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P F Hall

Publications and source records attributed to P F Hall.

At least 109 records · Page 6Linked to original sources
PubMed1984

Peptide alignment of the porcine 21-hydroxylase cytochrome P-450 using a cDNA sequence of the corresponding bovine enzyme.

The amino acid sequence deduced from a cDNA clone of the bovine adrenal steroid 21-hydroxylase cytochrome P-450 has been utilized to align peptide sequences derived from the corresponding porcine enzyme. Homology analysis revealed that only fifty percent of the amino acid sequence predicted by the cDNA clone overlapped with peptide sequences from the porcine enzyme. The homology in the remaining portions of the bovine sequence was restored by considering amino acid sequences predicted by the two additional DNA reading frames of the cDNA sequence. Forty eight percent of the bovine sequences predicted by the two alternate reading frames showed strong homology with the porcine peptide sequences. A minimum of 4 nucleotide sequencing errors account for the observed reading frame alterations and the approximate position of each error in the bovine cDNA sequence has been established.

Adrenal Glands↗
PubMed1984

C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 alpha-hydroxylase/C17,20-lyase cytochrome P-450.

The properties and the purity of a cytochrome P-450 (17 alpha-hydroxylase) from porcine adrenal microsomes have been examined following a report that the corresponding enzyme from bovine adrenocortical microsomes is inactive as a 17 alpha-hydroxylase and fails to show a high spin spectrum on addition of substrate, once the enzyme has been purified (Bumpus, J. A., and Dus, K. M. (1982) J. Biol. Chem. 257, 12696-12704). The purity of the porcine enzyme was demonstrated by electrophoresis on polyacrylamide with sodium dodecyl sulfate, immunoelectrophoresis, and NH2-terminal amino acid sequence (16 residues). The pure enzyme shows Mr = 54,000, heme content of greater than 0.8 nmol/nmol of protein, and absorption spectra typical of cytochrome P-450. The enzyme is active with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates as a 17 alpha-hydroxylase and with the corresponding 17 alpha-hydroxysteroids as a C17,20-lyase. All four substrates produce typical type I spectra with the enzyme (so-called high spin form). We conclude that: 1) porcine adrenal microsomes contain a 17 alpha-hydroxylase/C17,20-lyase which is a single protein molecule readily purified to an enzymatically active form; 2) the C17,20-lyase activity is largely suppressed in the microsomes; and 3) the enzyme closely resembles that found in testicular microsomes. We propose that this enzyme be referred to as the adrenal C21 steroid side chain cleavage enzyme.

Adrenal Glands↗
PubMed1984

Role of actin in the responses of adrenal cells to ACTH and cyclic AMP: inhibition by DNase I.

Erythrocyte ghosts were loaded with pancreatic DNase I and fused with Y-1 adrenal tumor cells to test the possibility that this enzyme might inhibit the steroidogenic responses of the cells to ACTH and cyclic AMP. Fusion of erythrocyte ghosts loaded with DNase I, but not those containing albumin, ovalbumin, boiled DNase I, or DNase I with excess G-actin, inhibited the increase in production of 20 alpha-dihydroprogesterone produced by ACTH and dibutyryl cyclic AMP; inhibition was concentration-dependent with 50% inhibition by 3 X 10(7) molecules of DNase I per cell. It was found that inhibition by DNase I was exerted at the step in the steroidogenic pathway at which cholesterol is transported to mitochondria where steroidogenesis begins. This was shown by measuring transport of cholesterol into the inner mitochondrial membrane, by measuring the production of pregnenolone by isolated mitochondria and by demonstrating that DNase I was without effect on the conversion of pregnenolone to 20 alpha-dihydroprogesterone (an end-product of steroid synthesis). The actin content of Y-1 cells was measured by two methods based upon inhibition of DNase I and by SDS gels following centrifugation. The cells were found to contain 2-3 X 10(7) molecules of actin per cell of which two-thirds is present as G-actin. Since DNase I is known to bind to G-actin to give a one to one complex, these and other findings suggest that at least some of the G-actin in the cells may be necessary for the steroidogenic responses to ACTH and cyclic AMP.

Actins↗
PubMed1984

Influence of follicle-stimulating hormone on glucose transport by cultured Sertoli cells.

