Modulation of Ca2+-activated, phospholipid-dependent protein kinase in platelets treated with a tumor-promoting phorbol ester

Incubation of human platelets with 12-0-tetradecanoylphorbol-13-acetate (TPA) caused a rapid decrease in soluble Ca2+, phospholipid-dependent protein kinase activity (protein kinase C) and an increase in protein kinase C associated with the particulate fraction. TPA also induced an increased activity of a Ca2+, phospholipid-independent protein kinase activity in both the soluble and the particulate fractions of platelets. This latter kinase eluted from DEAE cellulose columns at a higher salt concentration than protein kinase C, and was shown by Sephadex G-100 chromatography to have a MW of approx. 50,000 compared with an MW of 80,000 for protein kinase C. The data suggest that TPA treatment of platelets causes irreversible activation of protein kinase C by proteolysis of the enzyme to a form active in the absence of Ca2+ and phospholipid.     

Cytoplasmic localization of the transforming protein of Fujinami sarcoma virus: salt-sensitive association with subcellular components

Fujinami sarcoma virus (FSV) encodes a transforming protein of 130,000 daltons (P130) which is associated with a tyrosine-specific protein kinase activity. To elucidate mechanisms involved in cell transformation by FSV, we have studied the intracellular location of P130 in rat cells nonproductively infected with FSV. Immunofluorescent staining of several FSV-transformed rat cell lines with a tumor regressor antiserum specific against the fps sequences of P130 showed that the major staining was localized in the cytoplasm. Staining was also seen in cell ruffles and in some cases at areas of cell contact. The cytoplasmic location of P130 staining in cells infected with temperature-sensitive mutants of FSV was unchanged when they were grown at permissive or nonpermissive temperature. Cell fractionation of FSV-transformed cells under various conditions showed that the ionic strength used during cell fractionation had a striking effect on the distribution of P130. At 10 mM NaCl, 70% of P130 sedimented in the large granule fraction, whereas at 500 mM NaCl 70 to 90% of P130 was recovered in the cytosol fraction. Furthermore, a combination of ionic and nonionic detergents that effectively solubilized subcellular membranes was insufficient to solubilize P130 unless the salt concentration was raised. We conclude that the majority of P130 and its associated protein kinase activity are localized in the cytoplasm and that P130 is not an integral membrane protein.     

Separation of Assembly-Competent Tubulin from Brain Microtubule Protein Preparations Using a Fast-Performance Liquid Chromatography Procedure

Fast-performance liquid chromatography was used to purify assembly-competent tubulin from porcine brain microtubule protein prepared by two cycles of assembly-disassembly. Microtubule protein (1-100 mg at 1.5-2.5 mg/ml) in buffer consisting of 0.1 M 2-(N-morpholino)ethanesulfonic acid, 0.5 mM MgCl2, 1 mM EGTA, 0.3 M KCl, and 0.02 mM GTP (pH 6.6) was applied to the Mono Q column (anion exchanger). The microtubule-associated proteins, GTP and GDP, eluted in the void volume. The tubulin fraction eluted at 0.45-0.50 M KCl with 65-80% recovery. The tubulin fraction contained trace enzymatic activities when compared with the starting microtubule protein, i.e., less than 1 versus 60 mU/mg/min of nucleoside diphosphate kinase, 0.2 versus 7.0 nmol/mg/min of Mg-ATPase at pH 6.6, and 0.2 versus 88 mU/mg/min of adenylate kinase. Both the Mono Q-purified tubulin and the pelleted microtubules that were assembled in 0.5 mM [3H]GTP contained 0.77 mol of labeled nucleotide/tubulin dimer. The Mono Q-purified tubulin fraction was competent to assemble, i.e., the critical concentration was 0.1 mg/ml in the presence of 0.03 mM taxol and 1 mM GTP at 37 degrees C. The Mono Q-purified tubulin fraction showed trace high-molecular-weight components, which were removed on Mono S (cation exchanger) columns. Alternatively, microtubule protein in buffer was applied to the Mono S column. Tubulin, trace nontubulin proteins, and several enzymatic activities came off in the void volume. A combination of Mono Q-Mono S or Mono S-Mono Q chromatography resulted in highly purified protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of two adenosine 3':5'-phosphate-dependent protein kinase species from Chinese hamster ovary cells.

