Protein kinases from liver mitochondria of tumour-bearing rats.

The mitochondria of liver of Yoshida ascites tumour-bearing rats contained two forms of protein kinase distinguishable on the basis of their kinetic properties, substrate specificity and responses to cyclic adenosine 3',5'-monophosphate (cAMP). One of these (kinase I) was activated 2-3 fold by cAMP while the other form (kinase II) was insensitive to the action of cAMP. Kinase I which was selective towards histone F1 as substrate was obtained as a homogeneous preparation and was observed to have a molecular weight of 170 000 by Sephadex G-150 gel filtration. Protein kinase II appeared to be a smaller protein with molecular weight of 54 000 and was specific towards acidic proteins namely casein and phosvitin. Protein kinases isolated from liver mitochondria of normal rats showed variations in respect to elution profile of DEAE-cellulose and electrophoretic mobility. The preparation corresponding to kinase I did not show stimulatory responses to cAMP.     

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...     

cAMP-dependent protein kinase activation lowers hepatocyte cAMP

Rat hepatocyte protein kinase was activated by incubating the cells with various cAMP analogs. Boiled extracts were then prepared and Sephadex G-25 chromatography was carried out. The G-25 procedure separated the analogs from cAMP since the resin had the unexpected property of binding cyclic nucleotides with differing affinities. Separation was necessary because the analogs would otherwise interfere with the sensitive protein kinase activation method developed for assay of cAMP. The cAMP analogs, but not 5'-AMP, lowered basal cAMP by 50-70%. The effect was rapid, analog concentration-dependent, and occurred parallel with phosphorylase activation, suggesting that the cAMP analogs act through cAMP-dependent protein kinase activation. A cAMP analog completely blocked the cAMP elevation produced by relatively low concentrations of glucagon, but did not block the phosphorylase response, indicating that the cAMP analog substitutes for cAMP as the intracellular activator of protein kinase. One implication of the results is that elevation of cAMP and protein kinase activity by hormones has a negative feedback effect on the cellular cAMP level.     

Purification and characterization of ribonucleases from human seminal plasma.

Four ribonucleases (RNAases I-IV) have been purified to homogeneity from human seminal plasma by precipitation with 40-75%-satd. (NH4)2SO4, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose phosphocellulose, agarose-5'-(4-aminophenylphospho)uridine 2'(3')-phosphate (RNAase affinity column) and Sephadex G-75 or G-100. The homogeneity of these RNAases was confirmed by polyacrylamide-gel electrophoresis. Mr values for these purified RNAases were 78 000, 16 000, 13 300 and 5000 as estimated by gel filtration. Enzyme activities of RNAases I, III and IV were inhibited by Mn2+, Zn2+ and Cu2+ and activated by Na+, K+, Ba2+, Mg2+, Fe2+ and EDTA, whereas that of RNAase II was inhibited by Ba2+, Mg2+, Fe2+, Mn2+, Zn2+ and Cu2+ and activated by Na+, K+ and EDTA. RNAases I, II and IV demonstrated a higher affinity for poly(C) and poly(U) or yeast RNA, whereas RNAase III preferentially hydrolysed poly(U) over poly(C) and yeast RNA. In the presence of 5 mM-spermine, RNAase I was dissociated to a low-Mr (5000) enzyme with an increase in total RNAase enzymic activity. Xenoantiserum to each RNAase was raised and evaluated by immunoprecipitation and immunohistochemical methods. Anti-(seminal RNAase III) antiserum showed no immunological cross-reaction with RNAases of other human origin, whereas anti-(seminal RNAase I), -(RNAase II) and -(RNAase IV) antisera exhibited indistinguishable immunological reactions with serum RNAase and other human RNAases, except that anti-(seminal RNAase I) and -(RNAase antisera IV) did not react with pancreatic RNAases. Seminal RNAases I and IV were identical immunologically as shown by anti-(RNAase I) and anti-(RNAase IV) in immunodiffusion. Immunohistochemical study revealed that, among human tissues examined, only prostate expressed seminal RNAase III. These results suggested that human seminal RNAase I may be an aggregated molecule of RNAase IV and that seminal RNAases II and IV are similar to serum RNAases, whereas seminal RNAase III is a prostate-specific enzyme.     

