Effects of dietary vitamin D intake on plasma levels of parathyroid hormone and vitamin D metabolites in healthy Japanese

To clarify the nutritional status of vitamin D in Japanese, effects of dietary intake of vitamin D on plasma levels of intact and highly sensitive parathyroid hormone (I-PTH and HS-PTH), 25-hydroxyvitamin D (25-OH-D), 1,25-dihydroxyvitamin D (1,25(OH)2D), calcium (Ca) and phosphorus (P(i)) in 79 healthy Japanese were investigated. The plasma levels of 25-OH-D in men were significantly higher than those in women, whereas those of HS-PTH in men were significantly lower than those in women. The levels of 25-OH-D in men were generally higher than those in women. Significant correlations were observed between the dietary vitamin D intake and the plasma 25-OH-D or HS-PTH levels. Correlations between the plasma 25-OH-D levels and the plasma HS-PTH levels were also significant. These results suggest that dietary intake of sufficient amounts of vitamin D is effective for improving the vitamin D nutritional status through normalizing PTH levels.     

Human plasma transport of vitamin D after its endogenous synthesis

Transport of vitamin D3 from its sites of cutaneous synthesis into the circulation has been assumed to be via the plasma vitamin D binding protein (DBP). We studied vitamin D transport from the skin in seven healthy volunteers who received whole body irradiation with 27 mJ/cm2 dosage of ultraviolet B light (290-320 nm). Samples of venous blood were collected serially in EDTA and immediately chilled. In KBr, plasma samples were ultracentrifuged to provide a rapid separation of proteins of density < and > 1.3 g/ml. Upper and lower phases and serial fractions were analyzed for vitamin D3 (extraction, HPLC), cholesterol (enzyme assay), and human DBP (hDBP) (radial immunodiffusion). Total plasma vitamin D (basal level < 1 ng/ml) increased by 10 h and peaked at 24 h (9 +/- 1 ng/ml). 98% of the D3 remained at the density > 1.3 layers for up to 7 d, whereas cholesterol (> 85%) was detected at density < 1.3 and all of the hDBP was at density > 1.3. In three volunteers who each ingested 1.25 mg of vitamin D2, the total plasma D2 increased to 90 +/- 32 ng/ml by 4 h, and the D2 was evenly distributed between the upper and lower layers at 4, 8, and 24 h after the dose, indicating a continuing association of the vitamin with chylomicrons and lipoproteins, as well as with hDBP. Actin affinity chromatography removed D3 from plasma of irradiated subjects, indicating the association of the D3 with DBP. These findings indicate that endogenously synthesized vitamin D3 travels in plasma almost exclusively on DBP, providing for a slower hepatic delivery of the vitamin D and the more sustained increase in plasma 25-hydroxycholecalciferol observed after depot, parenteral administration of vitamin D. In contrast, the association of orally administered vitamin D with chylomicrons and lipoproteins allows for receptor-mediated, rapid hepatic delivery of vitamin D, and the reported rapid but less-sustained increases in plasma 25-hydroxycholecalciferol.     

Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein

A line of mice deficient in vitamin D binding protein (DBP) was generated by targeted mutagenesis to establish a model for analysis of DBP's biological functions in vitamin D metabolism and action. On vitamin D-replete diets, DBP-/- mice had low levels of total serum vitamin D metabolites but were otherwise normal. When maintained on vitamin D-deficient diets for a brief period, the DBP-/-, but not DBP+/+, mice developed secondary hyperparathyroidism and the accompanying bone changes associated with vitamin D deficiency. DBP markedly prolonged the serum half-life of 25(OH)D and less dramatically prolonged the half-life of vitamin D by slowing its hepatic uptake and increasing the efficiency of its conversion to 25(OH)D in the liver. After an overload of vitamin D, DBP-/- mice were unexpectedly less susceptible to hypercalcemia and its toxic effects. Peak steady-state mRNA levels of the vitamin D-dependent calbindin-D9K gene were induced by 1,25(OH)2D more rapidly in the DBP-/- mice. Thus, the role of DBP is to maintain stable serum stores of vitamin D metabolites and modulate the rates of its bioavailability, activation, and end-organ responsiveness. These properties may have evolved to stabilize and maintain serum levels of vitamin D in environments with variable vitamin D availability.     

