The site of 1α,25-dihydroxyvitamin D3 production in pregnancy

Metabolism of 25-hydroxyvitamin D₃ (25-OH-D₃) in pregnancy was investigated $\text{in}\text{vitro}$ in New Zealand White rabbits fed a rabbit chow. Kidney homogenates from pregnant mothers and fetuses were separately incubated with [³H]-25-OH-D₃. The homogenates from fetuses produced significant amounts of $\text{[}{}^3\text{H]-1α,25-dihydroxyvitamin}$ D₃ [1α,25-(OH)₂-D₃] from its precursor, while those from mothers predominantly produced [³H]-24,25-dihydroxyvitamin D₃ [24,25-(OH)₂-D₃]. The identity of the radioactive metabolites produced from [³H]-25-OH-D₃ was established by periodate cleavage and comigration with synthetic 1α,25-(OH)₂-D₃ or 24,25-(OH)₂-D₃ on high pressure liquid chromatography. These results clearly indicate that the fetal kidney is at least one of the sites of 1α,25-(OH)₂-D₃ synthesis in pregnancy.     

Elasnin,a new human granulocyte elastase inhibitor produced by a strain of Streptomyces

Elasnin, a new human granulocyte elastase inhibitor, has been isolated from $\text{Streptomyces}\text{noboritoensis}$ KM-2753. Elasnin is a neutral, lipophilic colorless and viscous oil ($\text{n}{}_{\mathrm{D}}{}^{17}\text{=1.4983}$, $\text{[α]}{}_{\mathrm{D}}{}^{18}\text{?0.9°}$, λ_max^EtOH 291 nm (ε, 7760)). The molecular formula was C₂₄H₄₀O₄ (M.W.: 392) as determined by its elemental analysis and mass spectrum. Elasnin inhibits markedly human granulocyte elastase, but is almost ineffective for pancreatic elastase, trypsin, chymotrypsin, thermolysin and papain.     

Nuclear and cytoplasmic binding components for vitamin D metabolites.

Specific binding of 1α,25-dihydroxyvitamin D₃ to macromolecular components of small intestinal nuclei and cytosol is demonstrated. The nuclear 1α,25-dihydroxyvitamin D₃ complex can be extracted from chromatin by 0.3 M KCl and sediments at 3.7S in sucrose density gradients. The cytoplasmic 1α,25-dihydroxyvitamin D₃-binding components also sediment at 3.7S, identically to the nuclear complex under the ultracentrifugation procedures employed.Macromolecular binding components with a high affinity for 25-hydroxyvitamin D₃ (K_d = 4.5 × 10⁻⁹ M) were also identified in intestinal cytosol which differ from the 1α,25-hydroxyvitamin D₃ receptor in that: 1) they sediment at 5–6S in sucrose gradients, 2) they are observed in organs other than the intestine, and 3) while they do bind 1α,25-dihydroxyvitamin D₃ at higher concentrations than 25-hydroxyvitamin D₃, they are not observed to transfer either 25-hydroxyvitamin D₃ or 1α,25-dihydroxyvitamin D₃ to the nucleus, $\text{in vitro}$.     

Equilibrium constants for association of guanidinium and ammonium ions with oxyanions: The effect of changing basicity of the oxyanion

Using guanidinium and n-butylammonium cations (C⁺) as models for the positively charged side chains in arginine and lysine, we have determined the association constants with various oxyanions by potentiometric titration. For a dibasic acid, H₂A, three association complexes may exist: $\text{K}{}_{\mathrm{<mtext>1M</mtext>}}\text{=}\text{[CHA]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[HA}{}^{\mathrm{?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>1D</mtext>}}\text{=}\text{[CA}{}^{\mathrm{?}}\text{]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[A}{}^{\mathrm{2?}}\text{]}$; $\text{K}{}_{\mathrm{<mtext>2D</mtext>}}\text{=}\text{[C}{}_2\text{A]}\text{[C}{}^{\mathrm{+}}\text{]}\text{[CA}{}^{\mathrm{?}}\text{]}$. For guanidinium ion and phosphate, K_1M = 1.4, K_1D = 2.6, and K_2D = 5.1. The data for carboxylates indicate that the basicity of the oxyanion does not affect the association constant: acetate, pK_a = 4.8, K_1M = 0.37; formate, pK_a = 3.8, K_1M = 0.32; and chloroacetate, pK_a = 2.9, K_1M = 0.43, all with guanidinium ion. Association constants are also reported for carbonate, dimethylphosphinate, benzylphosphonate, and adenylate anions.     

