An evaluation of the biologic activity and vitamin D receptor binding affinity of the photoisomers of vitamin D3 and previtamin D3

Skin is in the site of previtamin D3 and vitamin D3 synthesis and their isomerization in response to ultraviolet irradiation. At present, little is known about the function of the photoisomers of previtamin D3 and the vitamin D3 in skin cells. In this study we investigated the antiproliferative activity of the major photoisomers and their metabolites in the cultured human keratinocytes by determining their influence on 3H-thymidine incorporation into DNA. Our results demonstrated at both 10(-8) and 10(-6) M in a dose-dependent manner. Lumisterol, tachysterol3, 5,6-trans-vitamin D3, and 25-hydroxy-5,6-trans-vitamin D3 only induced significant inhibition at 10(-6) M. 25-Hydroxytachysterol3 was approximately 10- to 100-fold more active than tachysterol3. 7-Dehydrocholesterol was not active even at 10(-6) M. The dissociation constants of vitamin D receptor (VDR) for 25-hydroxytachysterol3, 25-hydroxy-5,6-trans-vitamin D3, and 5,6-trans-vitamin D3 were 22, 58, and 560 nM, respectively. The dissociation constants for 7-dehydrocholesterol, tachysterol, and lumisterol were greater than 20 microM. In conclusion, vitamin D3, its photoisomers and the photoisomers of previtamin D3 have antiproliferative activity in cultured human keratinocytes. However, the antiproliferative activity did not correlate with their binding affinity for VDR. The results suggest that some of the photoproducts may be metabolized to their 25-hydroxylated and 1 alpha,25-dihydroxylated counterparts before acting on VDR. Alternatively, a different receptor may recognize these photoproducts or another mechanism may be involved in modulating the antiproliferative activity of the photoisomers examined.     

1,25-Dihydroxyvitamin D3 binds specifically to rat vascular smooth muscle cells and stimulates their proliferation in vitro

Cultured vascular smooth muscle cells (VSMC) derived from rat aorta were found to contain a specific receptor for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Its Kd (5.0 x 10(-11) M) and capacity (22.9 fmol/mg of cytosol protein) for 1,25-(OH)2D3, its sedimentation coefficient on a sucrose density gradient (3.2 S), its relative affinities for various vitamin D3 metabolites [1,25-(OH)2D3 greater than 25-hydroxyvitamin D3 greater than 24,25-dihydroxyvitamin D3 greater than vitamin D3] and its affinity for DNA-cellulose were similar to those reported for the 1,25-(OH)2D3 receptor in other tissues. 1,25-(OH)2D3 at concentrations of more than 10(-10) M caused dose-dependent enhancement of the proliferation of VSMC in DMEM with 10% FCS. 25-Hydroxyvitamin D3 stimulated the proliferation of VSMC only at its highest concentration tested (10(-6) M). These data show that 1,25-(OH)2D3 stimulates the proliferation of VSMC after its binding to a cytoplasmic receptor of the cells in vitro, and support the possibility that VSMC are target cells of the hormone.     

Quantitative studies of the interaction of cholecalciferol ((vitamin D3) and its metabolites with different genetic variants of the serum binding protein for these sterols.

