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Biomedical subjects

J F Kuo

Publications and source records attributed to J F Kuo.

At least 109 records · Page 6Linked to original sources
PubMed1983

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide(W-7), a calmodulin antagonist, also inhibits phospholipid-sensitive calcium-dependent protein kinase.

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide(W-7), commonly regarded as a calmodulin antagonist, inhibited phospholipid-sensitive Ca2+ -dependent protein kinase and to a lesser extent cyclic GMP- and cyclic AMP-dependent protein kinases. Kinetic studies of the inhibition of the homogeneous spleen phospholipid-sensitive Ca2+ -dependent protein kinase indicated that W-7 inhibited the enzyme activity competitively with respect to phospholipid (Ki = 60 microM). N-(6-Aminohexyl)-1-naphthalenesulfonamide (W-5) was found to be much less potent than W-7. The findings indicate that W-7 was able to inhibit a variety of protein kinases, in addition to those requiring calmodulin previously reported.

Animals↗
PubMed1983

Phosphorylation of cardiac troponin inhibitory subunit (troponin I) and tropomyosin-binding subunit (troponin T) by cardiac phospholipid-sensitive Ca2+-dependent protein kinase.

Cardiac phospholipid-sensitive Ca2+-dependent protein kinase phosphorylated cardiac troponin inhibitory subunit (troponin I) and tropomyosin-binding subunit (troponin T), present either as the free form or as the troponin-tropomyosin complex. Exhaustive phosphorylation of troponin I and of troponin T revealed that 1.7 and 2 mol of phosphate was incorporated/mol of the subunits respectively. Cyclic AMP-dependent protein kinase, though incorporating 0.8 mol of phosphate/mol of troponin I, was unable to phosphorylate troponin T. Phosphorylation of troponin I (apparent Km = 3.4 microM; Vmax. = 2.6 mumol/min per mg of enzyme) or troponin T (apparent Km = 0.3 microM; Vmax. = 0.5 mumol/min per mg of enzyme) by the Ca2+-dependent enzyme was inhibited by various agents, such as adriamycin, palmitoylcarnitine, trifluoperazine, melittin and N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide (compound W-7). Ca2+ antagonists (such as verapamil), forskolin and ouabain were ineffective. These findings indicate that troponin I and troponin T were effective substrates for this species of Ca2+-dependent protein kinase, suggesting its potential regulatory role in the contractile activity of myofibrils modulated by troponin.

Animals↗
PubMed1983

beta-Adrenoceptor-adenosine 3',5-monophosphate system in human leucocytes before and after treatment for hyperthyroidism.

Modifications in characteristics of the beta-adrenoceptor-cAMP system were observed in leucocytes from 10 patients in the hyperthyroid state after antithyroid treatment. These include 1) an increased number of beta-adrenoceptors without a change in their affinity, 2) an increased magnitude of stimulation of adenylate cyclase by isoprenaline, without a change in the NaF-stimulated enzyme activity, 3) an increased cAMP-dependent protein kinase activity ratio, and 4) an increased activity of cAMP-phosphodiesterase. Moreover, the plasma cAMP levels were markedly elevated during the hyperthyroid state. It is suggested that the above changes may in part constitute the molecular basis for the reputed catecholamine supersensitivity in the hyperthyroid state.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Phospholipid-sensitive Ca2+-dependent protein phosphorylation system in various types of leukemic cells from human patients and in human leukemic cell lines HL60 and K562, and its inhibition by alkyl-lysophospholipid.

Phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK), its endogenous substrate proteins, and regulation of the enzyme system by an antitumor agent alkyl-lysophospholipid were investigated in various types of leukemic cells (chronic myelocytic, acute myelocytic, and acute monocytic) from patients and in two cultured human leukemic cell lines (HL60 and K562). Exceedingly high levels of PL-Ca-PK, largely localized in the particulate fraction, were found in all types and lines of leukemic cells; much lower levels of cyclic adenosine 3':5'-monophosphate-dependent protein kinase and cyclic guanosine 3':5'-monophosphate-dependent protein kinase were also found. Although numerous and similar endogenous substrates for PL-Ca-PK were found in all cell types and lines examined, substrates specific for certain leukemic cells appeared to be present. Substrate proteins for calmodulin-sensitive Ca2+-dependent protein kinase, cyclic adenosine 3':5'-monophosphate-dependent protein kinase, and cyclic guanosine 3':5'-monophosphate-dependent protein kinase, in comparison, were much fewer or undetected. The PL-Ca-PK activity and the phosphatidylserine-Ca2+-stimulated phosphorylation of endogenous proteins from leukemic cells were inhibited by alkyl-lysophospholipid, which acted as a competitive inhibitor of the phospholipid cofactor of the enzyme. The findings suggested that the PL-Ca-PK system is a predominant protein phosphorylation system in leukemic cells and that this enzyme system may represent a site of cytotoxic action of alkyl-lysophospholipid.

Calcium↗
PubMed1982

A homogeneous cyclic CMP phosphodiesterase hydrolyzes both pyrimidine and purine cyclic 2':3'- and 3':5'-nucleotides.

A homogeneous preparation of cyclic CMP phosphodiesterase (Helfman, D. M., Shoji, M., and Kuo, J. F. (1981) J. Biol. Chem. 256, 6327-6334) was found to catalyze the hydrolysis of both pyrimidine and purine cyclic 2':3'- and 3':5'-nucleotides. Hydrolysis of cyclic 2':3'-nucleotides resulted in the formation of both 2'- and 3'-nucleotides, although relative amounts of the products were variable. Hydrolysis of cyclic 2':3'-CMP or cyclic 2':3'-UMP yielded predominantly 3'-nucleotides. In contrast, hydrolysis of cyclic 2':3'-AMP produced equal amounts of 2'- and 3'-nucleotides, while the major product formed from cyclic 2':3'-GMP was 2'-nucleotide. When conventional pyrimidine and purine cyclic 3:5'-nucleotides were used as substrates, the enzyme hydrolyzed specifically the 3'-bond to yield only 5'-nucleotides. The relative rate of hydrolysis of cyclic 2':3'-nucleotides was cyclic CMP greater than cyclic UMP greater than cyclic GMP greater than or equal to cyclic AMP, respectively, whereas that for cyclic 3':5'-nucleotides was cyclic CMP greater than cyclic UMP greater than or equal to cyclic AMP greater than cyclic GMP, respectively. Furthermore, kinetic analysis suggested a single species of catalytic site on the enzyme may be involved in the hydrolysis of both pyrimidine and purine cyclic 2':3'- and 3':5'-nucleotides. These findings indicate that the present enzyme is the first multifunctional phosphodiesterase reported to date that is capable of hydrolyzing such a diversity of cyclic nucleotides.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1982

Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases.

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.

Adenosine Triphosphate↗
PubMed1982

Differential regulation of calcium-dependent and calcium-independent cyclic nucleotide phosphodiesterases from heart by palmitoylcarnitine and palmitoyl coenzyme A.

Regulation of Ca2+-dependent (peak I) and Ca2+-independent (peak II) phosphodiesterases from the heart by various fatty acyl esters and phospholipids were studied. DL-Palmitoylcarnitine stimulated the basal activity (in the absence of Ca2+) of peak I enzyme, while non-competitively inhibiting peak II enzyme with respect to cyclic AMP. It had no effect on other species of Ca2+-independent phosphodiesterases, including cyclic AMP- and cyclic GMP-specific enzymes from the lung, and cyclic CMP enzyme from the liver Palmitoyl-CoA and phosphatidylserine also stimulated the basal activity of peak I enzyme, but they were without effect on peak II enzyme. In comparison, DL-palmitoylcarnitine inhibited Ca2+-dependent activity of cardiac myosin light chain kinase, whereas phosphatidylserine was without effect. It is conceivable that differential regulation of phosphodiesterases by these lipids could profoundly alter the levels or effects, or both, of cyclic nucleotides and Ca2+ in the myocardium.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Differential effects of various phosphodiesterase inhibitors, pyrimidine and purine compounds, and inorganic phosphates on cyclic CMP, cyclic AMP and cyclic GMP phosphodiesterases.