Dipeptidyl carboxypeptidase from human seminal plasma.

Dipeptidyl carboxypeptidase (angiotensin I converting enzyme) was purified from human seminal plasma. The apparent relative molecular mass determined by gel filtration on Sephadex G-200 was 330 000. The pI in isoelectric focusing was 4.6--5.0 and the optimum pH 7.7--8.0. The enzyme is activated by chloride. These properties are similar to those reported for the lung enzyme. The specificity is that of a carboxypeptidase releasing dipeptides. A study of different substrates showed the activity to be highest with Z-Leu-Gly-Gly, followed by Z-Phe-His-Leu greater than bradykinin greater than Bz-Gly-Gly-Gly greater than Boc-Phe-Ala-Pro greater than Bz-Gly-His-Leu greater than angiotensin I.     

Purification and characterization of human seminal plasma aminopeptidase

A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.     

Characterization and regulation of heart adenosine 3':5'-monophosphate-dependent protein kinase isozymes.

There is broad species variation in the type of cAMP-dependent protein kinase isozyme present in supernatant fractions of heart homogenates as determined by DEAE-cellulose chromatography, Isozyme I, which elutes at less than 0.1 M NaCl, is predominant in mouse and rat hearts; while isozyme II, which elutes at greater than 0.1 M NaCl, is the predominant type in beef and guinea pig. Human and rabbit hearts contain about equal amounts of the two types. The type I heart kinases are more easily dissociated into free regulatory and catalytic subunits by incubation with histone than are the type II kinases, and the separated regulatory and catalytic subunits of isozyme II of rat heart reassociate more rapidly than the subunits of isozyme I under the conditions used. The data from several experiments using rat heart indicate that the basal activity ratio of the protein kinase in crude extracts (approximately 0.15) is due mainly to basal endogenous cAMP and that cAMP elevation accounts entirely for the epinephrine effect on the enzyme. Addition of epinephrine and 1-methyl-3-isobutylxanthine to the perfusate causes a rapid (1 min) increase in cAMP, active supernatant protein kinase, and active phosphorylase in perfused hearts of both rat (mainly isozyme I) and guinea pig (mainly isozyme II). The elevation percentage in cAMP is about the same in the two species, but the increase in active protein kinase is greater in rat heart. If hearts from either animal are perfused continually (10 min) with epinephrine (0.8 muM) and 1-methyl-3-isobutylxanthine (10 muM), the cAMP level, active protein kinase, and active phosphorylase remain elevated. Likewise, all parameters return rapidly to the basal levels when epinephrine and 1-methyl-3-isobutylxanthin are removed. Most of the epinephrine effect on the rat heart supernatant kinase is retained at 0 degrees if cAMP is removed by Sephadex G-25 chromatography, although this procedure completely reverses the epinephrine effect in the guinea pig heart. The epinephrine effect on the rabbit heart kinase (approximately equal amounts of isozymes I and II) is partially reversed by Sephadex G-25. These species differences can be accounted for by differences in association-dissociation behavior of the isozymes in vitro. The data suggest that epinephrine causes activation of both isozymes. The activity present in the particulate fraction comprises nearly half of the total cAMP-dependent protein kinase activity in homogenates of rabbit heart. Triton X-100 extracts of low speed particulate fractions from hearts of each species tested, including rat heart, contain predominantly or entirely the type II isozyme, suggesting differences in intracellular distribution of the isozymes. The binding of the protein kinase to the particulate fraction is apparently due to the properties of the regulatory subunit component. Differences in topographical distribution of the isozymes could provide for differences in either physiological regulation or substrate specificity.     

Evidence for involvement of protein kinase in the activation by adenosine 3':5'-monophosphate of brain tyrosine 3-monooxygenase.