Transport of 3-O-methyl-D-[14C]glucose by Sertoli cells cultured in plastic dishes, is competitively inhibited by glucose (Ki 4 microM). The glucose analogue was therefore used to study glucose transport in these cells in which it is not metabolized. Addition of follicle-stimulating hormone (FSH) (10 micrograms/ml) or dibutyryl cyclic AMP (1 mM) to the cells, increases transport of methylglucose by Sertoli cells. The increased transport results from increased influx and involves decrease in Km without change in Vmax. These changes in the kinetics of transport are seen with both FSH and dibutyryl cyclic AMP. FSH does not stimulate transport of methylglucose in peritubular fibroblasts nor in germ cells. In view of the importance of lactate as a substrate for spermatids (Mita and Hall, 1982) it is proposed that stimulation of the transport of glucose by Sertoli cells in response to FSH is important in the increased production of lactate by these cells in response to FSH and hence is one mechanism by which the tropic hormone enables the Sertoli cell to promote spermatogenesis.

3-O-Methylglucose↗
PubMed1984

The role of the cytoskeleton in hormone action.

The structure and functions of microfilaments and microtubules is briefly reviewed. Based on evidence from a variety of cells with various experimental approaches, it is proposed that the cytoskeleton is important in the functions of cells as follows: the cytoskeleton appears to be involved in attachment (to other cells and to substrata used in cell culture) and in movement; the cytoskeleton appears to be involved in the complex relationship between the surface and the interior of the cell. This is a two-way relationship in which the cell uses the cytoskeleton to influence the distribution of proteins within the plasma membrane and perhaps as a go-between to transmit information from the surface of the cell to the interior. Within the cell, the cytoskeleton appears to coordinate intracellular activities (e.g., between nucleus and cytoplasm) and to provide a compartmentation which restricts the movements of intracellular structures (from molecules to organelles), thereby facilitating productive encounters between such structures with an efficiency that is presumed to exceed that of random motion. The cytoskeleton may arrange the various components of the cell in space; it may direct intracellular traffic and promote intracellular movement. This loose picture of cytoskeletal activity may be more emphatic than the available data warrant. It should be regarded as informed extrapolation based upon fragments of information. When these ideas are applied to endocrine function, we can see that the secretion of hormones (exemplified by thyroid hormones and insulin) requires cytoskeletal activity to provide direction and perhaps to promote movement. In steroidogenic cells the movement of cholesterol from cytoplasm to mitochondria requires microfilaments. The antidiuretic hormone requires microfilaments and microtubules to promote transport of water through cells in response to osmotic gradients. Microfilaments adjust the compliance of the cell to provide a path for water and microtubules influence the permeability of the cell membrane to water.

Adrenocorticotropic Hormone↗
PubMed1984

The actions of insulin-like growth factors I and II on cultured Sertoli cells.

Cultured Sertoli cells prepared from young rats (13 days old) showed increased incorporation of [3H]thymidine into DNA, increased production of lactate, and increased incorporation of [3H]leucine into protein in response to micromolar concentrations of insulin and nanomolar concentrations of insulin-like growth factor II (IGF-II). The first of these responses was also seen with nanomolar concentrations of IGF-I. Receptor affinity labeling studies using Sertoli cell membranes and whole Sertoli cells showed that these cells possess abundant growth factor receptors of type I (mol wt, 350,000) that show high affinity for IGF-I, moderate affinity for IGF-II, and low affinity for insulin. Sertoli cell membranes also show abundant growth factor receptors of type II (mol wt, 230,000) that show high affinity for IGF-II, moderate affinity for IGF-I, and no detectable affinity for insulin. Moreover, the responses of the Sertoli cell to insulin were observed at concentrations of 100 nM or higher, whereas insulin receptors are known to be saturated by insulin at concentrations of 10 nM or less. It is, therefore, concluded that Sertoli cells possess receptors for IGF-I and that the responses observed to insulin may result from binding of these hormones to receptors for IGF-I.

Animals↗
PubMed1983

Purification and properties of 17 alpha-hydroxylase from microsomes of pig adrenal: a second C21 side-chain cleavage system.

We have purified a cytochrome P-450 from microsomes of pig adrenal glands to homogeneity (11.1 n moles heme/mg protein) as demonstrated by electrophoresis on polyacrylamide gels with sodium dodecyl SO4 and by line of identity with an antibody using double diffusion. The enzyme shows both 17 alpha-hydroxylase and C17,20-lyase activities and therefore constitutes a C21 side-chain cleavage system like that previously purified in this laboratory from neonatal pig testis. Antibody to the testicular enzyme cross-reacts (line of identity) with both enzymes. It is concluded that the adrenal enzyme is the same or very similar to the testicular enzyme, that each enzyme possesses two enzymatic activities, and that microsomes provide some regulatory device to limit the lyase activity of the enzyme in vivo. No evidence was found for the usually accepted existence of an adrenal steroid 17 alpha-hydroxylase without lyase activity.

