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Biomedical subjects

P F Hall

Publications and source records attributed to P F Hall.

At least 145 records · Page 8Linked to original sources
PubMed1978

Inhibition of steroidogenic response to corticotropin in mouse adrenal tumor cells (Y-1) by the ionophore A23187. Role of protein biosynthesis.

Addition of the ionophore A23187 to Y-1 mouse adrenal tumor cells in monolayer culture inhibits steroidogenesis and the steroidogenic response to corticotropin (50% inhibition at 1 . 10(-7)M). Inhibition is rapid in onset and is not overcome by addition of external Ca2+. The ionophore also inhibits stimulation of steroid synthesis by cyclic AMP. A23187 inhibits incorporation of the amino acid lysine into protein by Y-1 cells and the dose dependence of this inhibition closely resembles that of the inhibition of the steroidogenic response to corticotropin. Addition of A23187 to a subcellular system for protein synthesis prepared from Y-1 cells, inhibits incorporation of the amino acid phenylalanine into protein and this effect is not overcome by high concentrations of Ca2+. The inhibitory effect of A23187 on the response to corticotropin, like that response itself, takes place at some part of steroid synthesis after entry of cholesterol into the cells and before the side-chain cleavage of cholesterol. These studies confirm the importance of protein synthesis in the response to corticotropin and demonstrate that the effect of protein synthesized under the influence of corticotropin is exerted at some point in the events which bring substrate (cholesterol) to the mitochondrial side-chain cleavage enzyme system. It is also shown that A23187 inhibits protein synthesis, and hence the response to corticotropin, by a mechanism which is independent of the concentration of available Ca2+.

20-alpha-Dihydroprogesterone↗
PubMed1978

Cytochrome P-450 for 11beta- and 18-hydroxylase activities of bovine adrenocortical mitochondria: one enzyme or two?

The 11beta- and 18-hydroxylase activities of a highly purified cytochrome P-450 from bovine adrenocortical mitochondria have been examined in detail with a view to determining whether the two activities are shown by a single protein or by two distinct proteins. The purified enzyme shows a single N-terminal residue (glutamic acid) and its amino acid composition is reported. Both enzyme activities decay considerably during storage at 4 degrees C for 11 days and the rates of decay are similar for the two activities. Metyrapone inhibits both activities competitively (Ki = 1.50 and 1.43 micrometer for 11beta- and 18-hydroxylase, respectively). Carbon monoxide inhibits both activities and the ratio CO:O2 for 50% inhibition is similar for the two activities (K = 1.69 and 1.53). A variety of nonspecific inhibitors produce approximately the same inhibition of both activities. Finally, antiserum produced by rabbits to the purified enzyme on double diffusion in agarose gels gives a single band with the purified enzyme. Increasing concentrations of antiserum added to the assay system produce increasing and proportionate inhibition of both activities. The evidence strongly supports earlier suggestions that the two hydroxylase activities occur in a single protein.

Adrenal Cortex↗
PubMed1978

On the role of protein synthesis in the response of adrenal tumor cells to ACTH.

Y-1 adrenal tumor cells were incubated with aminoglutethimide with and without ACTH. Greater production of pregnenolone from endogenous cholesterol was observed (after washing to remove aminoglutethimide) in mitochondria from cells incubated with aminoglutethimide and ACTH than in those from cells incubated with aminoglutethimide alone. This response was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. ACTH increased the incorporation of [3H]tyrosine into protein associated with mitochondria but not into total cell protein or protein of postmitochondrial supernatant. This response did not require aminoglutethemide block and was inhibited by cycloheximide and puromycin but not by chloramphenicol or actinomycin D. Dibutyryl cyclic AMP produced both of these responses (increased production of pregnenolone and synthesis of protein associated with mitochondria). The concentration of cycloheximide required to cause 50% inhibition of the responses to ACTH and dibutyryl cyclic AMP was approximately the same for steroidogenesis by whole cells, for production of pregnenolone by isolated mitochondria, for incorporation of [3H]tyrosine into Y-1 cell protein and for the increase in synthesis of protein associated with mitochondria produced by ACTH (0.08--0.2 microgram/ml). Disc gel electrophoresis revealed that the increased incorporation of [3H]tyrosine involved two proteins corresponding to molecular weight of approximately 27,000 and 13,000 respectively. These observations suggest that ACTH promotes synthesis of protein(s) by cytoplasmic ribosomes on stable messenger RNA, that the protein(s) becomes associated with mitochondria and that the protein(s) includes one or more which are associated with the increase in production of pregnenolone produced in mitochondria by the addition of ACTH to adrenal cells.

Adrenal Gland Neoplasms↗
PubMed1978

The effects of temperature and glucose on protein biosynthesis by immature (round) spermatids from rat testes.

