Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3-25-hydroxylase

Solubilized components of the vitamin D₃-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D₃in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochrome c reductase and cytochrome P-450 or P-448. The P-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochrome P-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochrome c, was isolated free of cytochromes b₅ or P-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants: K_m(NADPH) = 3.14 μM, K_m(NADH) = 31.25 μM, and K_{m(cyt c)} = 12.34 μM. The NADPH-cytochrome c reductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of the P-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide, ³H-labeled vitamin D₃, and soluble cytosolic supernatant, full vitamin D₃-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochrome P-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D₃-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochrome c reductase and a cytochrome P-450 terminal oxidase, for the catalytic conversion of vitamin D₃ to 25-hydroxyvitamin D₃.     

Properties of ATPase of gastric mucosa V. Preparation of membranes and mitochondria by zonal centrifugation

Using zonal sucrose density gradient centrifugation methods to fractionate the subcellular components in gastric mucosal homogenates have been developed. Methods are described which give high yield or rapidity in fractionation. A procedure is described which enables large-scale preparations of smooth walled vesicular membranes containing HCO₃^?-ATPase activity free from mitochondrial contamination as assessed by electron microscopic morphology and undetectable succinic dehydrogenase or monoamine oxidase activity. A method to purify gastric mitochondria is also described.     

Hepatic microsomal N-oxidation and N-demethylation of N,N-dimethylaniline in red-winged blackbird compared with rat and other birds.

Hepatic microsomes prepared from red-winged blackbirds ($\text{Agelaius phoeniceus}$) and albino rats were incubated with $\text{N}$,$\text{N}$-dimethylaniline (DMA)_in complete incubation mixtures at pH 7.9 and 37°C for 10 min. Formaldehyde and $\text{N}$,$\text{N}$-dimethylaniline-$\text{N}$-oxide produced from DMA were measured. Redwings were found to have significantly lower $\text{N}$-demethylation activities than rats, and redwings had only marginal or no $\text{N}$-oxidation activities. Hepatic microsomes from redwings did not further metabolize the $\text{N}$-oxide. The $\text{N}$-oxidation and $\text{N}$-demethylation activities of brown-headed cowbirds ($\text{Molothrus ater}$), common grackles ($\text{Quiscalus quiscula}$), and starlings ($\text{Sturnus vulgaris}$) were similar to those of redwings.     

(Des-Histidine1) (Nϵ-phenylthiocarbamoyllysine12)-glucagon: Effects on glycogenolysis in perfused rat liver

(Des-Histidine¹) (N^?-phenylthiocarbamoyllysine¹²)-glucagon, synthesized by the one-step Edman degradation procedure is a competitive inhibitor of glucagon action in the rat liver plasma membrane adenylate cyclase system. However, in the perfused rat liver, the compound did not inhibit glucagon stimulated glycogenolysis even when used at a concentration 100-fold in excess of native glucagon. Instead, it showed a weak potency, but full agonist activity, stimulating liver glycogenolysis to 100% of the level obtained by glucagon. These results are discussed in terms of the possible mechanism(s) of glucagon action.     

Ascorbic acid decreases [3H]dopamine binding in striatum without inhibiting dopamine-sensitive adenylate cyclase

Ascorbic acid is found in very high concentrations in cells of neural crest origin such as the central nervous system and the adrenal gland. A variety of evidence has been marshalled to support a role for ascorbate as a chemical messenger. One of the first non-biosynthetic biochemical effects ascribed to ascorate in the CNS was its ability to inhibit dopamine-stimulated adenylate cyclase (DA-ACase) in homogenates from striata of Long Evans rats (J. Neurochem.28, 663, 1977). Using an adenylate cyclase assay based on preparative HPLC, we were unable to detect any inhibition of DA-ACase by ascorbate at concentrations as high as 1 mM. Moreover, this failure to find inhibitory effects of ascorbate on DA-ACase occurred not only when striatal homogenates from Long-Evans rats were used, but also when tissue from Sprague-Dawley rats of N.C. Board of Health mice was tested. Although ascorbate may play a neuromodulatory role, it does not appear that its effects are mediated through effects on cAMP biosynthesis. Despite our inability to detect effects of ascorbate on DA-ACase, we did confirm that ascorbate significantly altered the binding of [³H]dopamine to striatal membranes. Thus, it is clear that the sites binding [³H]dopamine that are affected by ascorbate are unlikely to be the same ones coupled to aenylate cyclase.     