Adrenal Glands↗
PubMed1983

Steroid 21-hydroxylase (cytochrome P-450) from porcine adrenocortical microsomes: microsequence analysis of cysteine-containing peptides.

The steroid 21-hydroxylase cytochrome P-450 from porcine adrenocortical microsomes was purified to homogeneity. The protein exhibited two NH2-terminal sequences, one of which was identical with the first but lacking the NH2-terminal methionine. The sequence was extremely hydrophobic but had little homology to the 17 alpha-hydroxylase/C17,20-lyase isolated from neonatal porcine testes or to rat or rabbit liver microsomal cytochromes P-450. The cysteine-containing fragments of the S-carboxymethylated protein were purified by high-performance liquid chromatography and sequenced. Three of the cysteine-containing peptides exhibited significant sequence homology with peptides from the major phenobarbital-induced rat liver cytochrome P-450 (P-450b) and two with peptides from cytochrome P-450cam (camphor methylene hydroxylase from Pseudomonas putida). The presence of conserved regions in the primary sequences of these proteins appears likely to provide clues to the nature of their heme-binding domains.

Adrenal Cortex↗
PubMed1983

Side-chain cleavage of C21 steroids by testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/lyase): involvement of heme.

The two steps in the side-chain cleavage of C21 steroids to give C19 steroids (i.e. 17 alpha-hydroxylation and C17,20 lyase activity) were examined using a highly purified cytochrome P-450 from microsomes of neonatal pig testis to determine the photochemical action spectra for the two reactions. Photochemical action spectra, using either 4-ene (progesterone) or 5-ene (pregnenolone) substrates, showed maximal reversal of inhibition by CO with light of 451 nm. Evidently the heme of cytochrome P-450 is involved in both 17 alpha-hydroxylation and in C17,20-lyase activity as in the case of the side-chain cleavage of cholesterol. Mechanisms proposed to account for enzymatic cleavage of the alpha-ketol side-chain of C21 steroids (C17,20 lyase activity) must be consistent with these findings.

Aldehyde-Lyases↗
PubMed1982

Sonographic detection of polyhydramnios: a five-year experience.

Between 1975 and 1980, 16,586 sonograms, including repeat scans, were obtained on pregnant women for various obstetric indications. The diagnosis of polyhydramnios was made 78 times by an experienced sonographer as a simple observer judgment of an excess amount of amniotic fluid. In pregnancies thus diagnosed, the fetus was then carefully scanned for anomalies, and repeat scans were done. Major clinical problems encountered are discussed.

Congenital Abnormalities↗
PubMed1982

Metabolism of round spermatids from rats: lactate as the preferred substrate.

Round spermatids were prepared from rat testes and incubated with various substrates (glucose, fructose, pyruvate, lactate and acetate) to measure utilization of substrates and production of ATP in the presence of saturating levels of each substrate. By both criteria lactate is the preferred substrate by a factor of 3 or 4. Production of more than half of the ATP with lactate is substrate is prevented by addition of an inhibitor of alpha-ketoacid dehydrogenase (5-methoxyindole-2-carboxylic acid) Pyruvate and lactate are interconverted and pyruvate inhibits production of ATP from lactate. Synthesis of ATP with lactate and with pyruvate is inhibited by rotenone, rutamycin or 2,4-dinitrophenol. Utilization of glucose is limited by aldolase activity. These findings suggest that exogenous lactate is oxidized by lactate dehydrogenase followed by pyruvate dehydrogenase and Krebs; cycle enzymes under conditions which do not allow pyruvate to inhibit lactate dehydrogenase. ATP is synthesized through electron transport. Post-mitochondrial supernate from spermatids showed that high concentration of pyruvate (greater than 1 mM) inhibit lactate dehydrogenase with pyruvate as substrate and that with lactate as substrate, pyruvate behaves as a competitive inhibitor of lactate dehydrogenase. Evidently lactate is the preferred substrate for round spermatids and energy production is most efficient when this substance is present in high concentrations and pyruvate is present in low concentrations. Reasons are given for suggesting that Sertoli cells may provide the relatively large amounts of lactate required by round spermatids.

Adenosine Triphosphate↗
PubMed1982

Stimulation by follicle-stimulating hormone of synthesis of lactate by Sertoli cells from rat testis.

