Specific regulation by steroid hormones of protein kinases in the endometrium. 1. Alteration by estrogen and progesterone in levels of protein kinases in rabbit endometrium

The alteration in activities of multiple protein kinases has been studied in the endometrium of a rabbit treated with estrogen and progesterone. The administration of estrogen or progesterone to the castrated rabbit resulted in a remarkable increase of total activity in the cytosol fraction of the endometrium. The administration of estrogen caused an increase of type I adenosine-3',5'-monophosphate-dependent (cAMP-dependent) protein kinase and a slight decrease of type II cAMP-dependent protein kinase. In contrast, the treatment with progesterone after priming administration of estrogen brought about an increase of type II cAMP-dependent protein kinase and a decrease of type I cAMP-dependent protein kinase. Therefore, the activity ratio of type II to type I decreased by estrogen and increased by progesterone. The simultaneous administration of cycloheximide abolished the stimulatory effect of respective hormones on the level of each protein kinase. The activity profile of protein kinases on DEAE-cellulose column after ovulation caused by the administration of human chorionic gonadotropin to a non-castrated rabbit was similar to that of the rabbit treated with progesterone. The results presented demonstrate the specific regulation by the steroid hormones of de novo synthesis of protein kinases in the target organ.     

Properties of soluble cyclic AMP-dependent protein kinase activity of renal inner medulla

Studies of the chromatographic distribution of soluble protein kinase in rat kidney demonstrated that the type I isoenzyme predominates in cortex, whereas activity in outer and inner medulla is almost exclusively the type II form. The type II isoenzyme also predominates (95% or greater) in human, canine, bovine, porcine and rabbit inner medulla. Compared to soluble type I activities from rat renal cortex or medulla, type II activity of inner medulla demonstrates a marked resistance to activation by NaCl and/or urea in subcellular preparations. However, with respect to solute activation, the resistance of the type II enzyme of inner medulla does not differ from that of type II activities from other tissues. In contrast to the effects on basal activity, NaCl and urea potentiated inner medullary type II activation by cyclic AMP and also delayed the rate of subunit reassociation after chromatographic removal of cyclic AMP. Incubation of inner medullary slices in high osmolality buffer (NaCl and urea) did not alone activate soluble protein kinase, an observation which implied that the enzyme was also resistant to solute activation in the intact cell system. Moreover, at 1650 mosM, vasopressin activation of soluble protein kinase was enhanced compared to responses at 750 mosM despite comparabel levels of cyclic AMP accumulation at the two osmolalities. However, a cyclic AMP-independent action of high osmolality to reduce the rate of inactivation of arginine vasopressin-stimulated protein kinase was not demonstrable in inner medullary slices.The present data suggest the possibility that the resistance of inner medullary protein kinase to solute activation could be related to the isomeric form of enzyme (type II) present in this tissue. The high concentrations of NaCl and urea routinely found in inner medulla during hydropenia also influenced protein kinase responses to arginine vasopressin, and may do so in part by directly potentiating the action of cyclic AMP on subunit dissociation.     

Selective expression of type I and type II cyclic AMP-dependent protein kinases in subcellular fractions of concanavalin A-stimulated rat thymocytes