Effects of 1α,25-,24R,25-, and 1α,24R,25-hydroxylated metabolites of vitamin D3on calcium and phosphate absorption by duodenum from intact and nephrectomized rats

The effects of 1α,25-dihydroxyvitamin D₃, 24R,25-dihydroxyvitamin D₃ and 1α,24R,25-trihydroxyvitamin D₃ on active calcium and phosphate transport by rat duodenum were studied in vitamin D-deficient rats that either underwent sham surgery or were bilaterally nephrectomized. Both 1α, 25-dihydroxy- and 1α,24R,25-trihydroxyvitamin D₃ markedly stimulated calcium and phosphate absorption with similar effects in shamoperated and nephrectomized rats. A 10-fold higher dose of 24R,25-dihydroxyvitamin D₃ was required for an equivalent stimulation of absorption in sham-operated rats, and this compound had no effect on duodena from nephrectomized rats. These data provide the first evidence that 24R,25-dihydroxy- and 1α,24R,25-trihydroxyvitamin D₃ can stimulate the active intestinal absorption of phosphate. The lack of response to 24R,25-dihydroxyvitamin D₃ in nephrectomized rats confirms prior results which indicated that renal metabolism of this secosteroid to 1α,24,25-trihydroxyvitamin D₃ is required for biological activity. In addition, we describe a simple bioassay technique which apparently reflects, with reasonable accuracy, the changes in duodenal calcium and phosphate absorption which occur under more rigorous short-circuited conditions and, in particular, can be used for screening putative 1α-hydroxyl analogs of vitamin D in nephrectomized rats.     

1Alpha,25-dihydroxyvitamin D3 receptor: competitive binding of vitamin D analogs

The intestinal nuclear receptor for lα,25-dihydroxyvitamin D₃ has been utilized to determine the ability of vitamin D-active sterols to compete with this hormone at the molecular level. 25-Hydroxyvitamin D₃ and lα-hydroxyvitamin D₃ must be present in 150 and 450 times the concentration respectively of lα,25-dihydroxyvitamin D₃, $\text{in}$$\text{vitro}$, to displace the physiologic hormone. These data indicate that: i) superphysiologic levels of 25-hydroxyvitamin D₃ may simulate lα,25-dihydroxyvitamin D₃ and act directly on isolated target organs and ii) the biologic potency observed for low doses of lα-hydroxyvitamin D₃, $\text{in}$$\text{vivo}$, is probably the result of 25-hidroxylation of the lα-derivative to form lα,25-dihydroxyvitamin D₃.     

Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate

(1) $\text{N-4-}\text{Azido}\text{-2-}\text{nitrophenyl}\text{-γ-[}{}^3\text{H]aminobutyryl-Ado}\text{PP[}\text{NH}\text{]P(}\text{NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P)}$ a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of ³H]NAP₄-AdoPP[NH]P to soluble ATPase from beef heart mitochrondria (F₁) was studied in the absence of photoirradiation, and compared to that of $\text{[}{}^3\text{H]Ado}\text{PP[}\text{NH}\text{]P}$. The photoactivable derivative of AdoPP[NH]P was found to bind to F₁ with high affinity, like AdoPP[NH]P. Once $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ had bound to F₁ in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP₄ or AMP. Furthermore, preincubation of F₁ with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ upon photoirradiation. (3) Photoirradiation of F₁ by $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}\text{mol F}{}_1$. Photolabeling by NAP₄-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl₂. (4) Bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was localized on the α- and β-subunits of F₁. At low concentrations (less than 10 μM), bound $\text{[}{}^3\text{H]NAP}{}_4\text{-}\text{Ado}\text{PP[}\text{NH}\text{]P}$ was predominantly localized on the α-subunit; at concentrations equal to, or greater than 75 μM, both α- and β-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (α or β), suggesting that each of the two subunits, α and β, is required for the activity of F₁. (6) The covalently photolabeled F₁ was able to form a complex with aurovertin, as does native F₁. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The percentage of inactivation of F₁ was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggesting that fluorescence quenching is related to the binding of ATP to the catalytic site of F₁.     

Studies on the molecular weight of the adenovirus type 2 hexon and its subunit

The molecular weight of the adenovirus type 2 hexon was calculated from sedimentation equilibrium, light scattering and sedimentation and diffusion experiments. The extinction coefficient, E_{1 cm}^1%, was determined to be 14.3 at 279 nm, from quantitative nitrogen and carbon analyses combined with the N,C content calculated from the amino acid composition. Other parameters determined were: the partial specific volume, $\text{}\text{?}\text{gn = 0.738}\text{cm}{}^3\text{g}{}^{\mathrm{?1}}$; the refractive index increment, $\text{(}\text{?n}\text{?c}\text{)}{}_{\mathrm{<mtext>T,P</mtext><mtext>,\mu </mtext>}}\text{= 0.193}\text{cm}{}^3\text{g}{}^{\mathrm{?1}}$ at 435.8 nm; the sedimentation coefficient, s_20,w⁰ = 13.0 S; and the diffusion constant, D_{20, w}⁰ = 3.32 × 10^?7 cm² s^?1. All molecular weights were between 355,000 and 363,000. Crystal density measurements were made on native and glutaraldehyde cross-linked crystals and the molecular weights calculated from these data were compared with the precise molecular weight determined by physico-chemical methods.Only one polypeptide of molecular weight 120,000 was found in reduced, or reduced and alkylated, hexon. Four or six organomercurial molecules were bound per 120,000 molecular weight of native hexon upon titration with 2-chloromercuri-4-nitrophenol and 2-chloromercuri-4,6-dinitrophenol, respectively. With 5,5′-dithiobis (2-nitrobenzoic acid) only one SH-group per 120,000 could be titrated in native hexon, but after denaturation in 1% sodium dodeeyl sulphate five more SH-groups reacted per 120,000 molecular weight. Thus there are three identical polypeptides of molecular weight 120,000 per hexon of total molecular weight 360,000.     

In vitro reaction of radioactive 7beta, 8alpha, --dihydroxy--9alpha, 10alpha-epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene and 7beta,8alpha--dihydroxy--9beta, 10beta--epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene with DNA.

The $\text{in vitro}$ reaction of bacteriophage T7-DNA with the radioactive diastereomeric benzo(a)pyrene-diol-epoxides, (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9β,19β-epoxy-7,8,9,10-tetrahydrobenzo(1)pyrene, was investigated. Chromatographic analysis of digests of the DNA allowed the distinction of characteristic deoxynucleoside adduct peaks for the two benzo(a)pyrene-diol-epoxides. Our results, together with data from the literature, allow the identification of these adducts as mostly N₂-(10-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine and N₂-(10-7β,8α,9β-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine, respectively. DNA-benzo(a)pyrene adducts with the same chromatographic properties were formed in mouse embryo fibroblasts upon treatment with benzo(a)pyrene.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 (PTH_1–34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [³H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylae activity appeared to follow Michaelis-Menten kintics, and 1,25-dihydroxyvitamin D-3 treatment increased the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 24-hydroxylase from 33 to 95 pmol/h per 10⁶ cells without affecting the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 · 10^?10 M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 μM cycloheximide. Treatment of the cells with PTH_1–34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 · 10^?9 M PTH_1–34. When 2.4 · 10^?9 M PTH_1–34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to $\text{70.7 ± 2.9\%}$ of control. Higher concentrations of PTH_1–34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH_1–34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH_1–34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

An hydroxylapatite batch assay for the quantitation of 1alpha,25-dihydroxyvitamin D3-receptor complexes.

