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Biomedical subjects

J F Kuo

Publications and source records attributed to J F Kuo.

At least 91 records · Page 5Linked to original sources
PubMed1985

Polyclonal antibodies to phospholipid/Ca2+-dependent protein kinase and immunocytochemical localization of the enzyme in rat brain.

Antisera against phospholipid/Ca2+-dependent protein kinase (protein kinase C) were raised in rabbits. Immunospecificity of the polyclonal antibodies, as determined by immunoblot and ELISA, was shown by their reactivity to the enzyme but not to other protein kinases or any of many other proteins tested. Immunocytochemical localization of the kinase in rat brains revealed that although the enzyme was distributed broadly in different brain regions, it was highly restricted to the periphery of the nucleus of neurons in cerebral cortex and to axons and cells strongly resembling oligodendroglia in white-matter regions. Initial electron microscopy of cerebral cortex revealed that the enzyme was highly concentrated in the presynaptic terminals, and only rarely were labeled postsynaptic specialization elements seen. It is suggested that the discrete localization of the enzyme, which is distinct from that of the calmodulin/Ca2+-dependent system, may be related to certain biological and functional aspects of brain that are regulated by Ca2+ at the level of protein phosphorylation.

Animals↗
PubMed1985

Enzyme immunoassay for detection of Neisseria gonorrhoeae antigens.

An enzyme immunoassay (EIA; Gonozyme, Abbott Laboratories) for the detection of Neisseria gonorrhoeae antigens was assessed. Clinical isolates of N. gonorrhoeae or specimens obtained from male urethra or female cervix were tested by EIA. EIA results were compared with results of conventional culture identification for N. gonorrhoeae. Fifty clinical isolates of gonococci reacted positively with Gonozyme. Five strains of E. coli showed negative reaction to Gonozyme. Five strains of nonpathogenic Neisseria reacted differently with Gonozyme depending upon the viable number of the test organisms. The sensitivity and specificity of EIA for specimens form both sex were 72.4% and 93.8%, respectively. The corresponding figures for the male specimens were 84.6% and 71.4%, and for the female specimens 62.5% and 95%, respectively.

Antigens, Bacterial↗
PubMed1984

Environmental pollutant Cd2+ biphasically and differentially regulates myosin light chain kinase and phospholipid/Ca2+-dependent protein kinase.

Cd2+ was found to mimic effectively, potentiate and antagonize the stimulatory action of Ca2+ on myosin light chain kinase (MLCK) and phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK, or protein kinase C). PL-Ca-PK, however, was slightly less sensitive to Cd2+ regulation than was MLCK. Cd2+ also biphasically regulates (i.e., stimulation followed by inhibition) phosphorylation, in the homogenates of the rat caudal artery, of myosin light chain and other endogenous proteins catalyzed by MLCK and PL-Ca-PK. The activation by Cd2+ of MLCK was inhibited by anticalmodulins (e.g., R-24571), whereas the inhibition by a higher Cd2+ concentration of MLCK and PL-Ca-PK was reversed by thiol agents (e.g., cysteine). The present findings may provide one mechanism underlying the vascular toxicity of Cd2+, a major environmental pollutant.

Animals↗
PubMed1984

Phosphorylation of skeletal-muscle troponin I and troponin T by phospholipid-sensitive Ca2+-dependent protein kinase and its inhibition by troponin C and tropomyosin.