Total protein kinase activity and the expression of the type I and type II cyclic adenosine 3′:5′-monophosphate-dependent protein kinases were studied in subcellular fractions of rat thymocytes and the effect of concanavalin A treatment on protein kinase activity was assessed. At a concentration of 100 μ/ml of concanavalin A a marked decline of total nuclear protein kinase activity occurred which lasted approximately 20 to 90 min. Concomitantly, a twofold increase of total protein kinase activity in the 900g supernatant fraction was observed which lasted from 5 to 30 min. Studies using the heat-stable protein kinase inhibitor revealed that the concanavalin A-mediated activity changes were primarily due to changes of cAMP-dependent protein kinase activity, whereas cAMP-independent protein kinase activity remained unchanged. Analysis of the type I and type II cAMP-dependent protein kinase isozyme pattern before and after concanavalin A treatment revealed a selective change of the relative expression of isozyme activities. Whereas type I protein kinase was the major nuclear isozyme before concanavalin A treatment, nuclear type II cAMP-dependent protein kinase increased markedly with a concomitant loss of type I isozyme expression. In the 900g supernatant fraction, containing primarily the type II isozyme in unstimulated cells, concanavalin A treatment caused an increase of the expression of the type I isozyme. The concanavalin A-mediated relative changes of cAMP-dependent protein kinase isozyme expression were confirmed by photoaffinity labeling of the regulatory subunits R^I and R^II before and after concanavalin A stimulation. The intracellular concanavalin A-mediated isozyme changes were time dependent, exhibiting maximal effects about 20 min after concanavalin A addition. These results indicate that selective regulation of intracellular cAMP-dependent protein kinase isozyme expression may be a mechanism related to isozyme-specific phosphorylation of specific intracellular substrates in concanavalin A-activated thymocytes.     

Increase in type I adenosine 3',5'-monophosphate-dependent protein kinase during isoproterenol-induced cardiac hypertrophy.

The amount of type I and type II cyclic AMP-dependent protein kinase present in the rat heart was determined at various times during isoproterenol-induced cardiac hypertrophy. Wistar rats were injected twice daily with isoproterenol (5 mg/kg, s.c.) for 2, 5 or 10 days. Cardiac weight increased gradually over the 10-day period of drug administration, and by day 10, heart weight was 156% of control. Following the cessation of isoproterenol administration, the cardiac weight regressed toward the control value by day 15. An increase in the specific activity of type I protein kinase to 197% of control occurred by day 10. The specific activity of type II protein kinase did not change significantly during either the hypertrophy or regression stage. The increase in the specific activity of type I protein kinase during a chemically-induced trophic response of the heart may indicate that type I cyclic AMP-dependent protein kinase plays a regulatory function in this process.     

The distribution and dissociation of cyclic adenosine 3':5'-monophosphate-dependent protein kinases in adipose, cardiac, and other tissues.

In crude extracts of adipose tissue the protein kinase dissociates slowly at 30 degrees into regulatory and catalytic subunits in the presence of 700 mug per ml of histone or 0.5 M NaCl. If the kinase is first dissociated by adding 10 muM adenosine 3':5'-monophosphate (cAMP), reassociation occurs instantaneously after removal of the cAMP by Sephadex G-25 chromatography. In contrast, in crude xtracts of heart, the protein kinase dissociates rapidly in the presence of 700 mug per ml of histone or 0.5 M NaCl and reassociates slowly after removal of cAMP. These differences are accounted for by the existence of two types of protein kinases in these tissues, referred to as types I and II. DEAE-cellulose chromatography of extracts of adipose tissue produces only one peak of cAMP-dependent protein kinase activity (type II) which elutes between 0.15 and 0.25 M NaCl. Similar chromatography of heart extracts resolves enzyme activity into two peaks; a type I enzyme which elutes between 0.05 and 0.1 M and predominates (greater than 75% of total activity), and a type II enzyme which elutes between 0.15 and 0.25 M NaCl. The dissociation properties of the types I and II enzymes from heart and adipose tissue are retained after partial purification by DEAE-cellulose and Sepharose 6B chromatography. Rechromatography of the separated peaks of the cardiac enzymes does not change the elution pattern. Sucrose density gradient centrifugation and gel filtration studies indicate that the molecular weights of these enzymes are very similar. The type II enzyme isolated by DEAE-cellulose chromatography of heart extracts resembles the adipose tissue enzyme, i.e. it undergoes slow dissociation at 30 degrees in the presence of histone or 0.5 M NaCl. The adipose tissue kinase and the heart type II kinase are not identical, however, since they do not elute at exactly the same point on DEAE-cellulose columns. A survey of several tissues indicates the presence of type I and II protein kinases similar to the enzymes in adipose tissue and heart as determined by DEAE-cellulose chromatography of crude extracts and by dissociation of the enzymes with histone. The presence of MgATP prevents dissociation of type I enzyme from heart by 0.5 M NaCl or histone. The profile of the enzyme on DEAE-cellulose, however, is not changed...     

