3-ethyl and 3-methyl ethers of 19-iodocholesterol as potential adrenal scanning agents

19-Iodocholesterol-¹³¹I, though useful as an adrenal scanning agent, was found to be unstable. However, the substitution of methoxyl and ethoxyl groups for the hydroxyl group in the 3 position rendered these derivatives much more stable in solid form than the parent compound. They showed concentration in the adrenals of dogs similar to that of 19-iodocholesterol itself and therefore may be useful as adrenal scanning agents.     

Identification of the octapeptide [Met]enkephalin -Arg6-Gly7-Leu8 in extracts of bovine adrenal medulla

The primary structure of the 5300 dalton adrenal enkephalin-containing polypeptide was shown to contain at its carboxyl terminus the sequence -Lys-Arg-Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu-COOH (Jones $\text{et al}$., (1981) Proc. Natl. Acad. Sci. USA, in press). From knowledge of the type of processing that occurs at paired basic amino acid residues such as -Lys-Arg-, it was predicted that the octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu should be produced and exist in free form in the adrenal gland. This octapeptide has now been purified from bovine adrenal chromaffin granules. Its structure was determined by amino acid analysis, carboxypeptidase Y time course hydrolysis and sequential digestion with trypsin and carboxypeptidase B. The octapeptide has 35% the opiate receptor binding activity of [Met]enkephalin.     

Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes

Synexin, a soluble protein which increases the specificity of Ca²⁺ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. $\text{253}$, 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca²⁺-promoted aggregation of granule membranes. This effect is Ca²⁺-specific. However in contrast to Creutz $\text{et}\text{al}$, we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz $\text{et}\text{al}$ that synexin specifically binds the granule to the plasma membrane prior to exocytosis $\text{in}\text{vivo}$.     

Isolation of micro glutamic acid-rich protein from bovine brain

An acidic protein, designated as micro glutamic acid-rich protein, was purified to homogeneity from bovine brain extract, and was characterized in its physicochemical properties. The protein had an isoelectric point of 3.9, a molecular weight of 10,000, and was composed of very limited amino acid constituents; $\text{Asp}\text{Asn}$, Thr, Ser, $\text{Glu}\text{Gln}$, Pro, Gly, Ala and Lys, with a relative abundance of glutamic acid/glutamine which accounted for 51 % of the total amino acid composition. The yield of the protein was 750 μg/kg of wet brain tissue. The amino-terminal sequence analysis suggested that the protein arose through proteolysis of the 58,000-dalton precursor protein, that had been reported in a previous paper [Ishioka $\text{et al}$, (1980) Biochim. Biophys. Acta, 625, 281–290].     

Effects of adrenal steroid activator protein on the conversion of various 20- and 22-hydroxycholesterols to pregnenolone by adrenal mitochondrial enzymes

A heat-stable protein activator from bovine adrenal cortex mitochondria stimulates the conversion of cholesterol to pregnenolone in crude extracts of adrenal mitochondria, and resembles in some of its properties, the sterol carrier protein of liver (Kan $\text{et}\text{al}$. $\text{Biochem}$. $\text{Biophys}$. $\text{Res}$. $\text{Commun}$. $\text{48}$, 423–429, 1972). We have shown that activator preparations also stimulate highly purified adrenal enzyme preparations comprising four components: cytochrome P-450 specific for side chain cleavage, adrenodoxin, adrenodoxin reductase, and an NADPH-generating system. Furthermore, this activator stimulates the conversion not only of cholesterol, but also of (20S)-20-hydroxycholesterol, (22$\text{R}$)-22-hydroxycholesterol, and (20$\text{R}$, 22$\text{R}$)-20,22-dihydroxycholesterol to pregnenolone. Our findings provide additional evidence that the steroid-activator complexes are the substrates for the side chain cleavage enzyme and that the monohydroxy and dihydroxycholesterols are true intermediates in the conversion of cholesterol to pregnenolone by bovine adrenal cortex mitochondria.     

Three new alpha-chains of collagen from a non-basement membrane source

Three new collagen α chains have been isolated from synovial membrane, gingiva and skin. Two of these have a similar chromatographic behaviour to the α[A] and α[B] chains described by Burgeson $\text{et al}$. (4) from a foetal basement membrane source but have been separated from another contaminating α chain, α[C]. The α[A] and α[B] chains are present in approximately equal amounts. They contain no detectable 3-hydroxyproline, are highly glycosylated and all sugar residues are present as the disaccharides. The percentage of hydroxylation of the lysine is of the order of 70%. Only a third of these hydroxylysine residues are glycosylated. The significance of these peptides, present in a tissue substantially free of basement membrane, is discussed.     