Cholecalciferol (vitamin D3) and its 25-hydroxy metabolite are transported in plasma bound to a specific protein, the binding protein for cholecalciferol and its metabolites (DBP). DBP is identical with the group-specific component (Gc) proteins, which are known to display genetic polymorphism. Studies were conducted to explore whether or not major differences in the transport of cholecalciferol and its biological metabolites might exist among persons with different Gc phenotypes. Detailed quantitative studies were first carried out on the interaction of 25(OH)D3 with DBP in 21 different samples of serum, representing eight different Gc phenotypes. The studies used a filter disc assay method that provided highly reproducible quantitative results with cholecalciferol-related sterols. The Gc phenotypes studied included the three common types (Gc 1-1, 2-1, and 2-2) and several uncommon genetic variants (Gc Ab-Ab, Ab-1, Ab-2, Chip-1, and Chip-2). The binding affinities for 25(OH)D3 observed with these different sera were all fairly similar to each other. More extensive studies were then conducted to compare the binding of four cholecalciferol-related sterols to each of three genetic variants of DBP, by using sera from homozygous persons with the Gc 1-1, Gc 2-2 and Gc Ab-Ab phenotypes. The ligands tested included cholecalciferol, 25(OH)D3, 1,25(OH)2D3, and 24(R) 25(OH)2D3. The affinities of the three genetic types of DBP/Gc protein were found to be similar for each of the four cholecalciferol-related sterols. The apparent association constants for 25(OH)D3 and 24,25(OH)2D3 were similar (approx. 1--2 x 10(8) M-1); lesser affinities were observed for 1,25(OH)2D3 (kA approx. 1 x 10(7) M-1) and for cholecalciferol (kA approx. 3--4 x 10(5) M-1). Thus the common genetic variants of DBP/Gc protein, and the uncommon genetic variants studied here, all appear to have similar binding properties for cholecalciferol and its several metabolites.     

Binding energy and catalysis by D-xylose isomerase: kinetic, product, and X-ray crystallographic analysis of enzyme-catalyzed isomerization of (R)-glyceraldehyde

D-Xylose isomerase (XI) and triosephosphate isomerase (TIM) catalyze the aldose-ketose isomerization reactions of D-xylose and d-glyceraldehyde 3-phosphate (DGAP), respectively. D-Glyceraldehyde (DGA) is the triose fragment common to the substrates for XI and TIM. The XI-catalyzed isomerization of DGA to give dihydroxyacetone (DHA) in D(2)O was monitored by (1)H nuclear magnetic resonance spectroscopy, and a k(cat)/K(m) of 0.034 M(-1) s(-1) was determined for this isomerization at pD 7.0. This is similar to the k(cat)/K(m) of 0.017 M(-1) s(-1) for the TIM-catalyzed carbon deprotonation reaction of DGA in D(2)O at pD 7.0 [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. The much larger activation barrier for XI-catalyzed isomerization of D-xylose (k(cat)/K(m) = 490 M(-1) s(-1)) versus that for the TIM-catalyzed isomerization of DGAP (k(cat)/K(m) = 9.6 × 10(6) M(-1) s(-1)) is due to (i) the barrier to conversion of cyclic d-xylose to the reactive linear sugar (5.4 kcal/mol) being larger than that for conversion of DGAP hydrate to the free aldehyde (1.7 kcal/mol) and (ii) the intrinsic binding energy [Jencks, W. P. (1975) Adv. Enzymol. Relat. Areas Mol. Biol. 43, 219-410] of the terminal ethylene glycol fragment of D-xylose (9.3 kcal/mol) being smaller than that of the phosphodianion group of DGAP (~12 kcal/mol). The XI-catalyzed isomerization of DGA in D(2)O at pD 7.0 gives a 90% yield of [1-(1)H]DHA and a 10% yield of [1-(2)H]DHA, the product of isomerization with incorporation of deuterium from solvent D(2)O. By comparison, the transfer of (3)H from the labeled hexose substrate to solvent is observed only once in every 10(9) turnovers for the XI-catalyzed isomerization of [2-(3)H]glucose in H(2)O [Allen, K. N., Lavie, A., Farber, G. K., Glasfeld, A., Petsko, G. A., and Ringe, D. (1994) Biochemistry 33, 1481-1487]. We propose that truncation of the terminal ethylene glycol fragment of d-xylose to give DGA results in a large decrease in the rate of XI-catalyzed isomerization with hydride transfer compared with that for proton transfer. An ultra-high-resolution (0.97 ?) X-ray crystal structure was determined for the complex obtained by soaking crystals of XI with 50 mM DGA. The triose binds to XI as the unreactive hydrate, but ligand binding induces metal cofactor movement and conformational changes in active site residues similar to those observed for XI·sugar complexes.     