Chinese hamster ovary cells exhibit several characteristic morphological and physiological responses upon treatment with agents which increase the intracellular level of adenosine 3':5'-phosphate (cyclic AMP). To better understand the mechanism of these cyclic AMP-mediated responses, we separated two cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) (protein kinase I and protein kinase II) from the cytosol of Chinese hamster ovary cells by DEAE-cellulose chromatography and studied their properties. Protein kinase I is eluted at a lower salt concentration than protein kinase II and is stimulable to 10 times its basal catalytic activity, while protein kinase II is stimulable only 2-fold. Both kinases are completely dissociated by cyclic AMP and inhibited by specific cyclic AMP-dependent protein kinase inhibitor. They have similar Km values for magnesium (approximately 1 mM), cyclic AMP (approximately 60 nM), and ATP (approximately 0.1 mM), and the dissociation constant (Kdis) for cyclic AMP (approximately 13 nM) is the same for both enzymes. However, they appear to have different substrate preferences and cyclic AMP-binding properties in that cyclic AMP bound to protein kinase II exchanges readily with free cyclic AMP, while that bound to protein kinase I is not exchangeable. The native enzymes have different sedimentation coefficients (6.4 S for protein kinase I and 4.8 S for protein kinase II), whereas those of the activated enzymes are the same (2.9--3.0 S). It appears that the two cyclic AMP-dependent protein kinases which differ from each other in their regulatory subunits may play different roles in the mediation of cyclic AMP action in Chinese hamster ovary cells.     

Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.

Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.     

Cytosolic double-stranded RNA-dependent protein kinase is likely a dimer of partially phosphorylated Mr = 66,000 subunits.

The work described in this report suggests the existence of two biochemically distinguishable forms of the interferon-inducible, double-stranded RNA-dependent protein kinase. Kinase isolated from the cytosolic fraction (S-100) and the ribosome salt wash fraction of interferon-treated cells differed in their chromatographic properties. S-100 kinase eluted from a gel filtration column with M(r) = 140,000-160,000 and was predominantly anionic in nature, whereas ribosomal kinase eluted with M(r) = 66,000 and was predominantly cationic in nature. Purified preparations of S-100 kinase contained the M(r) = 66,000 subunit, P1, as the only polypeptide present in stoichiometric amounts, and thus the S-100 kinase appears to be a dimer of P1 subunits. Dimerization of the S-100 kinase was dependent on the phosphorylation state of the enzyme. Kinase isolated from S-100 was partially phosphorylated. Dephosphorylation of the S-100 kinase by treatment with alkaline phosphatase resulted in a monomeric form of the enzyme with biochemical characteristics similar to that of the ribosome salt wash kinase.     

Angiotensin II but not potassium induces subcellular redistribution of protein kinase C in bovine adrenal glomerulosa cells

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) between cytosol and membrane fractions was examined in bovine adrenal glomerulosa cells treated with angiotensin II or potassium. Protein kinase C was isolated from cytosol and from detergent-solubilized particulate fractions by DEAE-cellulose chromatography. A major peak of activity for both the soluble and particulate forms of adrenal glomerulosa protein kinase C was eluted at 0.05-0.09 M NaCl. The soluble and particulate forms were found to constitute about 95 and 5%, respectively, of the total enzyme activity in unstimulated cells. A second peak of kinase activity was eluted with 0.15-0.19 M NaCl, which was not dependent on the presence of phospholipids. Exposure of isolated cells for 20 min to 10(-8) M angiotensin II resulted in a decrease in cytosolic activity to 30-40% of control values, and in a corresponding increase in protein kinase C activity associated with the particulate fraction. This hormone-induced redistribution was found to be dose-dependent with an ED50 of 2 nM for angiotensin II, and it occurred rapidly, reaching a plateau within 5-10 min. It was prevented by the specific antagonist [Sar1,Ala8]angiotensin II. By contrast, stimulation with 12 mM KCl did not change the subcellular distribution of protein kinase C activity. These results suggest that redistribution of protein kinase C represents an early step in the post-receptor activation cascade following angiotensin II, but not potassium stimulation of adrenal glomerulosa cells.     