Differential androgenic control of prostatic cytosolic cAMP-dependent and -independent protein kinases

Whether or not various cytosolic protein kinases (and especially the type I cAMP-dependent protein kinase) of rat ventral prostate are specifically regulated with respect to total activity or specific activity by androgen has been investigated. Following androgen deprivation, the total activity per prostate of cAMP-dependent protein kinase (with histone as substrate) changed little at 24 h, declining by about 20% at 96 h. Under these conditions, its specific activity remained unaltered at 24 h, but was markedly enhanced at 96 h postorchiectomy. Type II cAMP-dependent protein kinase in rat ventral prostate cytosol was the only form of cAMP-dependent protein kinases present as determined by measurement of catalytic activity as well as [32P]-8-N3-cAMP binding to the regulatory subunits. There was no alteration in the distribution of the isoenzymes of cAMP-dependent protein kinases or the response of these kinase activities to cAMP owing to castration of animals. The prostatic cytosol also contains free regulatory subunit (with molecular weight similar to that of regulatory subunit R1) which coelutes with type II cAMP-dependent protein kinase. This finding was confirmed by using [32P]-8-N3-cAMP photoaffinity labeling of cAMP-binding proteins. With respect to cAMP-independent protein kinase (measured with dephosphophosvitin as substrate), a decline of 31% in its specific activity was observed in cytosol of prostates from rats castrated for a period of 24 h without significant further change at later periods following castration. However, there was a marked progressive reduction in total activity of this enzyme per prostate (loss of 72% at 96 h postorchiectomy). The increase in specific activity of cAMP-dependent, but not cAMP-independent, protein kinase in the face of decreasing total activity in the cytosol at later periods of castration (e.g., at 96 h) may reflect a slower loss of the former enzyme protein than the bulk of the cytosolic proteins. Administration of testosterone to castrated animals prevented these changes. These data do not indicate a specific regulation by steroid of the type I cAMP-dependent protein kinase in the prostate. Rather, the cAMP-independent protein kinase (with dephosphophosvitin as substrate) appears to be modulated by the androgenic status of the animal.     

Cell cycle-specific activity of type I and type II cyclic adenosine 3':5'-monophosphate-dependent protein kinases in Chinese hamster ovary cells.

Types I and II cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinases have been studied during the cell cycle of Chinese hamster ovary cells. Chinese hamster ovary cells were synchronized by selective detachment of mitotic cells from monolayer cultures. Protein kinases were separated by DEAE-cellulose chromatography and were similar to the types of cAMP-dependent protein kinases studied in skeletal muscle and in heart extracts. The total amount of protein kinases activity per cell was substantial, both in mitosis and at the G1/S boundary. During mitosis, the relatively high activity of protein kinase was due to a predominance of type I protein kinase. During early G1, the activity of type I protein kinase decreased and there was little detectable type II activity. A rapid increase in the activity of type II was evident at the G1/S boundary. The administration of puromycin (50 mug/ml) from 1 to 5 hours after selective detachment of mitotic cells abolished the activity of type II cAMP-dependent protein kinase seen at the G1/S border, but had no observable effect on the activity of type I protein kinase. The data presented demonstrate cell cycle-specific activity patterns of type I and type II protein kinase Type I protein kinase activity is high in mitosis and is constant throughout the cell cycle. Increased type II protein kinase activity seems to be related to the initiation of DNA synthesis in S phase. The data suggest a translational control of type II cAMP-dependent protein kinase activity.     