Skeletal-muscle troponin I and troponin T were found to be rapidly phosphorylated by cardiac phospholipid-sensitive Ca2+-dependent protein kinase, with Km values of 6.66 and 0.13 microM respectively. Stoichiometric phosphorylation of skeletal troponin I (endogenous phosphate content 0.7 mol/mol) indicated that the Ca2+-dependent enzyme and cyclic AMP-dependent protein kinase incorporated 0.9 and 0.8 mol/mol respectively. The same experiments with skeletal troponin T (endogenous phosphate content 1.9 mol/mol) revealed a maximal phosphorylation of 2 mol/mol by the Ca2+-dependent enzyme, whereas the cyclic AMP-dependent enzyme was unable to phosphorylate troponin T. The Ca2+-dependent enzyme phosphorylated both serine and threonine residues in skeletal and cardiac troponin I or troponin T; the cyclic AMP-dependent enzyme, in comparison, phosphorylated only serine in skeletal and cardiac troponin I. Although an equimolar amount of skeletal or cardiac troponin C markedly inhibited (80-90%) phosphorylation of skeletal and cardiac troponin I by the Ca2+-dependent enzyme, these troponin C preparations inhibited only phosphorylation of skeletal troponin I, but not that of cardiac troponin I, by the cyclic AMP-dependent enzyme. Calmodulin and Ca2+-binding protein S-100a could mimic the inhibitory effect of troponin C. A tissue specificity appeared to exist for the skeletal troponin T-skeletal troponin C interaction. Inhibition of troponin T phosphorylation by an equimolar amount of troponin C was lower than that of troponin I phosphorylation; these findings might explain in part why troponin T was the major substrate for the Ca2+-dependent enzyme in the troponin complex. The present studies indicate that skeletal and cardiac troponin I and troponin T were effective substrates for phospholipid-sensitive Ca2+-dependent protein kinase, suggesting a potential involvement of this Ca2+-effector enzyme in the regulation of myofibrillar activity.

Animals↗
PubMed1984

Phospholipid-sensitive Ca2+-dependent protein kinase inhibition by R-24571, a calmodulin antagonist.

R-24571 (calmidazolium), a derivative of the antimycotic agent miconazole, inhibited phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK), with an IC50 (the concentration causing 50% inhibition) of 5.3 microM. It also inhibited the calmodulin/Ca2+-stimulated enzymes, with IC50 values of 1.6 and 0.1 microM for myosin light chain kinase (MLCK) and phosphodiesterase respectively. Analysis of inhibition by R-24571 of PL-Ca-PK and MLCK revealed complex kinetics, suggesting that the agent interacted with the cofactors, the enzyme, and/or the cofactor-enzyme complexes. At saturating concentrations of the cofactors, R-24571 inhibited PL-Ca-PK and MLCK noncompetitively with their respective cofactors. Inhibition of MLCK by R-24571 was completely overcome by phosphatidylserine, indicating a strong hydrophobic interaction between R-24571 and the phospholipid in the presence of calmodulin. R-24571 also inhibited phosphorylation of various endogenous proteins in brain stimulated specifically by phosphatidylserine/Ca2+ or calmodulin/Ca2+. The present findings inducated that R-24571 has little specificity in inhibiting two types of Ca2+-dependent protein kinases sensitive to phospholipid or calmodulin.

Animals↗
PubMed1984

Developmental studies of phospholipid-sensitive Ca2+-dependent protein kinase and its substrates and of phosphoprotein phosphatases in rat brain.

Ontogenetic changes in the protein phosphorylation/dephosphorylation systems in rat brain were investigated. It was found that the activity level of phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) in the particulate fraction of grey and white matter and the soluble fraction of grey matter increased rapidly and markedly after birth, reached the highest level at day 30, and declined slightly or remained unchanged thereafter. The enzyme level in the soluble fraction of white matter, in contrast, remained constant throughout the development and maturation of brain. Various ontogenetic changes in the substrate proteins for PL-Ca-PK were also noted. The levels of myelin basic protein and other substrates (notably the Mr 87,000, 58,000, 54,000, and 50,000 protein in grey matter) progressively increased during development, reaching the highest level at adulthood. The level of the Mr 66,000 protein from the particulate fraction of white and grey matter, on the other hand, increased rapidly after birth, reached a peak at day 18, and then declined to the initial neonatal level at the adult stage. The time scale for the increases in the levels of PL-Ca-PK and its many substrates paralleled that of brain development and maturation (synaptogenesis and myelinogenesis). The activity levels of phosphoprotein phosphatases (assayed using 32P-labeled myelin basic protein, histone, and protamine sulfate) were found to only slightly (up to 60%) increase or decrease in certain fractions from different brain regions during development, suggesting that phosphorylation, compared to dephosphorylation, may be more important in determining the phosphorylation state of cellular proteins.

Aging↗
PubMed1984

S-100 and other acidic proteins promote Ca2+-independent phosphorylation of protamine catalyzed by a new protein kinase from brain.