24,25,28-trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2: novel metabolites of vitamin D2

Understanding of the inactivation pathways of 25-hydroxyvitamin D2 and 24-hydroxyvitamin D2, the two physiologically significant monohydroxylated metabolites of vitamin D2, is of importance, especially during hypervitaminosis D2. In a recent study, it has been demonstrated that the inactivation of 24-hydroxyvitamin D2 occurs through its conversion into 24,26-dihydroxyvitamin D2 [Koszewski, N.J., Reinhardt, T.A., Napoli, J.L., Beitz, C.D., & Horst, R.L. (1988) Biochemistry 27, 5785]. At present, little information is available regarding the inactivation pathway of 25-hydroxyvitamin D2 except its further metabolism into 24,25-dihydroxyvitamin D2 [Jones, G., Rosenthal, A., Segev, D., Mazur, Y., Frolow, F., Halfon, Y., Rabinovich, D., & Shakked, Z. (1979) Biochemistry 18, 1094]. In our present study, we investigated the metabolic fate of 25-hydroxyvitamin D2 in the isolated perfused rat kidney and demonstrated its conversion not only into 24,25-dihydroxyvitamin D2 but also into two other new metabolites, namely, 24,25,28-trihydroxyvitamin D2 and 24,25,26-trihydroxyvitamin D2. The structure identification of the new metabolites was established by the techniques of ultraviolet absorption spectrophotometry and mass spectrometry and by the characteristic nature of each new metabolite's susceptibility to sodium metaperiodate oxidation. In order to demonstrate the physiological significance of the two new trihydroxy metabolites of vitamin D2, we induced hypervitaminosis D2 in a rat using [3 alpha-3H]vitamin D2 and analyzed its plasma for the various [3 alpha-3H]vitamin D2 metabolites on two different high-pressure liquid chromatography systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-specific NMR monitoring of cis-trans isomerization in the folding of the proline-rich collagen triple helix

Understanding the folding of the proline-rich collagen triple helix requires consideration of the effects of proline cis-trans isomerization and may shed light on the misfolding of collagen in connective tissue diseases. Folding was monitored in real time by heteronuclear 2D NMR spectroscopy for the (15)N labeled positions in the triple-helical peptide T1-892 [GPAGPAGPVGPAGARGPAGPOGPOGPOGPOGV]. In the equilibrium unfolded monomer form, each labeled residue showed multiple peaks with interconversion rates consistent with cis-trans isomerization of Gly-Pro and Pro-Hyp bonds. Real-time NMR studies on the folding of T1-892 showed slow decay of monomer peaks and a concomitant increase in trimer peaks. Gly25 in the C-terminal rich (Gly-Pro-Hyp)(4) domain folds first, consistent with its being a nucleation domain. Analysis of the kinetics indicates that the folding of Gly25 is biphasic and the slower step represents cis-trans isomerization of imino acids. This illustrates that nucleation is limited by cis-trans isomerization. Monitoring Gly6, Gly10, Ala12, and Gly13 monomer and trimer peaks captures the C- to N-terminal propagation of the triple helix, which is also limited by Gly-Pro cis-trans isomerization events. The zipper-like nature of the propagation process is confirmed by the slower rate of folding of Ala6 compared to Gly13, reflecting the larger number of isomerization events encountered by the more N-terminal Ala6. The cis-trans isomerization events at multiple proline residues is a complex statistical process which can be visualized by these NMR studies.     

Synthesis of [10S(19)-3H]-dihydrotachysterol2 from ergocalciferol and preliminary investigations into its metabolic fate in rats