zeta-Related protein kinase C in nuclei of nerve cells

To determine whether or not PKC is present in the nuclei of nerve tissue we made use of biochemical and immunocytochemical techniques. A 219-fold purification of rabbit brain nuclear protein kinase C was achieved by sequential steps of Triton X-100 extraction of isolated nuclei, DEAE-cellulose, Butyl-toyopearl and hydroxylapatite chromatography. The major peak of protein kinase C activity was eluted from the hydroxylapatite column at the KPO4 concentration of 0.3 M. Both Ca2+ and Ptd Ser were required for stimulation of the enzyme. Immunoblot analysis revealed that the kinase fraction was immunoreactive with a polyclonal antibody, PC-zeta, that had been raised against a peptide synthesized according to the deduced sequence of rat zeta protein kinase C. Light-microscopy revealed strong immunoreactivity in the nuclei of Purkinje cells in cerebellum and pyramidal cells in the rat cerebral cortex. These observations suggest that a zeta-related protein kinase C is present in the nuclei of nerve cells.     

Increased permeability and subsequent resealing of the host cell membrane early after infection of Escherichia coli with bacteriophage T1.

The addition of T1 to cells growing at 37 degrees C in a minimal medium at 0.4 mM Mg2+ rapidly induced an irreversible loss of K+ and Mg2+ and uptake of Na+ by the cells. Both the ATP pool of the cells and the transmembrane proton motive force were reduced. These cells did not lyse from within, since viral DNA replication and the maturation of the 36,000-molecular-weight phage head protein were inhibited. By contrast, cells lysed when infected at 5.4 mM Mg2+. In these cells, T1 initially induced K+ efflux and Na+ influx and lowered the cytoplasmic ATP concentration. After a few minutes, the cation gradients and ATP pool were restored to levels close to that of control cells. At 5.4 mM Mg2+, the shutoff of host protein synthesis was delayed and coincided with the restoration of the ATP pool. In an ATP synthase-negative mutant, infection with T1 did not affect the cytoplasmic ATP concentration but inhibited host protein synthesis with the same rate as it did in wild-type cells.     

The C gamma subunit is a unique isozyme of the cAMP-dependent protein kinase.

There are at least three isozymes (C alpha, C beta, and C gamma) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the C gamma and C alpha isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to C alpha, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for C gamma. Furthermore, C gamma histone kinase activity was not inhibited by the protein kinase inhibitor peptide (5-24 amide), which has been widely used as a PKA-specific inhibitor. The major C gamma peak (type I) eluted from DEAE-Sepharose at a higher NaCl concentration (120 mM) than the C alpha type I eluted (70 mM). C gamma and C alpha type II eluted between 220 and 240 mM NaCl. C gamma required higher concentrations of cAMP than C alpha did for dissociation from the mutant type I holoenzyme. These differences provided a basis for the separation of the mutant RI-associated isozymes on DEAE-Sepharose. Both C alpha (41-42 kDa) and C gamma (39-40 kDa) were identified by a C subunit antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Zinc induced the PKA-mediated rounding phenotype in C gamma and C alpha clones, thereby restoring the cells to the parent Y1 adrenal cell phenotype. Collectively, these data indicate that C gamma is an active PKA C subunit but suggest that C gamma and C alpha have different protein and peptide recognition determinants.     