A method is described for the preparation of highly purified fractions (greater than 80% pure) of immature spermatids (round, steps 1--8) from rat testes by centrifugal elutriation in sufficient yields for biochemical studies when four rat testes are used. Electron microscopy established the identity of the cells and demonstrated that the cell membrane is intact. Some cells develop nuclear and cytoplasmic vacuoles during the 2 h required for preparation. Immature spermatids prepared by this method use glucose with an increase in oxygen consumption, lactate production, and protein synthesis over control levels (no glucose). The testicular cell suspension from which spermatids are separated, like whole testis and spermatids themselves, show higher incorporation of amino acids into TCA-precipitable material at 34 degrees C than at 38 degrees C and in the presence of glucose. A subcellular system prepared from immature spermatids with excess ATP shows greater incorporation of amino acids into TCA-precipitable material at 34 degrees C than at 38 degrees C. This difference does not result from increased breakdown of protein. It is concluded that body temperature (38 degrees C) inhibits some aspect(s) of protein synthesis in addition to previously reported effects on amino acid transport and production of ATP (Means and Hall. 1969. Endocrinology. 84:285--297.).

Animals↗
PubMed1977

Side-chain cleavage P-450 from bovine adrenocortical mitochondria. Reconstitution of enzyme activity.

The subunit structure of the cytochrome P-450 from bovine adrenocortical mitochondria responsible for the conversion of cholesterol to pregnenolone (side-chain cleavage) has been studied. Isoelectric focusing in 6 M urea reveals two fractions of identical amino acid composition which differ in apparent isoelectric points and in phospholipid content: fraction SI shows 0.6-1.8 nmol phospholipid per 53 000 daltons and pI approx. 4.0; SII shows 6.6-8.9 nmol phospholipid per 53 000 daltons and pI approx. 7.0. SII can be made to behave on isoelectric focusing like SI by removal of phospholipid and SI like SII when the extracted phospholipid is added to the protein (SI). Enzymatic activity can be restored to SII by addition of heme and to SI by addition of heme together with the phospholipid extracted from P-450 from the fractions SI and SII. This phospholipid contains at least four classes of phospholipid of which two have been tentatively identified as phosphatidylcholine and phosphatidylethanolamine. A variety of phospholipids from commercial sources do not permit reconstitution of enzyme activity. Evidence is presented to show that minor contaminants seen on polyacrylamide SDS gels are not essential for enzyme activity nor do they appear greatly to influence enzymatic activity. The possible role of phospholipid in reconstituting cytochrome P-450 activity is considered.

Adrenal Cortex↗
PubMed1977

Preparation and properties of side-chain cleavage cytochrome P-450 from bovine adrenal cortex by affinity chromatography with pregnenolone as ligand.

A method is described for preparing cytochrome P-450 (side-chain cleavage) from bovine adrenocortical mitochondria, by affinity chromatography on pregnenolong-Sepharose beads. The cytochrome P-450 appears in two fractions, a large form of heterogeneous molecular weight (large P-450) and a form of molecular weight 850 000 composed of 16 apparently identical subunits (molecular weight 52 000-53 000); this form is referred to as protein 16. Electrophoresis on polyacrylamide gel yields one main band and two minor bands; the appearance of the gels is identical whether the starting material is large P-450 or protein 16 or protein 16 prepared by an entirely different method. Yields of protein 16 can be increased by rechromatography on pregnenolone-Sepharose of large P-450 made 0.1 mM in NADPH. Large P-450 shows greater than 10 heme groups per 16 subunits and is less active enzymatically than protein 16. Chromatography on Sepharose and analytical ultracentrifugation show that large P-450 is heterogeneous with respect to molecular weight. Protein 16 shows a heme content of 8 nmol/mg protein and for both large P-450 and protein 16 heme content by CO-difference spectroscopy is in agreement with values by pyridine hemochromogen. This method of preparing P-450 is convenient and both large P-450 and protein 16 are highly purified.

Adrenal Cortex↗
PubMed1977

Purification and properties of cytochrome p-450 (11beta- and 18-hydroxylase) from bovine adrenocortical mitochondria.

We describe an improved procedure for the preparation of a cytochrome P-450 from bovine adrenocortical mitochondria which catalyzes 11beta- and 18-hydroxylation of steroids. The preparation is based upon chromatography on DEAE cellulose which separates the enzyme from the side-chain cleavage P-450, which can also be prepared in highly purified form from the same tissue extracts. The enzyme behaves as a single compound in glycerol density gradients. The enzyme aggregates at protein concentrations greater than 1 mg/ml to a series of forms of various molecular weights. On Sepharose 4B the enzyme shows a molecular weight of 185 000, while on glycerol density gradients a molecular weight of 1 - 10(6) is observed. The subunit molecular weight determined by electrophoresis on polyacrylamide gels with sodium dodecyl sulfate is 47 500 and the protein appears as a single band. The ratio of 11beta-/18-hydroxylase activities does not change significantly during purification and is constant through the protein peak on glycerol density gradients. Since there appears to be only one subunit species, it seems likely that the two hydroxylase activities are catalyzed by one protein.