Regulation of human tyrosine hydroxylase activity. Effects of cyclic AMP-dependent protein kinase, calmodulin-dependent protein kinase II and polyanion

To determine the regulatory mechanism for human tyrosine hydroxylase, we examined modulations of the activity of the enzyme from human pheochromocytoma by cyclic AMP-dependent protein kinase, calmodulin-dependent protein kinase II and polyanion. The most remarkable activation was observed when the enzyme was assayed at physiological pH (pH 7) after being subjected to phosphorylation by cyclic AMP-dependent protein kinase. Calmodulin-dependent protein kinase II and polyanion also modulated the enzyme activity. The results suggest that tyrosine hydroxylase may be regulated similarly in both human and rat.     

Differentiation-associated increase of cAMP-dependent type II protein kinase in a murine preadipose cell line (ST 13)

The activity of cAMP-dependent protein kinase and cAMP binding activity were studied during the differentiation of ST 13 murine preadipocytes into adipocytes. We found that both activities were marginally detectable in preadipose cells and increased remarkably when the cells were induced to differentiate, preceding by several days the morphological adipose conversion. The increased cAMP-dependent protein kinase was identified as type II enzyme by means of DEAE-Sephacel chromatography and by photoaffinity labeling with 8-azido[3H]cAMP. We further showed that the increase of protein kinase activity was specific to cell differentiation with the aid of modulators of the adipose conversion (insulin, fetal bovine serum, retinoic acid and 5-bromodeoxy-uridine). We propose that the increased expression of type II cAMP-dependent protein kinase would be a biochemical index of differentiation in ST 13 preadipocytes.     

Tissue distribution and developmental expression of protein kinase C isozymes

Protein kinase C is a ubiquitous enzyme found in a variety of mammalian tissues and is especially highly enriched in brain and lymphoid organs. Based on biochemical and immunological analyses, we have identified three types of protein kinase C isozyme (designated types I-III) from rat brain. Monospecific antibodies against each of the protein kinase C isozymes were prepared for the determination of tissue distribution, subcellular localization, and developmental changes of these enzymes. The various protein kinase C isozymes were found to be distinctively distributed in different tissues: the type I enzyme in brain; the type II enzyme in brain, pituitary and pineal glands, spleen, thymus, retina, lung, and intestine; and the type III enzyme in brain, pineal gland, retina, and spleen. The rat brain enzymes were differentially distributed in different subcellular fractions. The type I enzyme appeared to be most lipophilic and was recovered mostly in the particulate fractions (80-90%) regardless of the EGTA- or Ca2+-containing buffer used in the homogenization. Significant amounts (30-40%) of the type II and III enzymes were recovered in the cytosolic fraction with EGTA-containing buffer. The expressions of different protein kinase C isozymes appear to be differently controlled during development. In rat brain, both type II and III enzymes were found to increase progressively from 3 days before birth up to 2-3 weeks of age and remained constant thereafter. However, the expression of the type I enzyme displayed a different developmental pattern; it was very low within 1 week, and an abrupt increase was observed between 2 and 3 weeks of age. In thymus, the type II enzyme was found to be maximal shortly after birth; whereas the same kinase in spleen was very low within 2 weeks of age, and a significant increase was observed between 2 and 3 weeks. These results demonstrate that protein kinase C isozymes are distinctively distributed in different tissues and subcellular locales and that their expressions are controlled differently during development.     