Phosphatidylinositol depletion in GH3 rat pituitary cells inhibits sustained responses to thyrotropin-releasing hormone. Reversal with myo-inositol

TRH stimulation of rat pituitary (GH3) cells causes biphasic changes in cytoplasmic free Ca2+ concentration [( Ca2+]i) and PRL secretion. It has been proposed, based primarily on indirect evidence, that the first phase effects are mediated by inositol 1,4,5-trisphosphate, which releases Ca2+ from cellular stores, and the sustained effects are mediated by 1,2-diacylglycerol, which activates protein kinase C. To determine more directly if inositol lipid hydrolysis leading to protein kinase C activation is involved in the sustained effects of TRH, GH3 cells were depleted of phosphatidylinositol (PtdIns) by prestimulation and incubation in myo-inositol-free, Li(+)-containing medium. Cells depleted of PtdIns (to 53 +/- 3.2% of control) had unchanged PtdIns 4,5-bisphosphate content, and responded to TRH with a rapid elevation of inositol trisphosphate, and a first phase (or burst) elevation of [Ca2+]i and PRL secretion that was not different from that found in control cells. In contrast, in PtdIns-depleted cells, the prolonged generation of inositol phosphates, which are produced in equimolar amounts with 1,2-diacylglycerol, caused by TRH was virtually abolished, and the second phase (or sustained) elevation of [Ca2+]i and PRL secretion were inhibited by 50% and 40%, respectively. The inhibition of both sustained effects was reversed by adding 100 mM myo-inositol to the medium, which allowed for synthesis of PtdIns. Last, in cells in which protein kinase C was down-regulated by pretreatment with a phorbol ester, the sustained effects of TRH were inhibited also.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3'':5''-cyclic monophosphate-dependent protein kinase.

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.     

Partial purification and characterization of a new phosphoprotein kinase from cells infected with pseudorabies virus

Cytoplasmic fractions from normal baby hamster kidney fibroblasts and from fibroblasts infected with pseudorabies virus were fractionated by DEAE-cellulose chromatography and fractions assayed for protein kinase activity. In preparations from uninfected and infected cells protein kinase activities identified as casein kinase I and II, the two isoforms of the cyclic-AMP-dependent protein kinase, protein kinase C, and a presumed proteolytic fragment of protein kinase C were present in comparable amounts. However in infected cells a new protein kinase activity was detected, appearing about 4 h after infection and increasing during the following 6 h at least. This new protein kinase was purified 100-fold by high-performance gel-permeation and ion-exchange chromatography, and characterized. It has an apparent relative molecular mass of 68 000 on the basis of gel-permeation chromatography, and a sedimentation coefficient of 4.3 S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent Km = 60 microM), but not GTP, as phosphoryl donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A distinguishing characteristic of the protein kinase was a high KCl concentration optimum with the persistence of activity up to 800 mM KCl, at least.     

Comparison of Enterococcus quantitative polymerase chain reaction analysis results from fresh and marine waters on two real-time instruments

Apoptin, a protein derived from chicken anemia virus (CAV), induces apoptosis selectively in human tumor cells as compared with normal cells. This activity depends on phosphorylation and relocation of apoptin to the nucleus of cancer cells. Here, we describe an in vitro kinase assay that allows the biochemical characterization of apoptin kinase activity in tumor cells. The kinase phosphorylates apoptin in a strictly ATP-dependent fashion and in a broad salt range. The kinase activity is present constitutively in both cytoplasm and nucleus of various human tumor cells. Q-column chromatography showed that both cytoplasmic and nuclear fractions have identical fractionation characteristics, suggesting that the same kinase is present in both cellular compartments. Kinase activity derived from positive Q-column fractions bound to amylose-maltose-binding protein (MBP)-apoptin and could be eluted with ATP only in the presence of the cofactor Mg(2+). Apparently, unphosphorylated apoptin interacts with the kinase and is released only after phosphorylation has occurred, proving that our assay recognizes the genuine apoptin kinase. This is further corroborated by the finding that apoptin is phosphorylated in vitro at positions Thr108 and Thr107, in concert with earlier in vivo observations. Our assay excludes cyclin-dependent kinase 2 (CDK2) and protein kinase C beta (PKC-β), previously nominated by two separate studies as being the genuine apoptin kinase.     

