Isolation and characterization of protein kinase C from Y-1 adrenal cell cytoskeleton.

The cytoskeletons of Y-1 mouse adrenal tumor cells contain a calcium and phospholipid-dependent protein kinase (protein kinase C) that is bound sufficiently tight to resist extraction by 0.5% Triton but not by 1.0% Triton. The enzyme has been purified to near homogeneity from cytoskeleton and cytosol. It shows features typical of this type of kinase, namely a requirement for Ca2+ and phospholipid, stimulation by tumor promoters but not by nontumor-promoting phorbol esters, and inhibition by trifluoperazine. The enzyme shows specificity for four substrates found in the cytoskeleton, namely 80, 33, 20, and 18 kD. The first three substrates are phosphorylated by the enzyme; the fourth is dephosphorylated and is therefore affected by the kinase indirectly. The 80-kD protein is the kinase enzyme itself which is autophosphorylated in vitro and in the cytoskeleton. The 20-kD protein is myosin light chain. The 33- and 18-kD proteins are unidentified. The same substrates were phosphorylated when Y-1 cells were permeabilized with digitonin and incubated with [gamma-32P]ATP and phorbol-12-myristate-13-acetate. Partly purified protein kinase C changes the extent of phosphorylation of the same substrates when added to cytoskeletons previously extracted to remove endogenous protein kinase C. Addition of Ca2+, phosphatidylserine, and phorbol-12-myristate-13-acetate to cytoskeletons, and addition of these three agents plus protein kinase C to extracted cytoskeletons, causes these structures to undergo a rapid and extensive rounding. A similar change is induced in intact cells by addition of phorbol ester. It is concluded that protein kinase C is capable of changing the shape of adrenal cells by an action that involves autophosphorylation and phosphorylation of myosin light chain. This response may in turn be related to the steroidogenic responses to ACTH and cyclic AMP.

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.

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.).

A role for calmodulin in the regulation of steroidogenesis.

TWO APPROACHES WERE USED TO STUDY THE POSSIBLE ROLE OF CALMODULIN IN THE REGULATION OF STEROID SYNTHESIS BY MOUSE ADRENAL TUMOR CELLS: trifluoperazine was used as an inhibitor of calmodulin and liposomes were used to deliver calmodulin into the cells. Trifluoperazine inhibits three steroidogenic responses to both ACTH and dibutyryl cyclic AMP: (a) increase in steroid production, (b) increased transport of cholesterol to mitochondria, and (c) increased side-chain cleavage by mitochondria isolated from cells incubated with ACTH or dibutyryl cyclic AMP. When calmodulin is introduced into the cells via liposomes, steroid synthesis is slightly stimulated. When calmodulin extensively dialyzed against EGTA, this stimulation is abolished. Ca(2+) introduced via liposomes was also without effect. However, when both calmodulin and Ca(2+) are introduced via liposomes (either in separate liposomes or in the same liposomes), steroid synthesis is stimulated. This stimulation does not occur when either anticalmodulin antibodies or EGTA is also present in the liposomes or when trifluoperazine is present in the incubation medium. Calmodulin and Ca(2+) presented together in liposomes to the cells stimulate transport of cholesterol to mitochondria, and side-chain cleavage activity is greater in mitochondria isolated from cells previously fused with liposomes containing calmodulin and Ca(2+) than in mitochondria from cells fused with liposomes containing buffer only. These observations suggest that calmodulin may be involved in regulating the transport of cholesterol to mitochondria, a process which is stimulated by ACTH and dibutyryl cyclic AMP and which may account, at least in part, for the increase in steroid synthesis produced by these agents.

Partial characterization of mitochondrial G proteins in adrenal cells.

Four low molecular mass G proteins have been identified in mitochondrial membranes from bovine adrenal cortex. These proteins (referred to as proteins 1 to 4) showed molecular masses of 28, 27, 26 and 24 kDa with isoelectric points (pI) of 8.1, 5.6, and 6.3 respectively for proteins 1, 2 and 4. Protein 3 was shown to be heterogeneous, with isoelectric points of 5.0-6.1. Proteins were identified by binding of [alpha-(32)P]guanosine triphosphate (GTP) after separation by 12% SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose. Competitive binding by unlabelled competing nucleoside phosphate ligands showed specificity for guanosine triphosphate (GTP) and guanosine diphosphate (GDP) with little binding of guanosine monophosphate and no detectable binding with adenosine nucleoside phosphates. Binding was less than 10% with 100-fold excess GDP and GTP which showed equal intensities of binding. Inhibition of binding by 1000-fold cytidine triphosphate and uridine triphosphate was approx. 10%. Magnesium (Mg(2+)) stimulated binding of GTP by all four proteins. The effect of Mg(2+) was essentially the same for proteins 1, 2 and 3, while protein 4 was less sensitive to Mg(2+) at concentrations <10(-3) M. Centrifugation of sonicated mitochondrial membranes through sucrose density gradients showed the presence of all four proteins in contact points. The presence of lower concentrations (expressed per mg protein) of the proteins in inner and outer membranes suggests that either small amounts of these membranes are part of contact points as presently prepared or that the proteins occur in contact points and to a much smaller extent in inner and outer membranes. It is proposed to examine a possible role for these proteins in transport of cholesterol from outer to inner mitochondrial membranes.