Addition of adenosine 3':5'-monophosphate (cAMP) to high speed supernatant preparations obtained from rat brain caused a 3- to 4-fold increase in tyrosine 3-monooxygenase (tyrosine hydroxylase) activity. The tyrosine 3-monooxygenase remained in an activated state upon removal of the cAMP by passing the enzyme through a Sephadex G-25 column. Substances which inhibit cAMP-dependent protein kinase, namely, EDTA, ADP, and adenosine, and protein kinase modulator, each antagonized the activation of tyrosine 3-monooxygenase produced by cAMP. Furthermore, addition of partially purified brain cAMP-dependent protein kinase caused a several-fold increase in tyrosin 3-monooxygenase activity. The activation of tyrosine 3-monooxygenase by added cAMP and protein kinase required the presence of ATP and Mg-2+. These data suggests that the cAMP activation of tyrosine 3-monooxygenase may be mediated by a cAMP-dependent protein kinase.     

Parietal cell protein kinases. Selective activation of type I cAMP-dependent protein kinase by histamine

cAMP-dependent protein kinases have been characterized in parietal cells isolated from rabbit gastric mucosa. Both Type I and Type II cAMP-dependent protein kinase isozymes are present in these cells. Type II isozymes were detected in 900, 14,000, and 100,000 X g particulate fractions as well as 100,000 X g cytosolic fractions; Type I isozymes were found predominately in the cytosolic fraction. When parietal cells were stimulated with histamine, an agent that elevates intracellular cAMP content and initiates parietal cell HCl secretion, cAMP-dependent protein kinase activity was increased in homogenates of these cells as measured by an increase in the cAMP-dependent protein kinase activity ratio. Histamine activation of cAMP-dependent protein kinase was correlated with parietal cell acid secretory responses which were measured indirectly as increased cellular uptake of the weak base, [14C]aminopyrine. These results suggest that cAMP-dependent protein kinase(s) is involved in the control of parietal cell HCl secretion. The parietal cell response to histamine may be compartmentalized because histamine appears to activate only a cytosolic Type I cAMP-dependent protein kinase isozyme, as determined by three different techniques including 1) ion exchange chromatography; 2) Sephadex G-25 to remove cAMP and allow rapid reassociation of the Type II but not the Type I isozyme; and 3) 8-azido-[32P]cAMP photoaffinity labeling. Forskolin, an agent that directly stimulates adenylate cyclases, was found to activate both the Type I and Type II isozymes. Several cAMP-dependent protein kinases were also detected in parietal cell homogenates, including a Ca2+-phospholipid-sensitive or C kinase and two casein kinases which were tentatively identified as casein kinase I and II. At least two additional protein kinases with a preference for serine or lysine-rich histones, respectively, were also detected. The function of these enzymes in parietal cells remains to be shown.     

Purification and characterization of human seminal plasma acid phosphatase

A 81-fold purification of human seminal plasma acid phosphatase was obtained by a three-step procedure, involving ammonium sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Homogeneity of the preparation during purification steps was tested by polyacrylamide gel electrophoresis and only one major band was obtained after the final step. The pH optimum for the activity of the purified enzyme was 5.6 and thermal stability was obtained even up to 40 degrees C. PNPP was the most specific synthetic substrate. The Km of purified seminal acid phosphatase towards PNPP was 1.5 X 10(-3) M. Among the metal ions tested, Hg+2 showed an I50 value of 4.2 X 10(-7) M. Studies with PCMB, PMSF and EDTA did not show any inhibition, whereas NaF and L(+)tartrate, at 1 mM concentration, inhibited the enzyme by 95% and 85%, respectively.     

Distribution of a seminal plasma-associated protein kinase inhibitor in normal, oligozoospermic, and vasectomized men

Human sperm-free seminal plasma contains an inhibitor, which is protein in nature, of the histone kinase present in seminal plasma. Since protein kinase inhibitors have been observed to be present in spermatozoa, the objective of the present study was to determine whether this seminal plasma-associated enzyme inhibitor originates from the sperm, or whether it is a component of accessory secretion(s) comprising the seminal plasma. Sperm-free seminal plasma from normospermic (greater than 20 X 10(6) sperm/ml), oligozoospermic (less than or equal to 20 X 10(6) sperm/ml), and vasectomized donors was obtained, and inhibitor-enriched fractions were prepared by (NH4)2SO4 fractionation and gel filtration. Contamination of the sperm-free seminal plasma by spermatozoa or spermatozoan components was negligible as assessed by light microscopy, polyacrylamide gel electrophoresis, and measurement of the activity of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase. Specific (inhibitory units/mg protein) and total inhibitory activities were determined in each of the donors by constructing linear inhibition curves using various concentrations of inhibitor. The results were correlated with the initial sperm concentration. There was no apparent relationship between the amount of inhibitory activity present and the initial sperm concentration. The histone kinase inhibitor also did not appear to be associated with testicular or epididymal secretions since it was observed in the seminal plasma of vasectomized donors. It is concluded that this inhibitor of histone kinase originates from the accessory secretions comprising the human ejaculate.     