Dihydrotachysterol2 (DHT2), a 5,6-trans derivative of vitamin D2, is very successfully used in the treatment of hypoparathyroidism and renal osteodystrophy. However, the metabolism and the action of DHT2 are poorly understood. Investigations into metabolism of DHT2 start at the synthesis of the radioactively labelled compound. This paper deals with a two-step synthesis of [10S(19)-3H]-dihydrotachysterol2 from vitamin D2. Vitamin D2 can be converted into 5,6-trans vitamin D2 by iodination under irradiation and by triplet-sensitized isomerization. The first method led to unwanted side-reaction products which were difficult to separate from 5,6-trans vitamin D2. Triplet-sensitized isomerization yielded merely 5,6-trans vitamin D2 which could be separated from the residual starting material by chromatography on silica gel and recrystallization. [10S](19)-3H]-Dihydrotachysterol2 with a specific radioactivity of 56 kCi/mol was prepared from 5,6-trans vitamin D2 via partial, homogeneous catalytic reduction of the 10S(19) double bond with tritium gas. It was purified by high-performance liquid chromatography (h.p.l.c.) and characterized by chromatography and ultra-violet absorption spectrophotometry. The biological usefulness of the material was demonstrated in rats following intragastric administration. Blood was collected after 24 and 48 h and fractionation of serum lipids on h.p.l.c. showed 5 peaks of radioactivity.     

Tuning the primary reaction of channelrhodopsin-2 by imidazole, pH, and site-specific mutations

Femtosecond time-resolved absorption measurements were performed to investigate the influence of the pH, imidazole concentration, and point mutations on the isomerization process of Channelrhodopsin-2. Apart from the typical spectral characteristics of retinal isomerization, an additional absorption feature rises for the wild-type (wt) on a timescale from tens of ps to 1 ns within the spectral range of the photoproduct and is attributed to an equilibration between different K-intermediates. Remarkably, this absorption feature vanishes upon addition of imidazole or lowering the pH. In the latter case, the isomerization is dramatically slowed down, due to protonation of negatively charged amino acids within the retinal binding pocket, e.g., E123 and D253. Moreover, we investigated the influence of several point mutations within the retinal binding pocket E123T, E123D, C128T, and D156C. For E123T, the isomerization is retarded compared to wt and E123D, indicating that a negatively charged residue at this position functions as an effective catalyst in the isomerization process. In the case of the C128T mutant, all primary processes are slightly accelerated compared to the wt, whereas the isomerization dynamics for the D156C mutant is similar to wt after addition of imidazole.     

1 alpha,25-dihydroxyvitamin D3 stimulates colony formation of chick embryo chondrocytes in soft agar

The effect of vitamin D metabolites on the growth of chick embryo chondrocytes in soft agar was examined. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] at 10(-8)-10(-7) M induced colony formation by chick embryo chondrocytes in soft agar in the presence of 10% fetal bovine serum. Furthermore, 1,25(OH)2D3 increased the number of colonies in the presence of a maximal dose of basic fibroblast growth factor, a potent mitogen for chondrocytes in soft agar. However, 24R,25 (OH)2D3 and other metabolites had little effect on the soft agar growth of chondrocytes in the presence or absence of basic fibroblast growth factor. These results suggest that 1,25(OH)2D3 is an active metabolite which may be involved in supporting cartilage growth.     

Stable isotope-labeled vitamin D, metabolites and chemical analogs: synthesis and use in mass spectrometric studies

Methods for the measurement of vitamin D and its metabolites using stable isotope-labeled internal standards and mass spectrometry are reviewed. The synthesis of both labeled and unlabeled standards is illustrated, and details of the synthesis of (26,26,27,27,27(-2)H5)-25,26-dihydroxyvitamin D3 and (28,28,28(-2)H3)-24,25-dihydroxyvitamin D2 are given. The use of in vitro biologic systems for the production of further metabolites of deuterated 25-hydroxyvitamin D3 is discussed. Use of deuterated 25-hydroxydihydrotachysterol3 as a substrate in the isolated perfused rat kidney has provided valuable data for the assignment of structure to a number of metabolites of 25-hydroxydihydrotachysterol3 formed in this system.     