Discrepancy of protein kinase C translocation and platelet aggregation--immunocytobiochemical evidence

We searched for possible relationships between platelet aggregation induced by 12-O-tetradecanoyl phorbol 13-acetate (TPA) or thrombin and the translocation of protein kinase C. Using monoclonal antibodies against subspecies of protein kinase C, we noted a predominant expression of the isozyme, type II, in human platelets (M. Watanabe, M. Hagiwara, K. Onoda, and H. Hidaka, 1988, Biochem. Biophys. Res. Commun. 152, 642). Analysis of the subcellular distribution of protein kinase C revealed that 65% of the kinase activity was present in the cytosolic fraction in unstimulated platelets, with the remaining activity in the membrane fraction. Treatment of platelets with 100 nM TPA resulted in a greater than 60% decrease in protein kinase C activity in the cytosolic fraction and a greater than 200% increase in the activity in the membrane fraction, within 10 min after treatment. Translocation of the enzyme was also found after treatment of platelets with thrombin, although the response was of lower magnitude than that induced by TPA. Similar results were obtained by immunoblotting using MC-2a, an anti-type II protein kinase C monoclonal antibody. We also examined localization of the enzyme, by electron microscopic immunocytochemistry. The presence of type II protein kinase C seemed to be localized mostly in hyaluromeres and not in granulomeres. When platelets were fixed just after the addition of TPA (within 1 min), protein kinase C was localized at the submembranal region with no remarkable change in shape but there was a decrease in the number of granules in the cytoplasma and the open canalicular system was dilated. We then investigated the effects of cytochalasin B, W-7, ML-9, and H-7 on TPA-induced platelet aggregation and the translocation of protein kinase C. W-7 and ML-9 potently inhibited platelet aggregation but none of these compounds hampered the translocation. Thus, activation of protein kinase C may not be a complete requirement for the initiation of platelet aggregation.     

Biochemical characterization of rat brain protein kinase C isozymes

Biochemical characteristics of three rat brain protein kinase C isozymes, types I, II, and III, were compared with respect to their protein kinase and phorbol ester-binding activities. All three isozymes appeared to be alike in their phorbol ester-binding activities as evidenced by their similar Kd for phorbol 12,13-dibutyrate and requirements for Ca2+ and phospholipids. However, differences with respect to the effector-mediated stimulation of protein kinase activity were detectable among these isozymes. The type I enzyme could be stimulated by cardiolipin to a greater extent than those of the type II and III enzymes. In the presence of cardiolipin, the concentrations of dioleoylglycerol or phorbol 12,13-dibutyrate required for half-maximal activation (A1/2) of the type I enzyme were nearly an order of magnitude lower than those for the type II and III enzymes. In the presence of phosphatidylserine, differences in the A1/2 of dioleoylglycerol and phorbol 12,13-dibutyrate for the three isozymes of protein kinase C were less significant than those measured in the presence of cardiolipin. Nevertheless, the A1/2 of these two activators for the type I enzyme were lower than those for the type II and III enzymes. At high levels of phosphatidylserine (greater than 15 mol %), binding of phorbol 12,13-dibutyrate to the type I enzyme evoked a corresponding stimulation of the kinase activity, whereas binding of this phorbol ester to the type II and III enzymes produced a lesser degree of kinase stimulation. For all three isozymes, the concentrations of phosphatidylserine required for half-maximum [3H]phorbol 12,13-dibutyrate binding were almost an order of magnitude less than those for kinase stimulation. Consequently, neither isozyme exhibited a significant kinase activity at lower levels of phosphatidylserine (less than 5 mol %) and phorbol 12,13-dibutyrate (50 nM), a condition sufficient to promote near maximal phorbol ester binding. In addition to their different responses to the various activators, the three protein kinase C isozymes also have different Km values for protein substrates. The type I enzyme appeared to have lower Km values for histone IIIS, myelin basic protein, poly(lysine, serine) (3:1) polymer, and protamine than those for the type II and III enzymes. These results documented that the three protein kinase C isozymes were distinguishable in their biochemical properties. In particular, the type I enzyme, which is a brain-specific isozyme, is distinct from the type II and III enzymes, both have a widespread distribution among different tissues.     