Some characteristics of cytosol binding protein for 1alpha,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol in rat intestinal mucosa

Cytosol binding protein for 1alpha,25-(OH)2D3 and 25-OHD3 was demonstrated in rat intestinal mucosa using sucrose density gradient ultracentrifugation analysis, Sephadex G-200 column chromatography and polyacrylamide disc gel electrophoresis. The binding protein has a sedimentation constant of 5-6S, a molecular weight of 100,000-120,000 daltons and a mobility in electrophoresis of Rf 0.42. Further analysis of binding behavior by DEAE-cellulose filter assays revealed that the bindig protein had a higher affinity for 25-OHD3 than for 1alpha,25-(OH)2D3, and that 1alpha,25-(OH)2D3 competed with 25-OHD3 for the same binding site on the protein. The apparent dissociation constant for 1alpha,25-(OH)2D3 and 25-OHD3 was 9.2 X 10(-9) M and 1.5 X 10(-9) M, respectively. The binding capacity was 3.1 pmoles/mg protein for both 1alpha,25-(OH)2D3 and 25-OHD3. The order of binding affinity was 25-OHD3 greater than 1alpha,25-(OH)2D3 greater than D3 = 1alpha-OHD3.     

Association of 1 alpha,25-dihydroxyvitamin D3-occupied vitamin D receptors with cellular membrane acceptance sites

We previously reported nongenomic activation of ROS 17/2.8 cells by vitamin D metabolites (1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], 25-hydroxyvitamin D3, 22-oxa-calcitriol, etc.). The hormone 1 alpha,25-(OH)2D3, or calcitriol, mediated rapid transient changes in intracellular free calcium levels and concomitant stimulation of inositol polyphosphate and diacylglycerol production. These effects resemble the mechanism of cell activation induced by ligands with plasma membrane (PM) receptors. As preliminary studies indicated that PM isolated from ROS 17/2.8 cells lacked specific binding sites for calcitriol alone, we studied the association between calcitriol-occupied vitamin D receptors (VDR) and ROS 17/2.8 cellular membranes. Saturable binding to the PM and the endoplasmic reticulum (ER) of calcitriol-occupied VDR was demonstrated. Binding of the VDR-[3H]calcitriol complex was displaceable by nonradioactive VDR/calcitriol, but not by the unoccupied VDR or by calcitriol alone. ER binding, but not PM binding, was competitively inhibited by a peptide from the VDR sequence recognized by an ER protein, calreticulin, and by an anticalreticulin antibody. The monoclonal antibody (9A7) against the VDR inhibited PM and ER binding of the hormone-occupied VDR. These results were substantiated by studies using baculovirus-expressed human VDR for binding studies with the PM and ER and for immunoblot analysis. We conclude that specific PM and ER sites of association for calcitriol-occupied VDR exist and suggest that these associations could participate in the nongenomic rapid actions of 1 alpha,25-(OH)2D3.     

Biologic activity of dihydroxylated 19-nor-(pre)vitamin D3

Vitamin D3 and its hydroxylated metabolites are normally in thermal equilibrium with their previtamin D isomers. To evaluate the biologic activity of 1 alpha, 25-dihydroxyprevitamin D3, we synthesized 19-nor analogs of 1 alpha, 25-dihydroxy(pre)vitamin D3 because the absence of a C19 methylene group prevents the isomerization of these analogs. The affinity of 1 alpha, 25-(OH)2D3-19-nor-D3 for the intestinal vitamin D receptor and plasma vitamin D binding protein was mildly decreased [30 and 20% of the affinity of 1 alpha, 25-(OH)2D3, respectively], but the affinity of 1 alpha, 25-(OH)2-19-nor-previtamin D3 was only 1 and 6% of that of 1 alpha, 25-(OH)2D3 for the receptor and DBP, respectively. The in vitro effects on human promyeloid leukemia (HL-60 cell) differentiation and osteocalcin secretion by human osteosarcoma (MG-63) cells by 1 alpha, 25-(OH)2-19-nor-D3 were nearly identical to those of 1 alpha-25-(OH)2D3, whereas 19-nor-previtamin D3 showed poor activity (2%). The in vivo calcemic effects of both analogs, studied in vitamin D-deficient chicks treated for 10 consecutive days with the analogs, showed no activity of the previtamin D3 analog and reduced calcemic effects (< or = 10%) of 1 alpha, 25-(OH)2-19-nor-D3. We conclude that the previtamin D form of 1 alpha, 25-(OH)2D3 has lost most of its biologic activity in vitro and in vivo.     