Classification of commercial cultivars of Humulus lupulus L. (hop) by chemometric pixel analysis of two dimensional nuclear magnetic resonance spectra

The development of fast and effective spectroscopic methods that can detect most compounds in an untargeted manner is of increasing interest in plant extracts fingerprinting or profiling projects. Metabolite fingerprinting by nuclear magnetic resonance (NMR) is a fast growing field which is increasingly applied for quality control of herbal products, mostly via 1D ¹H NMR coupled to multivariate data analysis. Nevertheless, signal overlap is a common problem in ¹H NMR profiles that hinders metabolites identification and results in incomplete data interpretation. Herein, we introduce a novel approach in coupling 2D NMR datasets with principal component analysis (PCA) exemplified for hop resin classification. Heteronuclear multiple bond correlation (HMBC) profile maps of hop resins (Humulus lupulus) were generated for a comparative study of 13 hop cultivars. The method described herein combines reproducible metabolite fingerprints with a minimal sample preparation effort and an experimental time of ca. 28 min per sample, comparable to that of a standard HPLC run. Moreover, HMBC spectra provide not only unequivocal assignment of hop major secondary metabolites, but also allow to identify several isomerization and degradation products of hop bitter acids including the sedative principal of hop (2-methylbut-3-en-2-ol). We do believe that combining 2D NMR datasets to chemometrics, i.e. PCA, has great potential for application in other plant metabolome projects of (commercially relevant) nutraceuticals and or herbal drugs.     

Delta12-prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes

Prostaglandin D2 (PGD2) is a lipid mediator produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of asthmatic patients. There are two receptors for PGD2, namely the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). The proinflammatory effects of PGD2 leading to recruitment of eosinophils and Th2 lymphocytes into inflamed tissues is thought to be predominantly due to action on CRTH2. Several PGD2 metabolites have been described as potent and selective agonists for CRTH2. In this study we have characterized the activity of delta12-PGD2, a product of PGD2 isomerization by albumin. Delta12-PGD2 induced calcium mobilization in CHO cells expressing human CRTH2 receptor, with efficacy and potency similar to those of PGD2. These effects were blocked by the TP/CRTH2 antagonist ramatroban. delta12-PGD2 bound to CRTH2 receptor with a pKi of 7.63, and a 55-fold selectivity for CRTH2 compared to DP. In Th2 lymphocytes, delta12-PGD2 induced calcium mobilization with high potency and an efficacy similar to that of PGD2. delta12-PGD2 also caused activation of eosinophils as measured by shape change. Taken together, these results show that delta12-PGD2 is a potent and selective agonist for CRTH2 receptor and can cause activation of eosinophils and Th2 lymphocytes. These data also confirm the selective effect of other PGD2 metabolites on CRTH2 and illustrate how the metabolism of PGD2 may influence the pattern of leukocyte infiltration at sites of allergic inflammation.     

Effect of tibolone (Org OD14) and its metabolites on aromatase and estrone sulfatase activity in human breast adipose stromal cells and in MCF-7 and T47D breast cancer cells

Tibolone (Org OD14) is a synthetic steroid used for post-menopausal hormone replacement therapy (HRT). Since HRT might increase breast cancer risk, it is important to determine the possible effects of tibolone on breast tissues. Tibolone and its metabolites Org 4094, Org 30126 and Org OM38 have been reported to inhibit estrone sulfatase activity in MCF-7 and T47D breast cancer cell lines, which suggest beneficial effects on hormone dependent breast cancer by reducing local production of free estrogens. Breast adipose stromal cells (ASCs) contain aromatase activity-an obligatory step in the biosynthesis of estrogens-and possibly contain sulfatase activity. We investigated the effects of tibolone, its metabolites and the pure progestin Org 2058 on PGE(2)-stimulated aromatase activity and on sulfatase activity in human ASC primary cultures and on sulfatase activity in MCF-7 and T47D cell lines. In MCF-7, tibolone and metabolites, but not Org 2058, were found to inhibit sulfatase activity. In T47D, tibolone inhibited sulfatase only at 10(-6)M, although weakly. ASC had high sulfatase activity, which was inhibited by 10(-6)M of tibolone, Org 4094 and Org 30126, but not by Org OM38 or Org 2058. Surprisingly, aromatase activity in ASC was increased by both tibolone and Org 2058 at 10(-6)M. As ligand binding assay results and immunohistochemistry indicated the absence of progesterone and estrogen receptors in ASC, these effects on aromatase and sulfatase activity in ASC likely take place by other routes. Because tibolone and its metabolites inhibit sulfatase activity, and because tibolone only increases aromatase activity at a high concentration, we conclude that effects of tibolone on the breast are probably safe.     