Radiobromine labeled norcholesterol analogs. Synthesis and tissue distribution study in rats of bromine-82 labeled 6β-bromomethyl-19- norcholest-5(10)-en-3β-ol

6β-Bromomethyl-19-norcholest-5(10)-en-3β-o1 (NCL-6-Br) was synthesized directly from cholest-5-ene-3β,19-diol 19-toluene-psulfonate $\text{via}$ homoallylic rearrangement with ammonium bromide or sodium bromide in acetonitrile. This method was applied to the radiolabeling of NCL-6-Br with bromine-82. Tissue distribution of bromine-82 labeled NCL-6-Br (NCL-6-Br-82) in rats was determined. The mean percent dose per gram uptake in adrenal at 24 and 120 h was 98 and 80 %/gm, respectively, which indicated a higher adrenal uptake as compared to iodine-131 labeled 19-iodocholest-5-en-3β-o1 (CL-19-I-131), but was at a lower level than that achieved with iodine-131 labeled 6β-iodomethy 119-norcholest-S(10)-en-3β-o1 (NCL-6-I-131). The ratio of radioactivity in the adrenal-to-liver concentration was also lower than that of CL-19-I-131 or NCL-6-I 131.     

Circular dichroism spectra of bradykinin analogs containing β-homoamino acids

The CD spectra of β-homoprolyl⁷-bradykinin (βHProB) and β-homophenylalanyl⁸-bradykinin (βHPheB) were compared to those of bradykinin. The spectra were analyzed in terms of models that have been proposed for the solution conformation of bradykinin. Cann $\text{et al}$. (1) proposed 3 → 1 hydrogen-bonding across Pro³, Pro⁷ and Phe⁸ in bradykinin, as shown by a 234 nm trough in the CD. The extra CH₂ groups in the chains of the two bradykinin analogs would be expected to facilitate the proposed hydrogen-bonding, but in the case of βHProB the 234 nm trough is eliminated, and is reduced in magnitude for βHPheB. Ivanov $\text{et al}$. (2) proposed a cyclic conformation for bradykinin, stabilized by ionic attraction between the side-chain of Arg¹ and the carboxylate terminal. The extra CH₂ groups of these two analogs would be expected to increase the stability of such a conformation, and there was some evidence that the ionic effects on the CD spectra of the two analogs were different from those on the bradykinin spectra. Alternatively, the effects could be attributed to cis-trans isomerizations around the prolyl peptide bonds.     

In vitro on an HCG responsive, testosterone secreting adrenal cortical adenoma

Clinical and biochemical investigation of a virilized woman has shown an adrenal cortical adenoma to be the source of elevated plasma testosterone levels and to be responsive to gonadotropin administration $\text{in vivo}$ (Givens et al.) (2). In the present study, the gonadotropin responsiveness and biosynthetic potential of the adenoma were evaluated $\text{in vitro}$. Incubation of minced adrenal tumor with hCG resulted in increased ¹⁴C-acetate incorporation into pregnenolone, progesterone, dehydroepiandrosterone (DHA), androstenedione, and testosterone. Androstenedione and testosterone were the major products, accounting for 27% and 20% respectively of the total radio-activity added, when ³H-pregnenolone was incubated with homogenized tissue. Estrogen synthesis was not observed in the tumor. The adenoma contained 9.0 μg/g testosterone and 1.9 μg/g androstenedione as determined by radio immunoassay. 17β-hydroxysteroid oxido-reductase was active in the adenoma. Androstenedione was reduced to testosterone at a rate of 0.6 μg/100mg/hr. Under the same conditions, reduction of estrone to estradiol was undetectable. The reductase activity was present in both the mitochondrial and microsomal fractions. NADPH was the required cofactor. When NADH was substituted, the rate was less than 10% of that with NADPH in both particulate fractions.The experimental results indicate the presence of steroid path-way(s) necessary to synthesize testosterone, and represent the first $\text{in vitro}$ demonstration of gonadotropin sensitive steroidogenesis in an adrenal cortical adenoma.     

Separation of pigeon liver apo- and holo- fatty acid synthetases by affinity chromatography.

Apo- and holo-fatty acid synthetases of pigeon liver were separated by affinity gel chromatography under conditions similar to, but not identical to, those used in separating subunits I and II of [¹⁴C]pantetheine-labeled fatty acid synthetase complex [Lornitzo $\text{et al.}$, J. Biol. Chem. $\text{249}$, 1654 (1974)]. When [¹⁴C]pantetheine-labeled fatty acid synthetases were separated, the enzymatically active holo form contained all of the [¹⁴C] label. Incubation of the apo-pigeon liver fatty acid synthetase complex with CoA, ATP and a partially purified pigeon liver soluble enzyme system, from which fatty acid synthetase had been removed, resulted in the formation of holo-enzyme. Activation of apo-fatty acid synthetase could also be achieved by replacing the apo-(4′-phosphopantetheine-less) acyl carrier protein with holo-acyl carrier protein. It is evident, therefore, that the inactive apo-fatty acid synthetase lacks a 4′-phosphopantetheine group.     