Some characteristics of new tissue-binding proteins for metabolites of vitamin D other than 1,25-dihydroxyvitamin D

Protein(s) have been found in a wide range of tissues which have a high affinity for 25-hydroxycholecalciferol. Of the tissues examined only erythrocytes do not have this protein. The properties of the protein have been examined and it has been found that the association constatns range from 2 - 10(9) to 5 - 10(9) M-1 and the sedimentation constants between 5.0 and 6.0 S. It was not possible to distinguish the proteins from the different tissues by their S values, mobility on gel electrophoresis or behaviour on ion-exchange chromatography. These techniques were all used, however, to show that the tissue 25-hydroxycholecalciferol binding protein is distinct from the main plasma binding protein for this steroid and from the intestinal 1,25-dihydroxycholecalciferol-binding protein. A protein has been in the plasma of rachitic animals but not of normals, which is apparently indistinguishable from this new tissue 25-hydroxycholecalciferol-binding protein. The steroid specificity of this new binding protein has been shown to be dependent upon a C-25 hydroxyl group, and an intact conjugated double bond system. Possible functions for this protein have been briefly discussed.     

Proteins in the heat shock-70 family specifically bind 25-hydroxyvitamin D3 and 17beta-estradiol

Most New World primates evolved to express a form of compensated resistance to steroid hormones from the gonads and adrenal glands as well as to the hydroxylated vitamin D3 prohormone, 25-hydroxyvitamin D3 (25OHD3), and the vitamin D hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] originating from the liver and kidney, respectively. We recently demonstrated that this form of resistance is associated with the overexpression of a novel member of the 70-kDa heat shock protein (hsp-70) molecular chaperone family, which we have termed the intracellular vitamin D binding protein (IDBP). In the current report we more closely examine the ligand-binding capability of purified IDBP and two other mammalian hsp-70 family members, heat-inducible (hsp-70) and constitutively expressed (hsc-70) hsp-70 proteins. Purified IDBP, hsp-70, and hsc-70 all bound 25OHD3 with relatively high affinity; the mean Kd for 25OHD3 ranged from 0.5-2.2 nmol/L (rank order: IDBP > or = hsp-70 > or = hsc-70). By Scatchard analysis, high affinity, specific binding of 1,25-(OH)2D3 was not reproducibly observed for any of the three members of the hsp-70 family. Unlike purified IDBP, hsc-70 and hsp-70 were also competent binders of the gonadal steroid 17beta-estradiol (mean Kd for 25OHD3, 2.5 and 6.6 nmol/L by hsc-70 and hsp-70, respectively), but not of two other gonadal hormones, progesterone and testosterone. These data suggest that IDBP is relatively specific for 25OHD3 and that additional hsp-70-like binding proteins are present in unpurified New World primate cell extracts that are specific for 1-hydroxylated vitamin D metabolites as well as other gonadal steroid hormones.     

Dietary vitamin D affects cell-mediated hypersensitivity but not resistance to experimental pulmonary tuberculosis in guinea pigs

Outbred, Hartley strain guinea pigs were fed purified diets varying only in their levels of vitamin D. The amounts of vitamin D in the diets were adjusted to represent 0, 25, 50, 100, or 200% of the recommended level (1,180 IU/kg of body weight) for guinea pigs. In some experiments, half of the animals in each diet group were vaccinated with Mycobacterium bovis BCG vaccine at the time the diets were introduced. Six weeks later, all guinea pigs were infected by the respiratory route with a low dose of virulent M. tuberculosis H37Rv. Vitamin D-deficient animals exhibited marked reductions in levels of the major vitamin D metabolite, 25-hydroxyvitamin D3, in plasma. Altered vitamin D intake was accompanied by changes in antigen (purified protein derivative)-induced, cell-mediated immune responses both in vivo (tuberculin hypersensitivity) and in vitro (lymphoproliferation). Dermal tuberculin reactivity developed more slowly in vitamin D-deficient guinea pigs but eventually achieved normal levels. The proliferation of splenocytes cultured with purified protein derivative was suppressed by both deficiency and excess of dietary vitamin D. Vitamin D status did not affect the abilities of naive guinea pigs to control primary, pulmonary tuberculosis, nor did it influence the protective efficacy of BCG vaccination. We conclude that changes in dietary vitamin D are associated with alterations in some cellular immune functions but may not be an important determinant of disease outcome in pulmonary tuberculosis, as has been suggested previously.     