A versatile hydroxylapatite batch assay for 1α,25-dihydroxyvitamin D₃-receptor complex from chick intestinal mucosa has been developed. The assay has been characterized with respect to time and temperature of incubations, protein concentration, amount of hydroxylapatite required to bind receptor-steroid complexes, pH, and effects of KCl and phosphate. Triton X-100 (0,5%, $\text{v}\text{v}$) was found to be essential for the removal of nonspecifically bound ligand. The hydroxylapatite was shown to bind the 1α,25-dihydroxy-vitamin D₃ receptor as demonstrated by the specificity and high affinity for 1α,25-dihydroxy-vitamin D₃ and the sedimentation properties of the phosphate-extracted hydroxylapatite-bound complex on sucrose density gradients. Binding appears to be nearly quantitative. The efficient separation of bound from free ligand utilizing this assay makes it possible to examine a number of aspects of the binding of this steroid hormone to its cytoplasmic receptor that has not previously been possible.     

An equilibrium and kinetic study of 1,25-dihydroxyvitamin D3 binding to chicken intestinal cytosol employing high specific activity 1,25-dehydroxy[3H-26, 27] vitamin D3

A reexamination of the equilibrium and the kinetics of 1,25-dihydroxy vitamin D₃ binding with its receptor in chick intestinal cytosol was performed because of the recent availability in our laboratory of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$]vitamin D₃ (160 Ci/mmol). Under saturating conditions at 25 °C, Scatchard analysis revealed an equilibrium dissociation constant (K_d) of 7.1 × 10^?11m which is several fold lower than previously reported for this binding reaction. Furthermore, an estimate of 1.8 × 10³ receptor sites per cell was obtained from the intercept of the line with the abscissa of the Scatchard plot. From a kinetic analysis of 1,25-dihydroxy vitamin D₃ binding with chick intestinal cytosol, association and dissociation rate constants were determined. Values that were obtained at 25 °C for these processes were 9.5 × 10⁸m^? min^? and 7.1 × 10^?3 min^?, respectively. Although these studies, such as for other steroid hormones, were carried out using a crude native cytosol preparation, we have been able to demonstrate unequivocally through the use of high specific activity 1,25-dihydroxy[${}^3\text{H-26,27}$] vitamin D₃ a truly high affinity binding site.     

Isolation and Structure of Sclerothionine,a Metabolite of Sclerotinia Fungus

A new sulfur-containing imidazole compound, m.p. 218~223°C (decomp.), ${[\alpha ]}_{\text{D}}^{24}+{7.4}^{°}$ in water), C₁₁H₁₉N₃O₃S was isolated from sclerotia of Sclerotinia libertiana and named sclerothionine. The chemical structure of sclerothionine was identified with 2-hydroxyethyl-ergothioneine which was synthesized from ethylene chlorhydrine and ergothioneine.     

Comformation and absolute configuration of -methyllanthionine

If the bicyclic peptide ring proposed by Gross $\text{et}$$\text{al}$. (1,2) does in fact exist in nisin and related antibiotics, then the unusual β-methyllanthionine component must be significantly distorted from its conformation in the free state, as determined by x-ray structure analysis. The torsion angles about the SC_β bonds are 50–100° from the torsion angles in models of the sulfur-bridged peptide ring proposed for nisin. The chirality of the methylated β-carbon atom is (S). The conformation of the amino acid differs from that of $\text{meso}$-lanthionine only by a 180° rotation of a carboxyl group about the $\text{C}{}_{\mathrm{\alpha }}{}^{\mathrm{D}}\text{C}{}_{\mathrm{\beta }}\text{(CH}{}_3\text{)}$ bond.     