A new protein kinase modulated by S-100 (tentatively referred to as protein kinase X) was partially purified from pig brain extracts. The activity of protein kinase X, which was independent of Ca2+, was demonstrated when protamine (free base), but not protamine sulfate and other proteins (including histone), was used as substrate. The enzyme activity, found to distribute in both soluble and particulate fractions and to occur at the highest level in brain compared with other tissues (heart, kidney, liver, skeletal muscle, spleen, and testis) of rats, was also modulated by other acidic proteins (calmodulin, troponin C, and stimulatory modulator) in a Ca2+ -independent manner. S-100 and other acidic proteins appeared to function as "substrate modifiers" by interacting with protamine (a highly basic protein), but not with the enzyme, thus rendering protamine in the complex a superior phosphate acceptor. The two isoforms of S-100 (i.e., a and b) were equally effective. Although the enzyme was not inhibited by many agents (trifluoperazine, melittin, cytotoxin I, polymyxin B, and spermine) shown to inhibit markedly phospholipid/Ca2+- or calmodulin/Ca2+ -stimulated protein kinase, gossypol was found to inhibit specifically protein kinase X. The present findings suggest that S-100, a major acidic protein specific to nervous system, may promote phosphorylation by protein kinase X of certain neural proteins resembling protamine or containing protamine-like domains, in addition to its presumed role of a low-affinity Ca2+ -binding protein.

Animals↗
PubMed1984

Phospholipid-sensitive Ca2+ -dependent protein kinase preferentially phosphorylates serine-115 of bovine myelin basic protein.

Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.

Amino Acid Sequence↗
PubMed1984

S-100 modulates Ca2+-independent phosphorylation of an endogenous protein (Mr = 19K) in brain.

A new brain enzyme (tentatively named protein kinase X), which catalyzes protamine phosphorylation modulated by S-100, was reported recently. An endogenous substrate protein (Mr = 19K) for protein kinase X was isolated from brain by means of S-100-Sepharose 4B affinity chromatography. S-100, but not calmodulin, promoted phosphorylation of the 19K Mr protein in a Ca2+-independent manner, and this reaction was inhibited by gossypol. The substrate protein, localized in the particulate fraction, was present at a much higher level in brain from adult than neonatal rats (2-day-old), a developmental change similar to that seen for protein kinase X. It is suggested that a protein phosphorylation system modulated by S-100 exists in brain, and that this process may be involved in regulation of certain neural functions.

Animals↗
PubMed1983

Phospholipid-sensitive Ca2+-dependent protein kinase phosphorylates the beta subunit of eukaryotic initiation factor 2 (eIF-2).

The ability of homogeneous phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) from pig spleen to phosphorylate eukaryotic initiation factor 2 (eIF-2) was examined. PL-Ca-PK phosphorylated the beta-subunit of eIF-2, whereas myosin light chain kinase (MLCK) and cyclic AMP- and cyclic GMP-dependent protein kinases (cA-PK and cG-PK) did not. PL-Ca-PK could incorporate a maximum of 1.6 mol phosphate/mol eIF-2. The app. Km and Vmax for PL-Ca-PK phosphorylation of eIF-2 were 0.13 microM and 0.02 mumol.min-1.mg enzyme-1, respectively. Phosphoamino acid analysis revealed that incorporation of phosphate into eIF-2 occurred almost exclusively at serine residues. These findings indicate that eIF-2 was an effective substrate for PL-Ca-PK, suggesting that this enzyme may play a role in the regulation of protein synthesis.

Amino Acids↗
PubMed1983

Polyamines inhibit phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases.