Parathyroidectomy reduces 25-hydroxyvitamin D3-1 alpha-hydroxylase activity in the hypocalcemic vitamin D-deficient chick

To test the hypothesis that in the vitamin D-deficient state the activity of 25-hydroxyvitamin D3-1 alpha-hydroxylase (25-OHD3-1 alpha-hydroxylase) is modulated by parathyroid hormone and the plasma concentration of phosphate only in the presence of small amounts of 1,25-dihydroxyvitamin D3 (or some other metabolite of vitamin D), we measured the activity of this enzyme 24 h after parathyroidectomy (PTX) in frankly hypocalcemic, vitamin D-deficient chicks that were not supplemented with vitamin D or one of its metabolites. The otherwise predictable complications of PTX in this metabolic setting (hypocalcemia of increasing severity, tetany, moribundity, and death) were prevented by continuous intravenous administration of calcium (as a solution of calcium chloride/calcium gluconate 1:1) through a catheter in the external jugular vein placed at the time of PTX. The findings were as follows: (a) The activity of 25-OHD3-1 alpha-hydroxylase was significantly less in the parathyroidectomized group than in the sham-operated control chicks (P less than 0.001). (b) The reductive effect of PTX on the activity of this enzyme was significantly attenuated when hypophosphatemia was increased in severity by administration of glucose. (c) In the post-PTX state the activity of 25-OHD3-1 alpha-hydroxylase and plasma concentration of phosphate were significantly, inversely related (P less than 0.001). (d) In the sham-operated control group the activity of this enzyme and the plasma concentration of phosphate were not significantly correlated. These findings indicate that in the vitamin D-deficient state, both circulating parathyroid hormone and the plasma concentration of phosphate can significantly modulate the activity of 25-OHD3-1 alpha-hydroxylase in the absence of vitamin D or its metabolites. The findings also suggest that in the vitamin D-deficient state the plasma concentration of phosphate modulates the activity of this enzyme only when the concentration of circulating parathyroid hormone is not increased.     

The lack of effect of chronic metabolic acidosis on 25-OH-vitamin D metabolism and serum parathyroid hormone in humans

We evaluated the turnover of the plasma 25-OH-vitamin D pool, acid, and mineral balances in paired balance studies of 6 normal subjects during normal acid base conditions and during stable chronic metabolic acidosis induced by NH4Cl. Positive acid balances and negative Ca balances due to hypercalciuria were observed as previously reported. Plasma 25-OH-D pool turnover averaged 6.1+/-0.4 nmol/day during control and did not change during acidosis (6.5 +/- 0.5 nmol/day) nor were any significant increments in net intestinal absorption of Ca, PO4, or Mg, the physiological expression of vitamin D action, observed during acidosis. In 3 other subjects, repetitive measurements of serum iPTH during 7 control days and 24 days of stable NH4Cl acidosis showed no changes. We interpret the data to support the hypothesis that neither PTH nor vitamin D and its metabolites mediates the increase in net bone resorption that must accompany chronic metabolic acidosis.     

Pantothenate synthetase of Escherichia coli B. I. Physicochemical properties

Using a new apparatus for preparative polyacrylamide gel electrophoresis, pantothenate synthetase (D-pantoate: beta-alanine ligase (AMP-forming), [EC 6.3.2.1] was purified about 500-fold from Escherichia coli B. It was found to be homogeneous in analytical disc gel electrophoresis and sedimentation ultracentrifugation (so20, w=4.9). From sedimentation equilibrium ultracentrifugation, a molecular weight of 70,100 was obtained, which is in good agreement with the value obtained by the Sephadex G-150 gel filtration method (69,000); the diffusion constant was calculated to be 5.88X10(-7) cm2/sec. The minimum molecular weight calculated from the amino acid composition of this enzyme protein was 19,700, a value in reasonable accord with molecular weight of the enzyme subunit, 18,000, obtained by gel electrophoresis in the presence of sodium dodecylsulfate. The partial specific volume, v, was calculated to be 0.71 cm3/g. The enzyme had an amino-terminal glycyl residue and a Leu-Ala-Ser-OH sequence at the carboxyl end. Electrophoresis of the enzyme with carrier ampholine gave an isoelectric point of pH 4.6.     