Distinct osmo-sensing protein kinase pathways are involved in signalling moderate and severe hyper-osmotic stress

Plant growth is severely affected by hyper-osmotic salt conditions. Although a number of salt-induced genes have been isolated, the sensing and signal transduction of salt stress is little understood. We provide evidence that alfalfa cells have two osmo-sensing protein kinase pathways that are able to distinguish between moderate and extreme hyper-osmotic conditions. A 46 kDa protein kinase was found to be activated by elevated salt concentrations (above 125 mM NaCl). In contrast, at high salt concentrations (above 750 mM NaCl), a 38 kDa protein kinase, but not the 46 kDa kinase, became activated. By biochemical and immunological analysis, the 46 kDa kinase was identified as SIMK, a member of the family of MAPKs (mitogen-activated protein kinases). SIMK is not only activated by NaCl, but also by KCl and sorbitol, indicating that the SIMK pathway is involved in mediating general hyper-osmotic conditions. Salt stress induces rapid but transient activation of SIMK, showing maximal activity between 8 and 16 min before slow inactivation. When inactive, most mammalian and yeast MAPKs are cytoplasmic but undergo nuclear transloca- tion upon activation. By contrast, SIMK was found to be a constitutively nuclear protein and the activity of the kinase was not correlated with changes in its intra-cellular compartmentation, suggesting an intra-nuclear mechanism for the regulation of SIMK activity.     

Different phosphorylated forms of an insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes

Labeling with [3H]galactose was employed to isolate a glycosylphosphatidylinositol from rat hepatocytes which might be involved in the action of insulin. The polar head group of this glycosylphosphatidylinositol was generated by phosphodiesterase hydrolysis with a phosphatidylinositol-specific phospholipase C from Bacillus cereus. By Dowex AG1 x 8 chromatography the polar head group could be separated into three radioactive peaks eluting at 100 mM (peak I), 200 mM (peak II) and 500 mM (peak III) ammonium formate, respectively. Peak III was the most active as an inhibitor of the cAMP-dependent protein kinase. Treatment of peak III with alkaline phosphatase markedly reduced its activity on cAMP-dependent protein kinase. When peaks I, II or III were treated with alkaline phosphatase and analyzed again by Dowex AG1 x 8 chromatography, the radioactivity eluted with the aqueous fraction. The above results indicate that the polar head group of the insulin-sensitive glycosylphosphatidylinositol from rat hepatocytes exists in three different phosphorylated forms and that the biological activity of this molecule depends on its phosphorylation state.     

Activation of the double-stranded RNA-dependent eIF-2 alpha kinase by cellular RNA from 3T3-F442A cells.