The effects of various compounds on homogeneous cyclic CMP phosphodiesterase (cyclic CMP-PDE) from pig liver were compared with the effects on cyclic AMP phosphodiesterase (cyclic AMP-PDE) and cyclic GMP phosphodiesterase (cyclic GMP-PDE). Of the conventional inhibitors for AMP-PDE and cyclic GMP-PDE, only Sch 15280 was found to inhibit cyclic CMP-PDE. Nucleoside monophosphates, orthophosphate, and 2':3'-cyclic nucleotides were rather specific and were more effective in inhibiting cyclic CMP-PDE, compared to their effects on cyclic AMP-PDE and cyclic GMP-PDE. On the other hand, nucleoside di-and triphosphates and pyrophosphate (PPi) were less effective in inhibiting cyclic CMP-PDE and were without marked effect on cyclic AMP-PDE and cyclic GMP-PDE. Orthophosphate (Pi) was more potent than CMP, CDP and CTP in inhibiting cyclic CMP-PDE, with a rank order of inhibitory potency of Pi greater than CMP greater than CDP greater than CTP. Of the 3' :5'-cyclic nucleotides examined, cyclic UMP was more specific in inhibiting cyclic CMP-PDE compared to its effect on cyclic AMP-PDE and cyclic GMP-PDE. In all experiments similar results were obtained when either cyclic CMP or cyclic AMP was used as a substrate for this multifunctional cyclic CMP-PDE, supporting the contention that a single catalytic site on the enzyme is responsible for the hydrolysis of both cyclic CMP and cyclic AMP. The present studies further support our original suggestion that cyclic CMP-PDE is a unique enzyme that is distinguishable from the conventional enzymes for purine cyclic nucleotides.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1982

Basic protein in brain myelin is phosphorylated by endogenous phospholipid-sensitive Ca2+-dependent protein kinase.

Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca2+, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 mM EGTA, with or without further fractionation, showed that Ca2+-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca2+ and phosphatidylserine; a protein kinase stimulated by Ca2+ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca2+-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca2+-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7).

Animals↗
PubMed1981

Stimulation by phospholipid of calcium-dependent phosphorylation of endogenous proteins from mammalian tissues.

The occurrence of endogenous substrate proteins for calcium-dependent protein kinase, augmented by either phospholipid or calmodulin, was examined in extracts of several rat tissues. Pancreas, vas deferens, adrenal and liver were found to contain substrate proteins for phospholipid-sensitive protein kinase. Under the conditions utilized, only vas deferens exhibited substrate proteins for calmodulin-sensitive protein kinase. Phosphorylation of pancreatic substrate protein for phospholipid-sensitive protein kinase was rapid and highly sensitive to Ca2+, being detectable within 15 s following exposure to Ca2+ and phosphatidylserine and at concentrations of Ca2+ as low as 0.5 muM. These findings suggest that phospholipid-sensitive protein kinase system may serve to mediate some effects of Ca2+ in a variety of mammalian cell types.

Adrenal Glands↗
PubMed1981

Inhibition by adriamycin of calmodulin-sensitive and phospholipid-sensitive calcium-dependent phosphorylation of endogenous proteins from heart.

Adriamycin, a lipid-interacting anti-cancer agent, was found to inhibit phospholipid-sensitive Ca2+-dependent phosphorylation of endogenous proteins from the cytosol of the guinea-pig heart. The drug, unexpectedly, also inhibited phosphorylation of separate endogenous proteins in the cardiac cytosol and membranes catalysed by the calmodulin-sensitive species of Ca2+-dependent protein kinase. In both phosphorylation systems, the inhibition by adriamycin was reversed by either phospholipid (phosphatidylserine or cardiolipin) or calmodulin respectively. Adriamycin also inhibited phosphorylation of histone (exogenous protein) catalysed by purified cardiac phospholipid-sensitive Ca2+-dependent protein kinase, but not that by cyclic AMP-dependent and cyclic GMP-dependent protein kinases. It appears that Ca2+-dependent protein phosphorylation systems, regulated either by phospholipid or calmodulin, may represent hitherto unrecognized sites of action of adriamycin. It remains to be seen whether inhibition by adriamycin of these systems is related to the severe cardiotoxicity, the major adverse effect of the drug that limits its clinical usefulness.

Animals↗