Sertoli cells from rats aged 16 days were cultured in defined medium for 2 days and then treated by addition of fresh medium containing various hormones (treated) or saline (control). The concentration of lactate in the medium was measured as a function of time. The production of lactate measured under these conditions was increased by addition of FSH to the medium. For NIH FSH (13 and 14), ED50 for stimulation of lactate production was approximately 0.05 micrograms/ml. Stimulation was also seen with LH and TSH (ED50, 0.8 micrograms/ml for both hormones). Reasons are given for believing that TSH may possess the inherent capacity to stimulate production of lactate in contrast to LH, which appears to act only by way of contaminating traces of FSH. Dibutyryl cAMP also stimulates lactate production by Sertoli cells. Other hormones tested, including androgens, were without effect. When production of lactate by Sertoli cells was compared with the maximal consumption by spermatids in vitro, it became apparent that Sertoli cells may provide the major source of metabolic substrate for spermatids in the form of lactate. Stimulation of lactate production by FSH was inhibited by puromycin, cycloheximide, and actinomycin D. Evidently this response requires synthesis of new protein and RNA. This effect of FSH may be important in the regulation of spermatogenesis.

Animals↗
PubMed1981

Microsomal cytochrome P-450 from neonatal pig testis: two enzymatic activities (17 alpha-hydroxylase and c17,20-lyase) associated with one protein.

Studies have been performed to test the hypothesis that cytochrome P-450 from testicular microsomes consists of a single protein with two enzymatic activities (17 alpha-hydroxylase and C17,20-lyase). Three lines of evidence to support the hypothesis were obtained. (1) The enzyme appears to be homogeneous by immunochemical criteria with anti-P-450 IgG (line of identity on immunodiffusion and a single band on immunoelectrophoresis), by demonstration of a single NH2-terminal amino acid (methionine) and the finding of 16 single amino acids at the NH2 terminus. (2) Optima for pH and temperature are the same for both enzymatic activities (pH 7.25 and 37 degrees C), and temperatures between 30 and 44 degrees C decreased both activities in such a way that the ratio of hydroxylase to lyase was the same at all temperatures tested. (3) A variety of inhibitors affect both activities to the same extent: Ki values for two competitive inhibitors (SU 8000, 0.04 microM; SU 10603, 0.3 microM) are the same for hydroxylase and lyase; partition coefficients for inhibition by carbon monoxide are similar for hydroxylase and lyase (20 +/- 2 and 27 +/- 3); anti-P-450 (serum and IgG) causes inhibition of both activities to the same extent, and the same is true of a variety of less specific inhibitors. It is concluded that a single heme protein (cytochrome P-450) from microsomes of neonatal pig testis catalyzes two reactions (hydroxylase and lyase) which are sequential steps in the synthesis of androgens by the testis leading to conversion of C21 precursors to C19 steroid hormones.

Aldehyde-Lyases↗
PubMed1981

Testicular microsomal cytochrome P-450 for C21 steroid side chain cleavage. Spectral and binding studies.

Kinetic and binding studies were performed with a purified microsomal cytochrome P-450 from neonatal pig testis, the C21 side chain cleavage system (17 alpha-hydroxylase/C17,20-lyase). Binding of substrates and inhibitors was measured by spectral methods and by equilibrium dialysis. Kinetic data revealed that pregnenolone inhibits lyase activity with 17 alpha-hydroxypregnenolone as substrate (Ki, 0.3 microM) and that progesterone inhibits lyase activity with 17 alpha-hydroxyprogesterone (Ki, 1.5 microM); inhibition is competitive in both cases. Binding and kinetic studies revealed that Km, Ks, and Kd (Michaelis constant and dissociation constants determined by spectral and dialysis methods, respectively) are all considerably lower for the delta 5 substrates than for the corresponding delta 4 compounds. Equilibrium dialysis shows that there is a single binding site for the substrates of both activities (hydroxylase and lyase). Spectral studies revealed a lag in the development of the spectral shift produced by the addition of steroids and gave results compatible with a single active site, although this spectral evidence is not conclusive by itself. It is concluded that (i) the powerful forward competitive inhibition by pregnenolone and progesterone may be important in regulating synthesis of androgens in vivo; (ii) the porcine enzyme uses delta 5 substrates in preference to delta 4 substrates, thereby accounting for extensive use of the delta 5 pathway by pig testis in vivo; (iii) the evidence presented suggests one active site for both hydroxylase and lyase activities.

17-alpha-Hydroxypregnenolone↗