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.

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+.

GTP-binding proteins in adrenocortical mitochondria.

We have identified two GTP-binding proteins in mitochondria from bovine adrenal cortex (fasciculata). Sub-mitochondrial particles were fractionated into inner membrane, contact point and outer membrane vesicles on sucrose density gradients. These sub-mitochondrial fractions were identified by the presence of enzyme markers and electron microscopy. Photoaffinity labelling with [gamma-32P]GTP identified a 45 kDa GTP-binding protein in outer mitochondrial membranes and a 19 kDa protein in the contact points. The molecular weight of 45 kDa and requirement for Mg2+ ions raise the possibility that this protein is an alpha subunit of a heterotrimeric GTP-binding protein or a novel GTP-binding protein. The specificity of nucleotide binding, the requirement for low concentrations of Mg2+ (0.1 mM) and molecular weight of 19 kDa suggest that this protein is a typical member of the so-called small GTP-binding protein family. The location of 45 kDa in the outer membrane and that of 19 kDa in the contact points suggest roles for these proteins in the interaction with the extramitochondrial environment and in the regulation of mitochondrial membranes, respectively.

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.

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.

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.

Cyclic AMP regulates expression of the rat gene for steroid 17 alpha-hydroxylase/C17-20 lyase P-450 (CYP17) in rat Leydig cells.

The upstream region of the rat CYP17 gene shows significant homology to the upstream regions of the bovine and human genes, 53 and 60 percent, respectively. The start site of transcription was determined by primer extension and S1 nuclease protection to be 41 base pairs (bp) upstream of the initiating methionine codon. Expression vectors were constructed by ligation of upstream sequences into promoterless chloramphenicol acetyl transferase (CAT) vectors. Transient transfection studies using primary cultures of rat Leydig cells indicate a strong cAMP-responsive element located within the -26/+65 region. Stimulation by cyclic AMP was abolished when sequences upstream of -264 were included in expression vectors. No significant expression was seen in Leydig cells in the absence of dbcAMP nor was there any expression in the presence or absence of dbcAMP in rat skin fibroblasts or in mouse adrenal (Y-1) cells in which CYP17 is not normally expressed. Three possible regulatory elements were found in the 5' upstream region: a CRE/ATF consensus sequence (GACGTCA) starting at position -57; a GRE consensus sequence (TGTTCT) starting at position -501; and a consensus sequence for AP-1 binding (TTAGTCA) starting at position -659. It was concluded that the CRE/ATF at -57 is not responsible for increased transcription in the presence of cyclic AMP.

Gene for 17 alpha-hydroxylase/C (17-20) lyase P-450: complete nucleotide sequence of the porcine gene and 5' upstream sequence of the rat gene.

We describe the isolation and characterization of a full-length clone for the porcine 17 alpha-hydroxylase/C(17-20) lyase (CYP17) gene. The complete exon and partial intron sequences are presented including approx. 1000 bp of the 5' upstream sequence. In addition we describe the isolation and characterization of the 5' upstream region of the rat CYP17 gene. The sequences of the first exon, part of the first intron, and approx. 3.5 kb of the 5' upstream region are presented.

Expression of the gene for cytochrome P-450 17 alpha-hydroxylase/C17-20 lyase (CYP17) in porcine Leydig cells: identification of a DNA sequence that mediates cAMP response.

Regulation of CYP17 gene expression in porcine Leydig cells was investigated in primary culture. We previously reported the sequence of the 5' upstream and much of the pig gene. (Zhang et al. (1992) Biochim. Biophys. Acta. 1131, 345-348). DNase I footprinting assays identified a region between -193 and -174 that was bound by nuclear proteins. Examination of the DNA sequence in this region revealed putative Sp1 and AP-2 binding sites, but gel retardation assays using an oligonucleotide from -198 to -168 as a probe revealed two specific DNA-protein complexes that were not Sp1 or AP-2. The oligonucleotide was cloned into a reporter gene containing a minimal porcine CYP17 promoter and the resultant construct was transiently transfected into porcine Leydig cells. This chimeric construct had both basal and cAMP-induced transcriptional activities. Southwestern blot identified a prominent binding of a nuclear protein around 68 kDa and a weaker binding of a nuclear protein around 110 kDa. Sequences between -250/+1 are highly homologous to those sequences from human, bovine and rodent CYP17 gene, but the -193/-174 region has no homology to those genes. Other regions of the porcine CYP17 were also important for the basal and cAMP-mediated regulation. Luciferase expression vectors were prepared with 5' flanking DNA from the porcine CYP17 gene and were expressed in primary culture of porcine Leydig cells. The region between -587/-325 was important for basal transcription, and a region of DNA between -325 and -140 was important for cAMP regulation.