Evidence for a single type of linkage in a fructofuranan from Lolium perenne

On extraction with water, rye grass (Lolium perenne) yielded a mixture of polysaccharides. Fractionation thereof led to the isolation of two fructofuranans, one of which preponderated. Analysis of the main polysaccharide by ¹³C-n.m.r. spectroscopy indicated that it is composed of fructofuranosyl residues linked only (2 → 6), and terminated by a glucosyl group linked as in sucrose.     

Properties of 3H-cimetidine binding in rat brain membrane fractions

In an attempt to characterize the brain histamine H₂ receptor, experiments were undertaken to study the binding properties of (N-methyl-³H) -cimetidine, an H₂ receptor antagonist, in rat brain membranes. Using a centrifugation assay, ³H-cimetidine binding having a K_d of 0.40μM and a Bmax of 3.9 pmoles/mg protein was detected. Of fourteen anions and cations tested, one, Cu⁺⁺, dramatically increased specific ³H-cimetidine binding, the increase being due mainly to a change in Bmax. Studies of substrate specificity for ³H-cimetidine binding revealed that Cu⁺⁺, while not significantly affecting the potency of H₂ receptor agonists and antagonists, dramatically decreases the potency of H₁ receptor substances on the ³H-cimetidine binding site. In addition, both the relative and absolute potencies of various H₂ receptor agonistsv and antagonists in displacing the ligand in the presence of Cu⁺⁺ parallels their potencies in biological systems. These findings suggest that, under these conditions, ³H-cimetidine may be labelling a biologically relevant H₂ binding site in brain and that Cu⁺⁺ may regulate the substrate specificity for this site. The brain regional distribution and kinetic analysis of the binding suggest that it is not localized solely to the synaptic receptor for histamine, but may also be associated with histamine receptors at other neuronal, glial or vascular sites.     

Location and amino acid sequence around the ADP-ribosylation site in the cholera toxin active subunit A1

Renatured, S-carboxymethylated subunit A₁ of cholera toxin possess the ADP-ribose transferase activity (Lai, et.al., Biochem. Biophys. Res. Commun. 1981, $\text{102}$, 1021). In the absence of acceptor self ADP-ribosylation of A₁ subunit was observed. Stoicheometric incorporation of ADP-ribose moiety was achieved in 20 min at room temperature in a 0.1 – 0.2M PO₄(Na) buffer, pH 6.6. On incubation of the complex with polyarginine, 75% of the enzyme-bound ADP-ribose moiety was transferred to the acceptor in 25 min. The ADP-ribosylated A₁ was stable at low pH, and on cleavage with BrCN, the ADP-ribose moiety was found associated with peptide Cn I, the COOH-terminal fragment of A₁ subunit. On further fragmentation with cathepsin D, a dodecapeptide containing ADP-ribose moiety was isolated whose structure was determined as: Asp-Glu-Glu-Leu-His-Arg-Gly-Tyr-Arg¹-Asp-Arg-Tyr. The Arg¹ in the peptide was indicated to be the site of ADP-ribosylation.     

The regulatory role of Mg2+ on rabbit skeletal muscle phosphorylase kinase

The stimulation of phosphorylase kinase by Mg²⁺ was studied. Both the nonactivated and activated kinases are stimulated by Mg²⁺ at concentrations that are 100- to 200-fold greater than ATP. This stimulation is observed at both pH 6.8 and 8.2 and results in a 10-fold increase in the activity of the nonactivated kinase. Mg²⁺ stimulation is additive with that observed by calmodulin. Both the Ca²⁺-dependent and -independent activities of the kinase are stimulated by high [Mg²⁺]. Kinetically this stimulation can be explained by a decrease in the K_m for both phosphorylase b and ATP or an increase in V. The pH $\text{6.8}\text{8.2}$ ratio (0.06) is unaffected by [Mg²⁺] between 5 and 20 mm, but increases when [Mg²⁺] is less than 5 mm or greater than 20 mm. The stimulation by high [Mg²⁺] is explained by a direct effect of this cation on the kinase molecule rather than on its protein substrate, phosphorylase. This activating effect of high [Mg²⁺] does not result in any permanent change in the kinase molecule and can be readily reversed by diluting [Mg²⁺] to a low value.     