Membrane-associated protein kinase activities in seminal plasma from vasectomized men

Differential centrifugation was used to prepare heavy and light membrane fractions from the seminal plasma of vasectomized men. The two membrane fractions combined contained half of the phosvitin and histone kinase activities but only 7% of the total protein content in vasectomy semen. These two kinase activities as well as phosphorylation of endogenous membrane proteins were optimally stimulated by Mg2+; Mn2+ could effectively substitute for Mg2+ only in endogenous phosphorylation reactions. Neither the phosvitin nor histone kinase responded to cAMP or cGMP, but the histone kinase was strongly inhibited by the heat-stable cAMP-dependent protein kinase inhibitor. The phosvitin kinase was not affected by this inhibitor. The phosphorylation of endogenous proteins in the heavy membrane fraction was not affected by the protein kinase inhibitor but protein phosphorylation in the light membrane fraction was partly (45%) inhibited. The differential effects of increased ionic strength, sulphydryl protecting agents, and the protein kinase inhibitor on protein kinase activity towards lysine-rich histones, phosvitin and endogenous proteins, as well as differential extractability and binding to an anion exchange column of histone kinase and phosvitin kinase activities, indicate that more than one kinase activity is present in these membrane subfractions. Electron microscopic examination showed that there are several kinds of membrane-limited components in vasectomy seminal fluid that vary in size, density, and ultrastructure. The association of type(s) of protein kinase to individual membrane components remains to be established.     

Proteinase inhibitors in aggregated forms of boar seminal plasma proteins

Boar seminal plasma proteins were separated by gel chromatography on Sephadex G-75 into five fractions (I–V). Serine proteinase inhibitors were found mainly in the protein fraction with relative molecular weight 5–25 kDa. Small amounts of these inhibitors were also found in the high molecular weight protein fraction (M_r>100 kDa). The protein fraction containing most of the proteinase inhibitory activity was further separated by RP HPLC. Isolated proteins were characterized by SDS electrophoresis and immunoblotting, N-terminal amino acid sequencing and by determination of the proteinase inhibitory activity. In the fraction containing proteinase inhibitors, also β-microseminoprotein (β-MSP), AQN 1 and lactoferrin were identified. The possible existence of complexes of protein components in the fraction with relative molecular weight 5–25 kDa was studied in detail using gel chromatographic separation on Sephadex G-50. A part of proteinase inhibitors with M_r 8 kDa was eluted together with AQN 1 spermadhesin. An interaction of isolated spermadhesin AQN 1 and proteinase inhibitor was shown.     

Isolation of basic acrosin inhibitor from bull seminal plasma (BUSI II).

An acrosin inhibitor was isolated from bull seminal plasma by gel filtration on Sephadex G-50 fine and ion-exchange chromatography on CM-Sephadex. The inhibitor is a basic polypeptide (pl greater than or equal to 10.5) of molecular weight 6 200 (calculated from amino acid composition). Its N-terminal amino group is blocked. The inhibitor is not strictly specific in its effect since it also inhibits trypsin and to a lesser degree chymotrypsin, in addition to bull and boar acrosin.     