Effects of polyamines on various protein kinases were investigated. It was found that both phospholipid-sensitive Ca2+-dependent protein kinase and myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) were inhibited to different degrees by polyamines, with an approximate order of inhibitory potency of spermine = 1, 12-diaminododecane greater than spermidine = 1, 10-diaminodecane much greater than cadaverine = putrescine. Kinetic analysis revealed that spermine inhibited the phospholipid-sensitive enzyme non-competitively with respect to Ca2+ (Ki = 0.84 mM) and phosphatidylserine (Ki = 0.90 mM); it also inhibited myosin light-chain kinase non-competitively with respect to Ca2+ (Ki = 1.82 mM) and calmodulin (Ki = 2.73 mM). 1, 12-Diaminododecane, in comparison, inhibited the phospholipid-sensitive enzyme competitively with respect to Ca2+ (Ki = 0.45 mM) and phosphatidylserine (Ki = 0.50 mM); it also inhibited myosin light-chain kinase competitively with respect to calmodulin (Ki = 0.63 mM) but non-competitively with respect to Ca2+ (Ki = 1.49 mM). Moreover, spermine (0.5 mM) was found to inhibit markedly phosphatidylserine/Ca2+- and calmodulin/Ca2+-stimulated phosphorylation of endogenous proteins in rat brain particulate fraction. All the polyamines tested were practically without effect on cyclic AMP-dependent and cyclic GMP-dependent protein kinases. Polyarginine, like spermine, was found to be a more selective inhibitor of Ca2+-dependent protein kinases, whereas polyglutamate preferentially inhibited the cyclic nucleotide-dependent enzymes. The present results indicated that, in addition to certain lipophilic compounds (such as trifluoperazine, palmitoylcarnitine, adriamycin and naphthalenesulphonamide) and polypeptides with hydrophobic regions (such as melittin and polymyxin B) previously reported, polycationic compounds (exemplified by polyamines) could also inhibit the two classes of Ca2+-dependent protein kinases requiring either phospholipid or calmodulin as a cofactor. Because of the high cellular concentration (up to 10 mM) and the differential effects of polyamines, it is suggested that spermine, and to smaller extents spermidine and putrescine, may be involved in the regulation of certain Ca2+-dependent protein-phosphorylation systems in vivo.

Animals↗
PubMed1983

Comparative abilities of lanthanide ions La3+ and Tb3+ to substitute for Ca2+ in regulating phospholipid-sensitive Ca2+-dependent protein kinase and myosin light chain kinase.

Although lanthanide ions La3+ and Tb3+ were only slightly able to substitute for Ca2+ to activate phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK), they potentiated the ability of a suboptimal concentration of Ca2+ to stimulate the enzyme. In comparison, the lanthanides were more effective Ca2+ substitutes for myosin light chain kinase, a calmodulin-sensitive Ca2+-dependent protein kinase. Both enzymes, however, were inhibited by high concentrations of lanthanides either in the presence or absence of Ca2+. Similar effects of the lanthanides were also noted on phosphorylation of endogenous substrates in the particulate fraction of rat brain stimulated by either phosphatidylserine/Ca2+ or calmodulin/Ca2+. The La3+- or Tb3+-stimulated activity of PL-Ca-PK, as the Ca2+-stimulated activity, was inhibited by various agents, such as trifluoperazine, polymyxin B, cobra cytotoxin I, melittin, and spermine.

Animals↗
PubMed1983

Modes of inhibition by acylcarnitines, adriamycin and trifluoperazine of cardiac phospholipid-sensitive calcium-dependent protein kinase.

Palmitoylcarnitine, adriamycin, and trifluoperazine competively inhibited, with respect to phosphatidylserine (a phospholipid cofactor), purified cardiac phospholipid-sensitive Ca2+-dependent protein kinase, with apparent Ki values of 3, 49 and 14 microM respectively. These compounds also inhibited the enzyme competitively with respect to Ca2+ (a metal activator), with corresponding apparent Ki values of 0.8, 140 and 9 microM. A synergistic inhibition was observed when palmitoylcarnitine and trifluoperazine were present in combination. A simple addition inhibition on the other hand, was observed for the combination of either palmitoylcarnitine and adriamycin, or trifluoperazine and adriamycin. 1,3-Diolein decreased the inhibitory effect of trifluoperazine by increasing the affinity of the enzyme for phosphatidylserine. The results indicate that the recently identified phospholipid-sensitive species of Ca2+-dependent protein kinase was inhibited by a variety of agents, probably via their abilities to interfere with a hydrophobic interaction between phospholipid and the enzyme, an interaction presumably required to confer upon the enzyme a Ca2+ sensitivity. Because other long-chain fatty acylcarnitines (stearoyl- and linoleoylcarnitine), short-chain fatty acylcarnitines (such as octanoylcarnitine) and palmitoyl CoA, compared to palmitoylcarnitine, were less active as inhibitors, it is further suggested that lipophilicity as well as other structural determinants are crucial for the ability of compounds to regulate the enzyme activity.

Animals↗
PubMed1983

Phospholipid-sensitive Ca2+-dependent protein kinase and its substrates in human neutrophils.

Phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) was found to be present at a high level in human neutrophils, with its activity localized in the particulate fraction. In contrast, cyclic AMP-dependent protein kinase (A-PK) and cyclic GMP-dependent protein kinase (G-PK), present at lower levels compared to PL-Ca-PK, were localized in the cytosolic fraction. Phosphorylation of several endogenous proteins (mol. wts. 89,000, 38,000, 34,000, 17,000 and 15,000), also localized in the particulate fraction, was stimulated specifically by a combination of phosphatidylserine and Ca2+, whereas no substrate proteins were observed for the calmodulin-sensitive Ca2+-dependent protein kinase system under the same incubation conditions. Although no substrate proteins for G-PK were detected, one substrate (mol. wt. 19,000) for A-PK was observed. Phosphorylation of substrates for PL-Ca-PK, but not that for A-PK and for enzymes independent of Ca2+ or cyclic AMP, was inhibited by a variety of agents, including trifluoperazine, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide], adriamycin, palmitoylcarnitine, and melittin. The present findings suggest that the phospholipid/Ca2+-stimulated protein phosphorylation system may be important in the membrane associated functions of human neutrophils.

Adult↗
PubMed1983

Cobra polypeptide cytotoxin I and marine worm polypeptide cytotoxin A-IV are potent and selective inhibitors of phospholipid-sensitive Ca2+-dependent protein kinase.

The effects of a number of polypeptide cytotoxins and neurotoxins on various protein kinases were examined. It was found that cobra cytotoxin I and marine worm cytotoxin A-IV effectively and specifically inhibited phospholipid-sensitive Ca2+-dependent protein kinase (PL-Ca-PK) relative to myosin light chain kinase and cyclic nucleotide-dependent protein kinases. Inhibition of PL-Ca-PK by these cytotoxins could be overcome by phosphatidylserine. Neurotoxins, in comparison, were much less effective inhibitors. The present findings indicated that these polypeptide cytotoxins, unlike other agents reported to date, were selective inhibitors of PL-Ca-PK and could be used to differentiate Ca2+-dependent events regulated by phospholipid or calmodulin.

Bee Venoms↗
PubMed1983

Purification to homogeneity, characterization and monoclonal antibodies of phospholipid-sensitive Ca2+-dependent protein kinase from spleen.

A phospholipid-sensitive Ca2+-dependent protein kinase was purified to homogeneity, for the first time, from extracts of pig spleen, employing the steps of DEAE-cellulose, octyl-agarose, Sephacryl S-200 and phosphatidylserine-Affigel 10 affinity chromatographies. The purified enzyme appeared as a single protein band on both analytical (non-denaturing) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, having a minimum mol.wt. of 68 000 +/- 200. The molecular weight of the enzyme was also determined to be 74 500 +/- 4600 by gel filtration and 80 000 based on its sedimentation coefficient (5.52 S) and Stokes radius (3.52 +/- 0.09 nm), indicating that the enzyme was a monomeric protein. The frictional ratio (f/f0) of the enzyme was 1.24, indicating it was non-globular in shape. The enzyme had a pI of 5.3, and a pH optimum of 6.5 for its reaction. Amino acid analysis indicated that the enzyme apparently was not similar to myosin light-chain kinase (a calmodulin-sensitive species of Ca2+-dependent protein kinase) or cyclic AMP-dependent and cyclic GMP-dependent protein kinases. The enzyme had an apparent Km for ATP of 7.5 microns. Histone H1 and myelin basic protein were effective substrates for the enzyme, with apparent Km values of 0.3 and 0.2 microns, and Vmax, values of 0.06 and 0.09 mumol/min per mg of enzyme respectively. The enzyme activity was dependent on both phosphatidylserine (apparent Ka = 6.25 micrograms/ml) and Ca2+ (apparent Ka = 160 microns). Calmodulin was unable to substitute for the phospholipid as a cofactor, nor was it a subunit of the enzyme. Sr2+ and Ba2+ could partially mimic Ca2+ to activate the enzyme in the presence of phosphatidylserine. An endogenous substrate protein (mol.wt. 41 000) for the enzyme was found in the total, solubilized fraction of pig spleen. Monoclonal antibodies against the enzyme interacted similarly with the homogeneous and impure enzyme; the antibodies, however, did not bind to cyclic nucleotide-dependent protein kinases.

Amino Acids↗