The interferon induced double-stranded-RNA-dependent eIF-2 alpha kinase has an established role in mediating part of interferons anti-viral effects. Several studies have suggested that it may have additional functions in cells not infected with virus. The mechanism of activation of the kinase and the consequences of its activity in uninfected cells remain to be determined. Our previous results have indicated that the activation (phosphorylation) of this kinase may be an important regulatory signal to the arrest of growth of mouse 3T3-F442A fibroblasts and their subsequent differentiation to adipocytes. We have found that the phosphorylation of the kinase occurred in vivo in the absence of viral infection and in vitro without the addition of dsRNA. We demonstrate here that total cytoplasmic RNA from 3T3-F442A cells contains a regulatory RNA(s) capable of activating dsRNA-dependent eIF-2 alpha kinase. Fractionation of the cytoplasmic RNA by oligo(dT)-cellulose indicated that the regulatory RNA eluted with the poly(A)-rich RNA fraction. It bound tightly to the dsRNA-dependent eIF-2 alpha kinase and was immune-precipitated with its antibodies as a complex of regulatory RNA and dsRNA-dependent eIF-2 alpha kinase. The regulatory RNA activity was further purified by phenol extraction of immune precipitates containing this complex. These findings indicated that the regulatory RNA forms a specific complex with the dsRNA-dependent eIF-2 alpha kinase. The activity of the regulatory RNA was sensitive to the dsRNA-specific RNase VI but not to proteinase K, DNase I or ssRNA-specific RNase T1. The activation of the dsRNA-dependent eIF-2 alpha kinase by regulatory RNA was prevented by addition of a high concentration of poly(I).poly(C). The regulatory RNA was also shown to activate partially purified dsRNA-dependent eIF-2 alpha kinase prepared from rabbit reticulocyte lysates and to inhibit protein synthesis in reticulocyte lysates. Our findings, that cellular RNAs can specifically activate the dsRNA-dependent eIF-2 alpha kinase, are consistent with a physiological role for the dsRNA-dependent eIF-2 alpha kinase and interferon during cell growth and differentiation. The relationship of the regulatory RNA activity to growth and differentiation of 3T3-F442A cells is discussed.     

Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.     

Extracellular calcium increases free cytoplasmic calcium and DNA synthesis in human osteoblasts.

A high concentration of extracellular calcium (8 mM) induced an increase in free cytoplasmic calcium, a lower cyclic AMP level and increased DNA synthesis in primary cultures of human osteoblast-like cells. Inhibition of protein kinase C with bisindolylmaleimide I inhibited the stimulatory effect of extracellular calcium on DNA synthesis in human osteoblast-like cells, whereas inhibition of protein kinase A with Rp-cAMPs had no effect on DNA synthesis. This indicates that protein kinase C, possibly via increased free cytoplasmic calcium, mediates the effect of extracellular calcium on DNA synthesis in osteoblast-like cells rather than a relative decrease in cyclic AMP and protein kinase A activity. Furthermore, a low concentration (0.5 mM) of extracellular calcium decreased DNA synthesis. In conclusion, these data suggest that a high extracellular calcium level may be a coupling factor that recruits osteoblasts in the bone remodeling process, and that a low level of extracellular calcium may also regulate osteoblast function.     

Adsorption of DNA to sand and variable degradation rates of adsorbed DNA

Adsorption and desorption of DNA and degradation of adsorbed DNA by DNase I were studied by using a flowthrough system of sand-filled glass columns. Maximum adsorption at 23 degrees C occurred within 2 h. The amounts of DNA which adsorbed to sand increased with the salt concentration (0.1 to 4 M NaCl and 1 mM to 0.2 M MgCl2), salt valency (Na+ less than Mg2+ and Ca2+), and pH (5 to 9). Maximum desorption of DNA from sand (43 to 59%) was achieved when columns were eluted with NaPO4 and NaCl for 6 h or with EDTA for 1 h. DNA did not desorb in the presence of detergents. It is concluded that adsorption proceeded by physical and chemical (Mg2+ bridging) interaction between the DNA and sand surfaces. Degradability by DNase I decreased upon adsorption of transforming DNA. When DNA adsorbed in the presence of 50 mM MgCl2, the degradation rate was higher than when it adsorbed in the presence of 20 mM MgCl2. The sensitivity to degradation of DNA adsorbed to sand at 50 mM MgCl2 decreased when the columns were eluted with 0.1 mM MgCl2 or 100 mM EDTA before application of DNase I. This indicates that at least two types of DNA-sand complexes with different accessibilities of adsorbed DNA to DNase I existed. The degradability of DNA adsorbed to minor mineral fractions (feldspar and heavy minerals) of the sand differed from that of quartz-adsorbed DNA.