Identification of ortho-octopamine and meta-octopamine in mammalian adrenal and salivary gland.

$\text{Ortho}$- and $\text{meta}$- octopamine have been identified in beef and rat adrenal gland and in rat salivary gland by means of gas chromatography-mass spectrometry. The tritrifluoroacetyl derivatives of $\text{ortho}$-, $\text{meta}$- and $\text{para}$- octopamine were resolved by gas chromatography and shown to produce two characteristic ions at $\text{m/e}$ 315 and $\text{m/e}$ 328. The di-O-trimethylsilyl-N-trifluoroacetyl derivatives of these three isomers were also resolved by gas chromatography and shown to produce a characteristic ion at $\text{m/e}$ 267. Biological samples were homogenized in formic acid:acetone, subjected to ion-exchange chromatography and then derivatized. When the derivatized biological extracts were examined for each characteristic ion, peaks were observed at the exact retention times of the standards. The three isomers are present in adrenal gland in concentrations of ～1 μg g^?1 and in rat salivary gland in concentrations of ～0.1 μg g^?1. This evidence confirms a previous report of the presence of $\text{m}$-octopamine in rat salivary gland measured by a radiochemical enzyme assay and is the first report of the presence of $\text{o}$-octopamine in biological tissue.     

Patterns of 3β-hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ activity (3Δ-HSDH) was examined in the rhesus monkey ($\text{Macaca mulatta}$) placenta and fetal adrenal at 135 and 155–162 days of gestation. Activity was evaluated in microsomes by the conversion of [³H]pregnenolone to [³H]progesterone. There was a 7-fold increase in enzyme activity in the whole adrenal (minus medulla) between the two stages of development. Combining data from both periods, enzyme activity was greater in the outer than in the inner region of the adrenal. No stage-dependent change in placental activity was evident. The temporal patterns in 3β-HSDH activity are consistent with corticoid and progesterone patterns in the circulation. Thus, the level of 3β-HSDH activity may be rate limiting in both the fetal adrenal and placenta.Enzyme activity was assessed in incubations which included unex-tracted, heat-treated, 100,000 g tissue supernatants. In both placental and adrenal incubations, competitive inhibition was noted. Ethyl ether extracts of 100,000 g tissue supernatants also inhibited 3β-HSDH in the respective tissues. GLC analysis of these extracts revealed the presence of putative dehydroepiandrosterone. Hormone levels and the nature of the inhibition that were observed are compatible with the conclusion that dehydroepiandrosterone can inhibit the conversion of pregnenolone to progesterone $\text{in vivo}$. The physiological importance of this remains to be determined.     

Raman spectrum of protocatechuate dioxygenase from Pseudomonas putida. New low frequency bands.

The Raman spectrum (441.6 nm excitation) of protocatechuate 3,4-dioxygenase (PCD) from Pseudomonas putida shows resonance enhanced bands at 1605, 1504, 1270, 858, and 830 cm^?1 which are due to the p-hydroxyphenyl group of tyrosine coordinated to iron. In addition, we observe strong resonance enhanced bands at 592 and 524 cm^?1 and weak (presumably iron-ligand) vibrations at 465, 423, and 371 cm^?1. Recent publications of the Raman spectrum of PCD from Pseudomonas aeruginosa (Tatsuno $\text{et al}$, J. Am. Chem. Soc. 100, 4614–4615 (1978) and Keyes $\text{et al}$, Biochem. Biophys. Res. Comm. 83, 941–945 (1978) using 488 and 514 nm excitation did not report these bands. Our 441.6 nm excitation Raman spectrum of human serum transferrin, another metalloprotein with an iron-tyrosine linkage, does not show the 592 and 524 cm^?1 bands and has only two very weak bands at about 423 and 364 cm^?1. We discuss several interpretations of these data.     

Calorimetric measurement of stepwise enthalpy changes for the binding of four oxygens to adult human hemoglobin

The successive enthalpy changes for the four steps of oxygen binding by diphosphoglycerate-free adult human hemoglobin have been measured by direct calorimetry at pH 7.4 and 6°. Average results in kcal/(mole O₂) are: ΔH₁ = ?25.1 ± 2.8; ΔH₂ = ?12.6 ± 3.0, ΔH₃ = ?12.5 ± 3.0, and ΔH₄ = ?10.1 ± 1.4. These results imply a substantial temperature dependence for the cooperativity of O₂ binding by the protein and generally resemble the van't Hoff results by Roughton $\text{et al}$. [Roy. Soc. of London Proc., $\text{B 144}$, 29 (1955)] for sheep hemoglobin at pH 9.1 and a temperature range of 2° to 19°.     