Phosphorylation of the EGF receptor from A431 epidermoid carcinoma cells by three distinct types of protein kinase C

Three distinct types of protein kinase C obtained from rat brain cytosol phosphorylated the EGF receptor of A431 epidermoid carcinoma cells at different rates. This receptor was phosphorylated most rapidly by type III protein kinase C, but slowly by type I enzyme. Type II enzyme showed intermediate activity. Chromatographic analysis indicated that A431 cells possessed only one of the three types found in rat brain, which apparently corresponded to type III enzyme. This type of protein kinase C, that is encoded by the alpha-sequence or a closely related sequence, appeared to be expressed commonly in many tissues and organs. The result implies that type III enzyme may play roles in growth promotion.     

Independent expression of cardiac type I and II cyclic AMP-dependent protein kinase during murine embryogenesis and postnatal development.

The amount of total cyclic AMP-dependent protein kinase and of the protein kinase isozymes present in mouse heart changes during development. During embryogenesis, the total cardiac protein kinase activity increases most markedly during the 6 days prior to birth. A maximum kinase level is achieved in the 7 day-old neonate, and then activity progressively declines to an adult level approximating that of the mid-embryo. The type II kinase exhibits a moderate increase during late embryogenesis which declines by the time of birth. The type I isozyme increases throughout embryogenesis and the first neonatal week to a maximum specific activity five-fold higher than the mid-embryogenesis level. The isozyme level then falls to an adult activity similar to the mid-embryonic. These changes in isozyme profile are reflected in a changing type I to type II kinase ratio of 1.1 at 13--14 days embryogenesis, 2.4 at birth, 3.0 in the 7 day-old neonates, and 1 in the adult heart. Thus, the two protein kinase isozymes change in association with the developmental process in an independent fashion.     

Advanced glycation end-product-induced mitogenesis and collagen production are dependent on angiotensin II and connective tissue growth factor in NRK-49F cells

Diabetic nephropathy (DN) is characterized by glomerulopathy and tubulointerstitial expansion followed by renal fibrosis. Angiotensin II (Ang II) and connective tissue growth factor (CTGF) are involved in the pathogenesis of DN, while Janus kinase 2 (JAK2) is important in advanced glycation end-product (AGE)-induced effects in renal interstitial (NRK-49F) fibroblasts. Thus, we studied the role of Ang II, CTGF, and JAK2 in AGE-induced effects in NRK-49F cells. We found that AGE (150 microg/ml) increased mitogenesis and type I collagen production at 7 days while Ang II (10(-7)M) increased mitogenesis and type I collagen production at 3 days. We also found that AGE (150 microg/ml) increased angiotensinogen protein at 2 days, which was attenuated by AG-490 (a JAK2 inhibitor). AGE (150 microg/ml) increased CTGF mRNA and protein expression at 3 and 5 days, respectively. Ang II (10(-7)M) increased CTGF mRNA and protein expression at 1 and 2 days, respectively, which were attenuated by AG-490. Moreover, losartan (a type I angiotensin receptor blocker) and captopril (an angiotensin converting enzyme inhibitor) attenuated AGE-induced CTGF mRNA/protein expression while attenuating AGE-induced mitogenesis and type I collagen production. AG-490 and CTGF antisense (but not sense) oligodeoxynucleotide (ODN) attenuated Ang II (10(-7)M) and AGE-induced mitogenesis and type I collagen production at 3 and 7 days, respectively. We concluded that AGE (150 microg/ml)-induced mitogenesis and type I collagen production are dependent on the Ang II-JAK2-CTGF pathway in NRK-49F cells. Moreover, Ang II-induced mitogenesis and type I collagen production are dependent on the JAK2-CTGF pathway.     

Protein kinases in human renal cell carcinoma and renal cortex. A comparison of isozyme distribution and of responsiveness to adenosine 3':5'-cyclic monophosphate.