Differential effects of prazosin and rauwolsin on the release of prostaglandins I2 and E2 from the perfused mesenteric arterial bed of the rabbit following nerve stimulation

Sympathetic nerve stimulation of the perfused mesenteric arterial bed of the rabbit, , increase the secretion of prostaglandin (PG)I₂ and PGE₂. Prazosin (4.8 × 10⁻⁶), and α₁ adrenergic receptor antagonist, inhibited this inrease in release of PGI₂ but not of PGE₂ whereas rauwolsin (10⁻⁷ M), an α₂ adrenergic receptor antagonist, inhibited the increase in release of PGE₂ but not of PGI₂. Prazosin (10⁻⁶ M) completely blocked the vasoconstrictor response to nerve stimulation, and to norepinephrine and phenylephrine administration, suggesting there to be little of an α₂ adrenergic receptor component in this response. It is concluded that the increase in PGI₂ release follows the activation of α₁ adrenergic receptors and is therefore post-junctional in origin, whereas the increase in PGE₂ release follows the activation of α₂ adrenergic receptors and may be pre- and/or post-junctional in origin.Indomethacin (2.8 × 10⁻⁷, 5.6 × 10⁻⁷ and 1.12 × 10^{−6 M} did not affect the vasoconstrictor responses to nerve stimulation at 10 Hz, whereas rauwolsin (10⁻⁷ M) in the presence of indomethacin substantially increased them. These results indicate that PGE₂ does not regulate norepinephrine release following nerve stimulation at 10 Hz to rabbit mesenteric arteries, and that the inhibition of norepinephrine release following stimulation of α₂ pre-junctional receptors is independent of PG involvement.     

The interferon-induced enzyme oligo-isoadenylate synthetase: rapid determination of its in vitro products

A procedure for rapid isolation of the products of the interferon-induced enzyme, oligoisoadenylate synthetase, is described. After incubation of [α-³²P]ATP with the poly(rI): (rC)-adsorbed fraction of cellular proteins, the products are treated with phosphatase. Aliquots of the nucleotides are then applied on small columns which contain 300 μl of alumina powder and eluted with 3 ml of 1 m glycine-HCl buffer, pH 2.0. The labeled free phosphate, released by the phosphatase treatment, is efficiently adsorbed by the alumina, while the phosphatase-resistant cores of the oligo-isoadenylates, up to the length of pentamer, are eluted. Larger oligomers are only partially recovered. We successfully applied this method for determination of the level of the enzyme in multiple samples of cell homogenates.     

Inhibition of yeast mitochondrial ATPase by concanavalin A

Concanavalin A binds to and inhibits enzyme activity of the energy transducing ATPase from yeast mitochondria. Activity loss is completely reversed by glucose or α-methyl-d-mannose. Concanavalin A reacts with the F₁ portion of the ATPase complex, suggesting that this enzyme unit may be a glycoprotein. A major concanavalin A binding site is associated with the largest subunit of the F₁ enzyme.     

Evidence for restricted oligosaccharide mobility at the erythrocyte membrane surface: A fluorescence study

The glycophorins of whole, human erythrocytes were labeled at their sialic acid residues with one of three fluorescent probes. After preparation of the erythrocyte ghosts, the mobility of each fluorescent probe on the intact membrane was compared with its mobility on the isolated, labeled glycopeptides dissolved in aqueous buffer. A four- to ninefold decrease in the rotational relaxation time, as defined by the Perrin equation, accompanied the proteolytic removal of the labeled glycopeptides from the membrane. This suggests that the fluorescent probes, and by extrapolation, the sugars to which they are immediately attached, are restricted in their mobility at the membrane surface. A crude model of the carbohydrate layer of the erythrocyte surface was constructed by incorporating the labeled, tryptic glycopeptides into agarose gels of different agarose content. A decrease in the probe's mobility was observed as agarose content was raised. This indicates that the high oligosaccharide density at the erythrocyte membrane surface may contribute to the observed immobilization of the fluorescent probes in situ.     

Inhibition of protein synthesis by carbonyl compounds (4-hydroxyalkenals) originating from the peroxidation of liver microsomal lipids

Carbonyl compounds released during the NADPH-Fe dependent peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (almost entirely as 4-hydroxynonenal) inhibit protein synthesis in a rabbit reticulocyte lysate. The ID₅₀ was 0.48 mM. The inhibitory effect was reproduced by synthetic 4-hydroxynonenal. The inhibition was already evident at 1–2 min of incubation. The addition of ?SH groups to the incubation medium afforded a marked protection against the inhibition of protein synthesis. The inhibitory effect seems to be due to an interaction of the carbonyl compound with ?SH groups essential for the cellular protein synthetic machinery.