Halothane attenuated haloperidol and enhanced clozapine-induced dopamine release in the rat striatum

The effect of halothane anesthesia on changes in the extracellular concentrations of dopamine (DA) and its metabolites (3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA)) induced by neuroleptics was studied using in vivo microdialysis techniques. Halothane attenuated haloperidol-induced dopamine release and enhanced clozapine-induced dopamine release in the rat striatum.A microdialysis probe was implanted into the right striatum of male SD rats. Rats were given saline or the same volume of 200 microg kg(-1) haloperidol (D(2) receptor antagonist), 10 mg kg(-1) sulpiride (D(2) and D(3) antagonist), or 10 mg kg(-1) clozapine (D(4) and 5-HT(2) antagonist) intraperitoneally with or without 1-h halothane anesthesia (0.5 or 1.5%). Halothane anesthesia did not change the extracellular concentration of DA, but increased the metabolite concentrations in a dose-dependent manner. The increased DA concentration induced by haloperidol was significantly attenuated by halothane anesthesia, whereas the metabolite concentrations were unaffected. Halothane had no effect on the changes in the concentrations of DA or its metabolites induced by sulpiride. The clozapine-induced increases in DA and its metabolites were enhanced by halothane anesthesia.Our results suggest that halothane anesthesia modifies the DA release modulated by antipsychotic drugs in different ways, depending on the effects of dopaminergic or serotonergic pathways.     

Cultured human keratinocytes cannot metabolize vitamin D3 to 25-hydroxyvitamin D3.

The metabolism of [3H]vitamin D3 was studied in cultured human keratinocytes (CHK). Intact CHK were incubated for 1, 6, 12, 24 and 48 h with [3H]vitamin D3 and the lipid soluble fractions from the media and cells were extracted by high-performance liquid chromatography (HPLC). Vitamin D3 and its metabolites, 25-OH-D3, 24,25(OH)2D3 and 1,25(OH)2D3 were added to the extracts, as markers, prior to HPLC. HPLC analysis of the lipid extracts did not reveal any monohydroxylated metabolites. CHK incubated for one hour with [3H]25-OH-D3 showed a 10 +/- 4% conversion to [3H]1,25(OH)2D3 whereas no conversion to [3H]1,25(OH)2D3 was observed in control CHKs that were boiled prior to incubation with [3H]25-OH-D3. These findings suggest that cultured neonatal keratinocytes are incapable of metabolizing vitamin D3 to 25-OH-D3.     

The quantitative determination of vitamin D3 and its metabolites in plasma

A method is described which enables determination of vitamin D3 and its physiologically most important metabolites, i.e. 25-OHD3, 24,25-(OH)2D3, 25,26-(OH)2D3 and 1,25-(OH)2D3 in a plasma sample of about 2 to 4 ml. The whole procedure involves two preparative and one analytical steps: Extraction with methanol/methylene chloride (2:1), chromatographic separation on Lipidex 5000 using a stepwise gradient of n-hexane and chloroform and finally HPLC separation on Zorbax-Sil columns with n-hexane isopropanol mixtures and subsequently reversed phase separation on RP 18-columns and mixtures of methanol and water. Except for 1,25-(OH)2D3 all D compounds were quantified by UV-detection with 1.4 ng of substance being the lowest detectable amount. 1,25-(OH)2D3 was measured by radioimmunoassay. Prior to HPLC analysis the extract was separated into three fractions on Lipidex 5000 which contained 1) vitamin D3, 2) 25-OHD3 and 3) the dihydroxy metabolites. The three fractions were separated by HPLC using different mixtures of isopropanol/n-hexane and methanol/water, respectively. Retention times of the individual D-components longer than 10 min appeared to be essential to separate these compounds from accompanying material. Overall recoveries of the individual metabolites were for vitamin D3 48.9%, for 25-OHD3 54.2%, for 24,25-(OH)2D3 50.9% and for 1,25-(OH)2D3 52.5%. Application of the methods to plasma samples from pigs with pseudovitamin D deficiency rickets, typ I, revealed a reduced concentration of 1,25-(OH)2D3 and 24,25-(OH)2D3 and an elevated level of 25-OHD3 in these animals. The results obtained by this method contributed substantially to a better understanding of the aetiological factors associated with this disease.     