Two major isozyme forms of cyclic AMP-dependent protein kinase (termed protein kinase I and II according to their order of elution from DEAE-cellulose) were resolved by DEAE-cellulose chromatography of extracts from human renal cortex and renal cell carcinoma. The ratio between protein kinase I and protein kinase II in carcinoma extracts was about twice that in extracts of renal cortex. The total soluble cyclic AMP-dependent protein kinase activity was similar in extracts from the normal and malignant tissue. Protein kinase isozymes prepared from renal cortex or carcinoma were highly dependent on cyclic AMP for activity under appropriate assay conditions, were activated to the same degree by various concentrations of cyclic AMP, and had similar affinity for the nucleotide, indicating that the mechanism for regulation of protein kinase activity by cyclic AMP was intact for the tumor kinases. The kinetics of endogenous phosphorylation of protein kinase II was similar for enzyme derived from normal or malignant tissue.     

Characterization and androgenic regulation of soluble protein kinases and protein phosphorylation in rat ventral prostate gland

The soluble protein kinase activities for protamine and casein, the histone kinases modulated by cAMP or Ca2+ and phospholipid, as well as the phosphorylation patterns of endogenous proteins were measured in rat ventral prostates from normal adults, castrates, and dihydrotestosterone-treated castrates. In normal prostate, the ratio of cAMP-dependent type I and II kinases was approximately 1:5. After a 3-week period of castration-induced regression, the concentrations of both enzymes were increased, but on a total organ basis, type I was decreased to 56%, while type II was reduced to 20% of normal levels. Casein kinase activity in unfractionated cytosol was not significantly altered by castration but when partially resolved into type I and II enzymes, there appeared to be a selective reduction in the type I component. In contrast, the total organ activities of protamine kinase or Ca2+-activated, phospholipid-dependent kinase, two measures of protein kinase C enzyme, were significantly increased (64 and 71%, respectively) above sham controls in regressed organs of castrates. All of the castration-induced changes in protein kinases were restored toward normal by dihydrotestosterone treatment. Castration effects on protein kinase C and the cAMP-dependent kinases appeared to be manifest in the phosphorylation of endogenous proteins. Castration resulted in a qualitative shift in the cAMP-dependent phosphorylation patterns as measured by gel electrophoresis, with increases in four major bands and decreases in two others, whereas the Ca2+-activated, phospholipid-dependent phosphorylation patterns were all enhanced. It is concluded that the androgenic regulation of protein kinase C differed qualitatively from that of other kinases, and its activation upon withdrawal of the androgenic stimulus may be involved in autophagic mechanisms in the prostate.     

Expressions of two types of soluble cAMP-dependent protein kinases of the rabbit myometrium in different functional states

Two types of soluble cAMP-dependent protein kinase (I and II) were isolated from rabbit myometrium cytosol at functional rest and characterized. In pregnancy, protein kinase is represented by type II alone. Upon delivery, one isoform of the enzyme was detected, which was eluted from a DEAE-cellulose column with 0.15-0.22 M. NaCl. During the postnatal period, the elution profile of the enzyme is made up of two protein bands, one fraction being eluted with 0.15-0.22 M NaCl (93% of total enzyme content), and the other one being represented by a minor component eluted with 0.07-0.09 M. NaCl (7%). In terms off isoenzyme activity, main kinetic properties, ability to autophosphorylate and Kass for cAMP, the protein kinase isolated during delivery and the major protein kinase fraction obtained in the postnatal period can be related to protein kinases type II. Quantitative and qualitative expression of two types of soluble cAMP-dependent protein kinase from rabbit myometrium isolated at different functional states may be due to differences in their biological activity.     

Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.

Deoxythymidine kinase activities were induced in HeLa TK- (deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography. Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg2+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45 degrees and 4 degrees. The deoxycytidine kinase activity present in the purified preparations was inactivated at the same rate as the deoxythymidine kinase activity. In the presence of the other substrate, deoxythymidine, herpes simplex virus type I deoxythymidine kinase was more stable than herpes simplex virus type II kinase. The purified herpes simplex virus type I and II deoxythymidine kinase had different activation energies when Mg2+ATP and deoxythymidine were used as substrates, but showed the same sensitivity toward ammonium sulfate inhibition.     

