

The effect of 1α-hydroxy-vitamin D-3 on the release of calcium (⁴⁰Ca, ⁴⁵Ca), inorganic phosphate and lysosomal enzymes, on glucose consumption and lactate production was studied in a bone organ culture system using half calvaria from 6–7-day-old mice. 1α-Hydroxy-vitamin D-3 stimulated the mobilization of minerals and increased the release of β-glucuronidase, β-N-acetylglucosaminidase and acid phosphatase, while no effect on the release of lactate dehydrogenase was seen. 1α-Hydroxy-vitamin D-3 also caused a significant increase in the total activities of acid phosphatase in the bones after culture, indicating increased enzyme synthesis. The stimulatory effect of the release of P_i and β-glucuronidase was also obtained after a temporary exposure to 1α-hydroxy-vitamin D-3. The stimulation by 1α-hydroxy-vitamin D-3 on the release of Ca²⁺, P_i and β-glucuronidase was suppressed by a protein synthesis inhibitor cycloheximide. No effect by 1α-hydroxy-vitamin D-3 on glucose consumption and lactate production was registered, suggesting that increased mineral mobilization does not require increased lactate production. It is concluded that although the data in the present paper do not prove a cause-and-effect relationship between lysosomal enzyme release and bone resorption, they give further support to the concept that the processes are intimately associated.     

Serum-free culture of Japanese quail kidney cells regulation of vitamin D metabolism

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 · 10⁶ cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25–30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromotographically separated on Sephadex LH-20. Three have been identified as 1α,25-dihydroxyvitamin D-3 (1,25(OH)₂D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)₂D-3) and 1α,24,25-trihhydroxyvitamin D-3 (1,24,25(OH)₃D-3). The activities of the 25-OH-D-3:1α- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)₂D-3 decreased 1,25(OH)₂D-3 synthesis, and increased both 24,25(OH)₂D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1α-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25-(OH)₂D-3 synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.     

Genome-independent effects of 1,25-dihydroxy vitamin D-3 on membrane potential

Cell membrane potential, $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, was monitored in rabbit hypertrophic cartilage metatarsals, amphibian proximal tubule and muscle cells during application of 1,25-dihydroxy vitamin D-3, 25-hydroxy vitamin D-3 or cholesterol (10^?10M). 1,25-Dihydroxy vitamin D-3 elicited quick variations of $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ (in less than 1 min) in proximal tubular cells (whether injected in the lumen or in peritubular capillaries) and in cartilage. The precursor 25-hydroxy vitamin D-3 and cholesterol produced a small shift of $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$ in proximal tubule only when applied from the luminal side, but this change was significantly smaller than that observed with 1,25-dihydroxy vitamin D-3. Muscle cells were unresponsive to both metabolites and cholesterol. It is concluded that rapid effects of 1,25-dihydroxy vitamin D-3 on $\text{V}{}_{\mathrm{<mtext>m</mtext>}}$, in target cells, are specific, most likely due to permeability changes and not related to nuclear protein synthesis; they may contribute to early modulation of cell function.