25-hydroxy vitamin D deficiency with reduction of intestinal calcium absorption and bone density in chronic alcoholism

Plasma levels of 25-hydroxycalciferol (25-OH-D), 47-calcium intestinal absorption, bone mineral content and the biologic parameters of phospho-calcium metabolism were studied in 30 chronic alcoholics, 15 with Laennec's cirrhosis (group A) and 15 without (group B). These patients were compared with 27 normal subjects. In group A, the mean 25-OH-D plasma level was 23.7 +/- SD 18.5 microgram/l and in group B 35.2 +/- SD 21.8 microgram/l. These mean levels were lower than those of the control group, which were 57.2 +/- SD 22.5 microgram/l (p less than 0.001). The mean value of the 47Ca intestinal absorption, measured as the percentage of the ingested dose per litre of plasma and multiplied by the body weight, was also significantly lower in group A, which was 140 +/- SD 47 (p less than 0.01), and in group B, which was 145 +/- SD 69 (p less than 0.05), compared with the normal subjects whose average was 182 +/- SD 45.6. Similarly, the total plasma calcium was low: 1.99 +/- SD 0.24 mmol/l in all the alcoholics, while that of the control group was 2.22 +/- SD 0.18 mmol/l (p less than 0.001). For the 30 chronic alcoholics there was a positive correlation between 25-OH-D and 47Ca intestinal absorption, (r = 0.484; p less than 0.004). This suggests that in chronic alcoholism the deficiency of 25-OH-D induces a diminution of the intestinal absorption of calcium which, in the long term, can result in bone demineralization evidenced in the patients studied by a bone mineral content lower than normal (p less than 0.001).     

The influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol, 25-hydroxycholecalciferol, and 1,25 dihydroxycholecalciferol in young broiler chickens

Three experiments were conducted to determine the influence of vitamin A on the utilization and amelioration of toxicity of cholecalciferol (vitamin D3), 25-hydroxycholecalciferol [25-(OH)D3], and 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in young broiler chicks. Two levels of vitamin A (1,500 and 45,000 IU/kg or 450 and 13,500 microg) were fed in all experiments. In Experiment 1, chicks were fed six levels of vitamin D3 (0, 5, 10, 20, 40, and 80 microg/kg). High dietary vitamin A decreased bone ash (P < 0.001), and increased the incidence of rickets (P < or = 0.02). Linear and quadratic responses to vitamin D3 levels were significant (P < 0.01) for body weight, bone ash, incidence and severity of rickets, and plasma calcium. In Experiment 2, six levels of 25-(OH)D3 (0, 5, 10, 20, 40, and 80 microg/kg) were added to the basal diet. Adding 25-(OH)D3 increased (P < 0.001) body weight, bone ash, and plasma calcium, and decreased rickets and plasma vitamin A. Adding 25-(OH)D3 overcame the reduction in bone ash produced by high dietary vitamin A showing a significant (P < 0.02) interaction. In Experiment 3, six levels of 1,25-(OH)2D3 (0, 2, 4, 8, 16, and 32 microg/kg) were added to the basal diet. High dietary vitamin A increased (P < 0.01) the incidence and severity of rickets. Adding 1,25-(OH)2D3 increased (P < 0.01) body weight, bone ash, plasma calcium, and reduced rickets and plasma and liver vitamin A. Adding 1,25-(OH)2D3 overcame the reduction in bone ash, and the increase in rickets produced by high vitamin A was significant (P < or = 0.05). These results indicate that high dietary vitamin A (45,000 IU/kg) interferes with the utilization of vitamin D3, 25-(OH)D3 and 1,25-(OH)2D3, increasing the requirement for each of them. Moreover, 45,000 IU/kg of dietary vitamin A ameliorated the potential toxic effects of feeding high levels of vitamin D3, 25-(OH)D3 and 1,25-(OH)2D3 to young broiler chickens. Further work is necessary to find the minimum levels of these vitamins needed to cause these effects.     