The biosynthesis of blood group B determinants by the blood group A gene-specified alpha-3-N-acetyl-D-galactosaminyltransferase

The blood group $\text{A}{}^1$ gene-specified α-3-N-acetyl-D-galactosaminyl-transferase in human plasma, when concentrated by adsorption onto group O red cell ghosts or Sepharose 4B, catalyses the transfer of D-galactose in α-linkage to low-molecular-weight H-active acceptors. The product synthesised with 2′-fucosyllactose is chromatographically indistinguishable from the blood group B-active tetrasaccharide, Galα1→3[Fucα1→2]Galβ1→4Glc. The optimum pH for the transfer of D-galactose by the $\text{A}{}^1$-transferase is 7. At this pH the V_max for the transfer of N-acetyl-D-galactosamine is about 300 times higher than that for the transfer of D-galactose. These results indicate that an $\text{A}{}^1$-transferase can, under centain conditions, synthesise B determinant structures.     

Possible interrelationship of multiple alpha-bungarotoxin binding components in the brain of the horseshoe crab, Limulus polyphemus.

Three $\text{[}{}^{125}\text{I]α-bungarotoxin}$ binding components have been detected in solubilized extracts of $\text{Limulus}$ brain tissue. These components have sedimentation coefficients of 9.0S, 15.4S and 17.4S. All three components were degraded by α-chymotrypsin. The addition of butyl alcohol to brain extract suggests an interrelationship of the toxin binding proteins by promoting a simultaneous decrease in the 9S component and increase in the 15.4S and 17.4S components. This transition was also demonstrated by the readdition of isolated fractions of each component to brain extract.     

Acid dissociation of cyclohexaamylose and cycloheptaamylose

Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pK_a's are ΔH⁰ = 8.4 ± 0.3 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?28. ± 1}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 6-Cy and ΔH⁰ = 10.0 ± 0.1 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?22.4 ±0.3}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 7-Cy. Intrinsic ¹³C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D₂O ($\text{v}\text{v}$). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 2⁰-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding.     

The addition of bisulfite to 5-fluorouracil. Evidence for a change in rate determining step

The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of $\text{k}{}_{\mathrm{obsd}}\text{[}\text{SO}{}_3{}^{\mathrm{=}}\text{]}$ measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO₃^? or (OHCH₂CH₂)₂N⁺C(CH₂OH)₃ HCl (BisTris⁺HCl). A small kinetic hydrogen-deuterium isotope effect ($\text{k}{}_{\mathrm{HS}}\text{k}{}_{\mathrm{DS}}\text{= 1.10}$) was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase.     

Purification of cytochromes P-448 from β-naphthoflavone-treated rainbow trout

Rainbow trout were treated with β-naphthoflavone and the hepatic microsomal cytochrome P-450 solubilized with 3-[(3-cholaminopropyl)dimethylammonio]-1-propanesulfonate. Chromatography on tryptamine-Sepharose 4B gave a single cytochrome P-450 peak which was further resolved into three components by elution from DEAE-Sepharose. The two main peaks were then chromatographed on hydroxyapatite and a total of four fractions obtained. Two of these fractions had similar properties and significantly metabolized $\text{[}{}^{14}\text{C}\text{]}\text{benzo}\text{[a]}\text{pyrene}$ in a reconstituted system containing rat cytochrome P-450 reductase. This activity was inhibited by α-naphthoflavone but not by metyrapone or SKF-525A. Purified cytochromes P-448 from 3-methylcholanthrene-treated rat had similar spectral properties and activity towards $\text{[}{}^{14}\text{C}\text{]}\text{benzo}\text{[a]}\text{pyrene}$ suggesting similarities between these forms.