Adrenal Cortex↗
PubMed1977

Response of adrenal tumor cells to adrenocorticotropin: site of inhibition by cytochalasin B.

The ability of cytochalasin B to inhibit the steroidogenic response of mouse adrenal tumor cells (Y-1) to adrenocorticotropin (ACTH) was examined with two aims: to consider the specificity of the inhibitor and to determine at what point(s) in the steroidogenic pathway it acts. Cytochalasin B did not inhibit protein synthesis or transport of [3H]-cholesterol into the cells nor did it alter total cell concentration of ATP. Together with previous evidence, this suggests that the effects of cytochalasin observed are relatively specific in these cells. Cytochalasin inhibits the increase in conversion of [3H]cholesterol to 20alpha-[3H]dihydroprogesterone (20alpha-hydroxypregn-4-en-3-one: a major product of the steroid pathway in Y-1 cells) produced by ACTH but does not inhibit conversion of cholesterol to pregnenolone by mitochondrial and purified enzyme preparations from Y-1 cells and bovine adrenal, respectively. Cytochalasin does not inhibit the conversion of pregnenolone to 20alpha-dihydroprogesterone but was shown to inhibit increased transport of [3H]cholesterol to mitochondria resulting from the action of ACTH. These findings indicate that cytochalasin acts after cholesterol has entered the cells and before it is subjected to side-chain cleavage in mitochondria. In view of the known action of cytochalasin on microfilaments, it is proposed that these organelles are necessary for the transport of cholesterol to the mitochondrial cleavage enzyme and that at least one effect of ACTH (and cyclic AMP) is exerted upon this transport process. The specificity of the effects of cytochalasin is considered in relation to this conclusion.

20-alpha-Dihydroprogesterone↗
PubMed1976

Inhibition by 5-thio-D-glucopyranose of protein biosynthesis in vitro in spermatids from rat testis.

The effect of 5-thio-D-glucopyranose (thioglucose) upon protein biosynthesis in vitro was examine in testes from mature rats. Thioglucose in vitro is without demonstrable effect upon incorporation of L-[U-14C]phenylalanine into protein by whole testis but inhibits this incorporation by a purified fraction of immature spermatids (stages 1--8) prepared by centrifugal eluctriation; the inhibition is observed with or without glucose added in vitro and is concentration dependent in the range 1--50 mM. Similar inhibition is observed with three other 14C-labeled amino acids (leucine, lysine and glutamate). Mature spermatids (greater than stage 8) and other heterogenous fractions of testicular cells prepared by the same method also show inhibition by thioglucose of incorporation of phenylalanine into protein so that it is not possible to say that the effect is confined to spermatids although it is most pronounced in these cells. Inhibition of protein synthesis in vitro is also observed when thioglucose was administered in vivo (33 mg/kg body wt./day). This change occurs at the minimal dose observed by other workers to produce arrest of spermatogenesis and hence infertility.

Animals↗
PubMed1976

The role of cytochrome P-450 in the regulation of steroid biosynthesis.

A cytochrome P-450 from bovine adrenocortical mitochondria has been purified to near homogeneity. The protein catalyzes side-chain cleavage of cholesterol (cholesterol leads to pregnenolone) but neither 11beta- nor 18-hydroxylation. It consists of 16 subunits of two species (MW 52,000) and contains 8 heme groups. The enzyme has been used to determine the stoichiometry of side-chain cleavage with the following results: (TPNH and O2 consumed/mole of cleavage), cholesterol 3:3:1, 20S-hydroxycholesterol 2:2:1 and 20S,22R-dihydroxycholesterol 1:1:1. These findings support the occurrence of the proposed pathway for the side-chain cleavage of cholesterol. Cleavage of the diol is inhibited by CO and shows a characteristic P-450 photochemical action spectrum. Evidently the diol is cleaved in a typical monoxygenase reaction. The active form of the enzyme contains 16 subunits (protein 16); forms consisting of 8 (protein 8) and 4 (protein 4) subunits can be isolated and are enzymatically active only by prior conversion to protein 16.

Adrenal Cortex↗
PubMed1976

Differences in cytochrome P-450 of various strains of rats following chronic administration of pentobarbital.

Cytochrome P-450 levels were analyzed in rats of two pigmented (black Long-Evans and ACI) and two albino strains (Fischer 344 and Sprague-Dawley) following the administration of pentobarbital sodium and physiological saline. Differences between the albino vs pigmented strains were observed following injections of saline. The Fischer 344 albino strains responded similarly to the pigmented strains following a progressively increasing dose schedule of pentobarbital sodium.

Animals↗