Localization and quantitation of the vitamin D binding protein (Gc-globulin) in human neutrophils

Plasma-derived vitamin D binding protein (DBP) is an important physiologic regulator of the neutrophil chemotactic response to activated complement. A cell-associated form of DBP has been observed in numerous cell types. We now report that mature, circulating human neutrophils also contain cell-associated DBP. Immunofluorescence studies of normal untreated neutrophils showed the presence of DBP on the cell surface. Western blotting of detergent-soluble neutrophil lysates with a polyclonal anti-DBP showed two major immunoreactive bands, one with an apparent molecular weight of 56 Kd (identical to purified plasma-derived DBP) and a second less prominent band at 12 to 14 Kd. Quantitation of the immunoreactive bands by video densitometry indicated that normal human neutrophils contain 1.5 +/- 0.8 ng DBP/10(6) cells (n = 9). Immunoprecipitation of detergent-soluble lysates with the polyclonal anti-DBP showed only the 56-Kd form by Western blotting. In contrast, a monoclonal anti-DBP immunoprecipitated the 12 to 14 Kd form of DBP from lysates of surface-radioiodinated cells. Western blots of subcellular fractions showed that immunoreactive bands were found in the specific (secondary) granule and plasma-membrane fractions. In addition, pretreatment of neutrophils with 10 nmol/L phorbol myristate acetate (PMA) resulted in approximately a 50% reduction in the amount of DBP in both the specific granule and plasma-membrane fractions. Finally, analysis of the cell-free supernates showed that DBP was spontaneously released into the extracellular milieu: moreover, this release was enhanced if the cells were first stimulated with C5a, formyl-norleucyl-leucyl-phenylalanine (fNLP) or PMA.     

Purification of C3b inactivator and demonstration of its two polypeptide chain structure

C3b inactivator (C3bINA) was isolated from plasma by sequential ammonium sulfate gradient solubilization, anion exchange chromatography, gel filtration, and repeat ammonium sulfate gradient solubilization. The final product was pure as assessed by alkaline disc gel electrophoresis, isoelectric focusing, and SDS polyacrylamide gel electrophoresis, and elicited a monospecific rabbit antiserum. The normal serum concentration of C3bINA was found to be 53 +/- 9 microgram/ml (mean +/- S.D.). Heterogeneity of purified C3bINA was apparent on alkaline disc gel electrophoresis and isoelectric focusing, but not with SDS polyacrylamide gel electrophoresis and thus is attributed to forms of C3bINA that differ in charge rather than in size. SDS polyacrylamide gel electrophoresis of unreduced, alkylated C3bINA yielded a single stained band with an apparent m.w. of 93,000, whereas the reduced protein demonstrated two bands of 55,000 and 42,000 m.w., thereby establishing a composition of two disulfide-linked polypeptide chains for C3bINA.     

Triiodothyronine restricts myofibrillar growth and enhances beating frequency in cultured adult rat cardiomyocytes

Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.     

Physical properties and chemical compositions of cytoplasmic and mitochondrial malate dehydrogenase from Physarum polycephalum

The malate dehydrogenase isoenzymes from Physarum polycephalum have been purified to homogeneity as confirmed by gel filtration chromatography, polyacrylamide gel disc electrophoresis and analytical ultracentrifugation. Certain physical and chemical parameters of the malate dehydrogenase isoenzymes reported here include sedimentation, molecular weight and subunit molecular weight. Most unique of the differences between the isoenzymes were the widely separate isoelectric points of 9.83 for mitochondrial malate dehydrogenase and 6.14 for the supernatant malate dehydrogenase. The amino acid analyses of each form were done revealing the isoenzymes were unquestionably unique proteins differing in the content of ten amino acids.     

Human pancreatic alpha-amylase. I. Purification and characterization

alpha-Amylase was extracted from human pancreas and purified by using ammonium sulfate fractionation, Sephadex G-100 and DEAE-Sephadex A-50 column chromatography. The enzyme was shown to be homogenous by three different criteria: polyacrylamide disc gel electrophoresis, SDS polyacrylamide gel electrophoresis and analytical ultracentrifugation. The values of SO20,w, D20,w, v, and frictional ration of the enzyme were calculated to be 5.01S, 7.56D, 0.718 ml g-1 and 1.10, respectively. The molecular weight of the alpha-amylase was determined by three different methods: sedimentation velocity-diffusion, conventional sedimentation equilibrium and SDS polyacrylamide gel electrophoresis and was found to be 57,850; 50,100 and 53,200 g mole-1, respectively (average value 53,700). The amino acid composition of the enzyme was determined and compared with those of alpha-amylases from various other sources.