Response of human and chick kidney adenylate cyclase to biologically active synthetic fragments of human parathyroid hormone

The 34-amino acid NH₂-terminal fragment of human parathyroid hormone synthesized according to the sequence described by Niall $\text{et al.}$ (1) is approximately 140 times more potent than the fragment synthesized according to Brewer $\text{et al}$ (2) in activating human renal cortex adenylate cyclase. The potencies of the two peptides, relative to the effect of MRC standard bovine parathyroid hormone preparation $\text{67}\text{342}$ in this system, were 5600 ± 600 (S.E.M.) units/mg and 40 ± 5 units/mg respectively. The potencies of the more active peptide and the corresponding bovine parathyroid hormone sequence were similar in this system and also in assays based upon the production of cyclic AMP by chick kidney both $\text{in vivo}$ and $\text{in vitro}$.     

Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in vitro

One juvenile hormone was isolated from culture medium containing isolated corpora allata of the grasshopper $\text{Schistocerca vaga}$ (Orthoptera: Acrididae) and was shown by microchemical methods to be methyl (2$\text{E}$, 6$\text{E}$) - (10$\text{R}$) - 10, 11-epoxy-3, 7, 11-trimethyldodeca-2, 6-dienoate. This compound (JH III), which occurs in a sphingid moth $\text{Manduca sexta}$, is the first juvenile hormone identified in an insect order other than the Lepidoptera. Grasshopper organs incorporate both [2?¹⁴C] acetate and [methyl-¹⁴C] methionine into JH III showing $\text{de novo}$ biosynthesis, but no indication of the synthesis of JH I or JH II was seen.     

Steric considerations regarding the biodegradation of cholesterol to pregnenolone.-exclusion of (22S)-22-hydroxycholesterol and 22-ketocholesterol as intermediates

(22$\text{S}$)-[22-³H₁]-Cholesterol was incubated with an adrenocortical preparation and the isolated (22$\text{R}$)-[22-³H₁]-22-hydroxycholesterol had a small loss of radioactivity, proving that direct replacement of the hydrogen from the now hydroxylated position occurred.In addition [1-³H₁]-4-methylpentanol was isolated, which also had incurred a relatively small loss of its specific activity, thereby excluding (20$\text{R}$)-3β,20-dihydroxycholest-5-en-22-one as an important metabolite in the degradation of cholesterol to pregnenolone by adrenal tissue.     

Urinary excretion of a novel hexasaccharide and glycopeptide analogue in fucosidosis.

Repeated Biogel P6 chromatography of the urine from a patient with fucosidosis yielded several fractions containing fucosyloligosaccharides and glycopeptides. Two of these were shown by ¹H nuclear magnetic resonance (¹H-n.m.r.) spectroscopy and permethylation analysis to have the following structures respectively: (I) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) glcNAc and (II) αfuc (1→3) [βgal (1→4)] βglcNAc (1→2) αman ($\text{1→}\text{3}\text{6}$) βman (1→4) βglcNAc (1→4) [αfuc ($\text{1→}\text{3}\text{6}$)] βglcNAc-Asn.     

Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.

Mitochondrial protein synthesis was analyzed in the yeast mit^? mutants of $\text{Saccharomyces}$$\text{cerevisiae}$ which specifically lack cytochrome $\text{c}$ oxidase. [³H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [³H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome $\text{c}$ oxidase subunit I.     

Impaired negative cooperativity of the semisynthetic analogues human [LeuB24]- and [LeuB25]-insulins

Several semisynthetic analogues of human insulin were prepared by enzyme-assisted coupling of synthetic octapeptides to the C-terminal of porcine desoctapeptide insulin. We report the receptor-binding and biological properties of [Leu^B24]- and [Leu^B25]-insulins, one of which has the same sequence as a “mutant” insulin recently found in a diabetic patient (Tager, H. et al.(1979) Nature $\text{28}$:121–125). [Leu^B24]- and [Leu^B25]-insulins had, respectively, 8–12% and 0.9–1.1% of the binding affinity of human insulin, and 11% and 2.7% of its potency in stimulating lipogenesis in isolated rat fat cells. Neither one was an antagonist of the biological effects of native insulin. While the ability of [Leu^B24]-insulin to induce negative cooperativity was clearly impaired, that of [Leu^B25]-insulin was almost abolished. [Leu^B25]-insulin was also a potent antagonist of the negative cooperativity induced by native insulin.