Structure, function and tissue-specific gene expression of 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase enzymes in classical and peripheral intracrine steroidogenic tissues

The membrane-bound enzyme 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase (3β-HSD) catalyses an essential step in the transformation of all 5-pregnen-3β-ol and 5-androsten-3β-ol steroids into the corresponding 3-keto-4-ene-steroids, namely progesterone as well as all the precursors of androgens, estrogens, glucocorticoids and mineralocorticoids. We have recently characterized two types of human 3β-HSD cDNA clones and the corresponding genes which encode type I and II 3β-HSD isoenzymes of 372 and 371 amino acids, respectively, and share 93.5% homology. The human 3β-HSD genes containing 4 exons were assigned by in situ hybridization to the p11-p13 region of the short arm of chromosome 1. Human type I 3β-HSD is the almost exclusive mRNA species present in the placenta and skin while the human type II is the predominant mRNA species in the adrenals, ovaries and testes. The type I protein possesses higher 3β-HSD activity than type II. We elucidated the structures of three types of rat 3β-HSD cDNAs as well that of one type of 3β-HSD from bovine and macaque ovary λgt11 cDNA libraries, which all encode a 372 amino acid protein. The rat type I and II 3β-HSD proteins expressed in the adrenals, gonads and adipose tissue share 93.8% homology. Transient expression of human type I and II as well as rat type I and II 3β-HSD cDNAs in HeLa human cervical carcinoma cells reveals that 3β-ol dehydrogenase and 5-ene-4-ene isomerase activities reside within a single protein. These expressed 3β-HSD proteins convert 3β-hydroxy-5-ene-steroids into 3-keto-4-ene derivatives and catalyze the interconversion of 3β-hydroxy and 3-keto-5α-androstane steroids. By site-directed mutagenesis, we demonstrated that the lower activity of expressed rat type II compared to rat type I 3β-HSD is due to a change of four residues probably involved in a membrane-spanning domain. When homogenates from cells transfected with a plasmid vector containing rat type I 3β-HSD is incubated in the presence of dihydrotestosterone (DHT) using NAD? as co-factor, 5α-androstanedione was formed (A-dione), indicating an intrinsic androgenic 17β-hydroxysteroid dehydrogenase (17β-HSD) activity of this 3β-HSD. We cloned a third type of rat cDNA encoding a predicted type III 3β-HSD specifically expressed in the rat liver, which shares 80% similarity with the two other isoenzymes. Transient expression in human HeLa cells reveals that the type III isoenzyme does not display oxidative activity for the classical substrates of 3β-HSD. However, in common with the type I enzyme, it converts A-dione and DHT to the corresponding 3β-hydroxysteroids, thus showing an exclusive 3-ketosteroid reductase activity. When NADPH is used as co-factor, the affinity for DHT of the type III enzyme becomes 10-fold higher than that of the type I. Rat type III mRNA was below the detection limit in intact female liver. Following hypophysectomy, its concentration increased to 55% of the values measured in intact or hypophysectomized male rats, an increase which can be blocked by administration of ovine prolactin (oPRL). Treatment with oPRL for 10 days starting 15 days after hypophysectomy markedly decreased ovarian 3β-HSD mRNA accumulation accompanied by a similar decrease in 3β-HSD activity and protein levels. Treatment with the gonadotropin hCG reversed the potent inhibitory effect of oPRL on these parameters and stimulated 3β-HSD mRNA levels in ovarian interstitial cells. These data indicate that the presence of multiple 3β-HSD isoenzymes offers the possibility of tissue-specific expression and regulation of this enzymatic activity that plays an essential role in the biosynthesis of all hormonal steroids in classical as well as peripheral intracrine steroidogenic tissues.