Calcium/calmodulin induces phosphorylation of vimentin and myosin light chain and cell rounding in cultured adrenal cells.

In previous reports on adrenal cells we have shown that calcium/calmodulin regulates cholesterol transport to mitochondria and induces phosphorylation of cytoskeleton homogenates. In this study, we have used bovine fasciculata cells permeabilized in situ to identify the phosphorylated proteins and to investigate the manner in which the cytoskeleton components may act together in any subsequent reorganization of the cell. The main cytoskeletal proteins namely vimentin, tubulin, actin, and the associated protein myosin light chain were identified on polyacrylamide gel electrophoresis by their molecular weights and by Western blotting using affinity-purified monoclonal antibodies. In permeabilized cells, calcium/calmodulin promoted phosphorylation of vimentin and myosin light chain within the first 10 min. When incubation time was extended in the presence of 1 mM non-radiolabeled ATP, cell contraction was seen after 15 min. Immunofluorescent microscopy showed that actin microfilaments and myosin light chain displayed a similar pattern of distribution which indicates the actomyosin. Electron microscopy revealed the actomyosin as a dense ring around the cell beneath the plasma membrane. Intermediate filaments (10 nm) were seen within this ring which gave rise to a mixed network in which microfilaments appeared to interconnect intermediate filaments. Immunogold electron microscopy revealed that the 10-nm filaments, found within the actomyosin ring, are vimentin intermediate filaments. It is proposed that calcium/calmodulin causes phosphorylation of the myosin light chain which triggers contraction, and this process involves the intermediate filament protein vimentin. The redistribution of the cytoskeleton and hence the cell rounding is due, in part to the interconnection between vimentin intermediate filaments and actin microfilaments.(ABSTRACT TRUNCATED AT 250 WORDS)

The organelles containing porcine 17 beta-estradiol dehydrogenase are peroxisomes.

Porcine 17 beta-estradiol dehydrogenase was recently purified and cloned. It catalyzes the NAD(+)-dependent oxidation of estradiol to estrone 360-fold more efficiently than the reverse reaction with NADPH. Immunogold electron microscopy localizes 17 beta-estradiol dehydrogenase in organelles of 120 to 500 nm with moderate electron-dense matrices bounded by single membranes. Antibodies against the peroxisomal markers catalase and acyl-CoA oxidase recognize the same organelles in double-labeling studies. This is the first report on the participation of peroxisomes in the metabolism of estradiol.

Calmodulin-binding proteins in plasma membranes from adrenocortical cells.

Highly purified plasma membranes from Y-1 mouse adrenal tumor cells and those from bovine fasciculata cells were shown by [125I]iodocalmodulin overlay to contain five calmodulin-binding proteins of 240,000, 150,000, 66,000, 60,000, and 51,000 mol wt (Mr). Three of these proteins were also detected by affinity chromatography on calmodulin-Sepharose. Calmodulin binding was inhibited by competition with unlabeled calmodulin and by an inhibitor of calmodulin (trifluoperazine). Binding to each of the proteins was Ca2+ dependent. The relative proportion of binding to each of the five proteins was very different for Y-1 and bovine membranes. In Y-1 membranes as much as 50% of total binding was to the 51,000 Mr protein, whereas in bovine membranes more than 50% of binding occurred with the 150,000 Mr protein. Three of the five proteins were tentatively identified as follows: the 240,000 Mr protein is alpha-spectrin, the 60,000 Mr protein is the A subunit of the Ca2+/calmodulin-dependent protein phosphatase called calcineurin and the 51,000 Mr protein is the major subunit of a Ca2+/calmodulin-dependent protein kinase. The kinase was shown to act on specific substrates. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the plasma membranes of adrenal cells, and by binding to alpha-spectrin it may influence the cytoskeletons of these cells. These effects of calmodulin are likely to be important in the regulation of steroid synthesis in the adrenal cortex.

Vectorial (transcellular) transport of potassium (86Rb+) by cultured Sertoli cells.