Stimulation of the insulin-sensitive cAMP phosphodiesterase by an ATP-dependent soluble factor from insulin-treated rat adipocytes

The insulin-sensitive cAMP phosphodiesterase (PDE) in the microsomal fraction (Fraction P-2) from basal (-insulin) rat adipocytes was stimulated upon incubation with 2 mM ATP plus the soluble fraction from insulin-treated adipocytes (Fraction S-2+). Fraction S-2+ was prepared in the presence of p-nitrophenylphosphate, sodium vanadate, and EGTA. The ATP-dependent stimulation of PDE was routinely 60-70%. The unknown factor in Fraction S-2 was water-soluble, heat-labile, excluded by Sephadex G-50, mostly retained by Sephadex G-100, and not inhibited with 1 microgram/ml heparin, 3 mM CaCl2, or 30 mM NaF. The soluble factor may be a mediator of insulin action on PDE, possibly a protein kinase.     

Adenylyl cyclase-cAMP system inhibits thrombin-induced HSP27 in vascular smooth muscle cells

We previously reported that thrombin stimulates the induction of heat shock protein (HSP) 27 via p38 mitogen-activated protein (MAP) kinase activation in aortic smooth muscle A10 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on the thrombin-stimulated induction of HSP27 in A10 cells. Forskolin, a direct activator of adenylyl cyclase, reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the HSP27 accumulation. Furthermore, dibutyryl-cAMP (DBcAMP), a permeable analog of cAMP, significantly suppressed the accumulation of HSP27. On the other hand, calphostin C, an inhibitor of protein kinase C (PKC), reduced the thrombin-induced p38 MAP kinase phosphorylation, and significantly suppressed the thrombin-stimulated accumulation of HSP27. Moreover, forskolin reduced the p38 MAP kinase phosphorylation induced by the 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, and significantly suppressed the TPA-stimulated accumulation of HSP27. These results indicate that adenylyl cyclase-cAMP system has an inhibitory role in thrombin-stimulated HSP27 induction in aortic smooth muscle cells, and the effect seems to be exerted on the thrombin-induced PKC- p38 MAP kinase signaling pathway.     

Phosphoprotein Phosphatase of Soybean Hypocotyls: PURIFICATION, PROPERTIES, AND SUBSTRATE SPECIFICITIES

A soybean histone-type protein kinase was used to prepare ³²P-labeled histone H1 as substrate for purification and characterization of a phosphoprotein phosphatase (EC 3.1.3.16) from soybean hypocotyls. The phosphatase has been purified 169-fold by ammonium sulfate fractionation, ethanol precipitation, and chromatography on Sephadex G-150, DEAE-Sephadex A-25 and Sephadex G-100. The activity of the phosphoprotein phosphatase is distinct from that of acid and alkaline phosphatases (EC 3.1.3.1) as well as from that of nucleotidases. The final enzyme preparation does not contain histone protease activity, although it can be detected during the early stages of purification. The protease(s) apparently can attack phosphorylated histone H1, indicating that phosphorylation does not protect the protein against proteolytic degradation.     

Evidence for the identity of nuclear and cytoplasmic adenosine-3':5'-monophosphate-dependent protein kinase from porcine ovaries and nuclear translocation of the cytoplasmic enzyme.

Protein phosphokinase activity from a 0.5 M NaCl extract of purified porcine ovary nuclei has been resolved by Sephadex G-200 gel filtration into three forms of kinase, protein kinase I and III, both independent of adenosine 3':5'-monophosphate (cyclic AMP), and cyclic-AMP-dependent protein kinase II. Cyclic AMP-binding activity was associated with protein kinase II but not with protein kinases I and III. Protein kinases I, II, and III exhibited different cyclic nucleotide dependency and substrate specificity. Protein kinase II was inhibited by a heat-stable protein from rabbit skeletal muscle, whereas protein kinases I and III were not inhibited. According to previously established criteria [Traugh, J.A., Ashby, C.D. and Walsh, D.A. (1974) nuclear protein kinase II can be classified as cyclic-AMP-dependent protein kinase consisting of regulatory and catalytic subunits. Nuclear protein kinases I and III are cyclic-AMP-independent enzymes. Evidence for the identity of nuclear cyclic-AMP-dependent protein kinase II with cytosol (105 000 X g supernatant fraction) cyclic-AMP-dependent protein kinase was obtained in several ways. Nuclear and cytosol cyclic-AMP-dependent protein kinases exhibited identical elution characteristics on DEAE-cellulose and Sephadex G-200 indicating that both kinases are of similar molecular size and possess similar ionic charge. Both kinases exhibited an identical Km for ATP of 8 muM, showed similar substrate specificity, and revealed similar antigenic properties. Cyclic-AMP-dependent protein kinase II was also identified in nuclei isolated in nonaqueous media, eliminating the possibility that the cyclic-AMP-dependent protein kinase activity identified in nuclei isolated in aqueous media may have arisen as the result of cytoplasmic contamination. After incubation of neonatal porcine ovaries which lack nuclear cyclic-AMP-dependent protein kinase with 0.1 muM 8-p-chlorophenylthio cyclic AMP, considerable cyclic-AMP-dependent protein kinase II activity was identified in nuclei isolated in nonaqueous media. From these data it is concluded that the nuclear cyclic-AMP-dependent protein kinase II is related to or identical with the ovary cytoplasmic cyclic-AMP-dependent protein kinase, supporting the concept that nuclear cyclic-AMP-dependent protein kinase is of cytoplasmic origin.     