Isomerization of (3S)-2,3-dihydro-5-fluoro-L-tryptophan and of 5-fluoro-L-tryptophan catalyzed by tryptophan synthase: studies using fluorine-19 nuclear magnetic resonance and difference spectroscopy

We are exploring the active site and the mechanism of the pyridoxal phosphate dependent reactions of the bacterial tryptophan synthase alpha 2 beta 2 complex by use of substrate analogues and of reaction intermediate analogues. Fluorine-19 nuclear magnetic resonance studies and absorption spectroscopy are used to study the binding and reactions of the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)- and (3R)-2,3-dihydro-5-fluorotryptophan. Tryptophan synthase specifically and tightly binds the 3S diastereoisomer of both 2,3-dihydro-5-fluoro-D-tryptophan and 2,3-dihydro-5-fluoro-L-tryptophan, whereas it binds 5-fluoro-D-tryptophan more tightly than 5-fluoro-L-tryptophan. Unexpectedly, we find that the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)-2,3-dihydro-5-fluorotryptophan are slowly interconverted by isomerization reactions. Since these isomerization reactions are 10(3)-10(5) times slower than the beta-replacement and beta-elimination reactions catalyzed by tryptophan synthase, they have no biochemical significance in vivo. However, the occurrence of these slow reactions does throw some light on the nature of the active site of tryptophan synthase and its requirements for substrate binding. Our results raise the interesting question of whether tryptophan synthase itself serves a catalytic role in these slow reactions or whether the enzyme simply binds the substrate and pyridoxal phosphate stereospecifically and thus promotes the intrinsic catalytic activity of pyridoxal phosphate.     

Responses of rachitic cartilage cells to metabolites of vitamin D3

Responses of cultured cartilage cells to metabolites of vitamin D3 were studied. Cells were obtained from the epiphyseal growth plate of rachitic chicks and were exposed to physiological and pharmacological concentrations of three metabolites of vitamin D3, 25 hydroxyvitamin D3 (25(OH)D3), 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 was found to reduce L-[U-14C]leucine incorporation into proteins and Na2 35SO4 incorporation into proteoglycans. The synthesis of 24,25(OH)2D3 from 25(OH)D3 was stimulated upon addition of 1,25(OH)2D3 to the cultures. Physiological concentrations of 24,25(OH)2D3 stimulated protein and proteoglycan synthesis. These findings support the notion that vitamin D3, through its active dihydroxylated metabolites, is directly involved in cartilage cells metabolism and healing of rickets.     

Photosynthesis of vitamin D in the skin: effect of environmental and life-style variables

Exposure to sunlight continues to play a major role in providing adequate vitamin D nutrition for most of the population of the world, including those who live in countries that practice fortification of dairy, margarine, and cereal products with vitamin D. During exposure to sunlight, the high-energy UV photons (290-315 nm) penetrate the epidermis and photolyze 7-dehydrocholesterol (provitamin D3) to previtamin D3. Once formed, previtamin D3 undergoes a thermally induced isomerization to vitamin D3 that takes 2-3 days to reach completion. Melanin effectively competes with provitamin D3 for the UV radiation that enters the epidermis and limits its photolysis to previtamin D3. However, this is not the major factor that prevents excess production of vitamin D in the skin of people who are constantly exposed to sunlight. During the initial exposure to sunlight, provitamin D3 is efficiently converted to previtamin D3. However, because previtamin D3 is photolabile, continued exposure to sunlight causes the isomerization of previtamin D3, principally to lumisterol. Thus, no more than 10-20% of the initial provitamin D3 concentrations ultimately end up as previtamin D3. Aging, sunscreens, seasonal changes, time of day, and latitude also significantly affect the cutaneous production of this vitamin-hormone.     

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.