Sertoli cells from rats aged 25 days were grown on Millipore filters (pore diameter 0.5 micron) for 7 days and were then used for determination of transport of 86Rb+ through the cells (base to apex); this procedure is referred to as measuring transcellular or vectorial transport. Sertoli cells were also used to measure apical efflux of 86Rb+ by loading the cells with the isotope to steady state and then incubating cells so that the apical surfaces were in contact with medium not containing 86Rb+, from which samples were taken. Basal efflux was measured in the same way except that the opposite surface of the cells was in contact with the medium. Cells grown on filters treated with collagen IV plus fibronectin showed transcellular transport of 86Rb+; t1/2 for equilibration across the cells was 9-12 min. The rate of transport was accelerated by addition of (Bu)2cAMP, forskolin, or FSH to the incubation medium. Half-maximal responses were seen with (Bu)2cAMP at 0.2 mM and with forskolin at 20 microM. Apical efflux (t1/2 9.8 +/- 2.1 min) was not influenced by the presence or absence of K+ in the medium nor by azide or (Bu)2cAMP. Basal efflux showed similar values for t1/2 in the presence of K+ (9.7 +/- 1.9 min) and values of 21.4 +/- 4.2 min in the absence of K+. Vectorial transport of 86Rb+ by these cells may account for the K+ gradient seen in the seminiferous tubule and appears to result from a basolateral potassium pump together with an apical membrane that is permeable to K+.

Adenosine 3',5'-monophosphate-dependent protein kinase associated with the cytoskeleton of adrenal tumor cells.

Preparations of cytoskeleton from Y-1 cells were found to phosphorylate various cytoskeletal proteins when incubated with [gamma-32P]ATP. When cAMP was added to the cytoskeleton, a rapid increase in phosphorylation of cytoskeletal protein was observed, and changes were seen in the phosphorylation of individual proteins; four additional proteins were phosphorylated (mol wt, 165,000, 92,000, 45,000, and 24,000) and three proteins were more intensely phosphorylated than without cAMP (mol wt, 125,000, 51,000, and 38,000). In addition, one protein (mol wt, 96,000) that was intensely phosphorylated without cAMP was not phosphorylated with the cyclic nucleotide, and a second (mol wt, 48,000) was less phosphorylated. The increased level of total phosphorylation returned to the unstimulated level within 10 min. The increased phosphorylation of proteins produced by cAMP was inhibited by protein kinase inhibitor. cAMP-dependent protein kinase activity was closely associated with the cytoskeleton, since it was not removed by Triton X-100 (1%, wt/vol), although some activity could be extracted with buffer containing high concentrations of salt. When the cytoskeleton of Y-1 cells was subjected to treatments that disrupt the cytoskeleton before the cells were extracted (cytochalasin B, colchicine, and sonication), no change was seen in cAMP-dependent protein kinase activity. However, cytochalasin B increased phosphorylation of two proteins that were not phosphorylated by cAMP-dependent kinase (mol wt, 63,000 and 43,000). Sonication of the cytoskeleton before addition of [gamma-32P]ATP caused a number of changes in cAMP-independent phosphorylation, but did not affect cAMP-dependent phosphorylation. cAMP-dependent phosphorylation required Mg2+ and was inhibited by Ca2+. It is concluded that the cytoskeleton of Y-1 cells contains bound cAMP-dependent protein kinase that phosphorylates certain cytoskeleton proteins. The cytoskeleton also contains one or more cAMP-independent kinase systems. It is suggested that the cAMP-dependent protein kinase described here may be important in the cytoskeletal responses to ACTH.

Inhibition of testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/C-17,20-lyase) by estrogens.

Highly purified cytochrome P-450 from neonatal pig testicular microsomes is capable of catalyzing both 17 alpha-hydroxylation and C-17,20-lyase activity. Estradiol was found to inhibit both activities of the purified enzyme with delta 4 and with delta 5 substrates (progesterone, pregnenolone, and the corresponding 17 alpha-hydroxysteroids). For the delta 4 series, inhibition of lyase is competitive and that of 17 alpha-hydroxylase is noncompetitive; for the delta 5 series, inhibition was noncompetitive for both activities. Ki values for lyase activity were determined from enzyme kinetics (5.0 microM for the delta 4 substrate and 20 microM for the delta 5 substrate). Estradiol produces a typical type I spectral shift with the pure enzyme (Ks = 3.0 microM where Ks is the concentration of steroid required to give half maximal spectral shift), so that Ki values were also determined directly from binding studies by using substrate-induced difference spectroscopy. Fifty per cent inhibition of the maximal spectral shift induced by the 17 alpha-hydroxysubstrates (Ki) are 3.8 and 7.6 microM for the delta 4 and delta 5 substrates, respectively. Values for Ki are higher with the substrates of 17 alpha-hydroxylase (progesterone and pregnenolone), by either method, than the corresponding Ki values for the lyase substrates. The concentration of estradiol in Leydig cells of neonatal pig testis is approximately 1.5 nmol/g. It is proposed that estradiol may influence testicular steroidogensis under physiological conditions by competitive inhibition of lyase activity.