Activation of protein kinase in thyroid slices by thyroid-stimulating hormone.

Protein kinase activity in homogenates of control thyroid slices and those incubated with thyroid-stimulating hormone (TSH) and prostaglandin EI was assayed and correlated with changes in cyclic adenosine 3':5'-monophosphate (cAMP) concentrations and binding of [3H]cAMP. Both TSH and prostaglandin E1 (25 mug/ml) increased protein kinase activity and the activity ratio (expressed as activity - cAMP to activity plus cAMP). It is unlikely that such activation reflects effects of the increased cAMP liberated at the time of homogenization. Hormone-induced activation of protein kinase persisted even after the homogenate had been diluted so that its cAMP concentration would be insufficient to achieve maximal activation of the enzyme. In contrast to the previous results of J. D. Corbin, T. R. Soderling, and C. R. Park ((1973 J. Biol. Chem. 248, 1813) using adipose tissue, homogenization of thyroid tissue in 0.5 M NaCl and chromatography using Sephadex G-100 did not seem to stabilize dissociation of protein kinase into its receptor and catalytic subunits. However, increasing amounts of NaCl in the homogenizing buffer were associated with an increase in the cAMP independence of enzyme activity. Dilution of the homogenate did not change the protein kinase activity ratio whether the homogenizing buffer contained NcCl or not. Increasing concentrations of NaF inhibited protein kinase activity. Within 1 to 3 min of incubation of thyroid slices with TSH, protein kinase activity and the activity ratio were increased significantly. This correlated quite well with increased cAMP concentrations in the slices and inhibition of [3H]cAMP binding to the homogenates. Maximal activation of the enzyme was achieved by 10 min which corresponds to the time of maximal effect on cAMP concentrations. Activation of protein kinase was achieved by 0.125 milliunit/ml of TSH and maximal effects with 0.5 to 1.25 milliunits/ml. These amounts agree well with those required for other effects of TSH. Although larger amounts of TSH produced even greater increases in cAMP concentrations this was not always associated with augmented inhibition of [3H]cAMP binding. These results are compatible with the concept that the TSH-mediated increase in cAMP is associated with activation of protein kinase in the intact cell. They also suggest that not all of the intracellular cAMP is available for activation of protein kinase.     

Identification of insulin-like growth factor-II in human seminal and follicular fluids

Antisera raised in rabbits against synthetic insulin-like growth factor-II (IGF-II) were used to develop a specific radioimmunoassay (RIA) for IGF-II. Affinity purified antibodies showed 6% cross-reactivity with IGF-I but failed to recognize insulin even at 10 micrograms/tube. Utilizing this RIA system, immunoreactive IGF-II was identified in the pooled samples of human follicular fluid and seminal plasma. The acid-ethanol precipitates of human seminal and follicular fluids were chromatographed on Sephadex G-50 column and the IGF-II immunoreactive fractions were subjected to reversed-phase high performance liquid chromatography. It was found that immunoactive IGF-II was eluted in the same location as that of synthetic IGF-II. The data indicate for the first time that human seminal plasma and follicular fluid contain significant amounts of IGF-II.