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S Kitajima

Publications and source records attributed to S Kitajima.

At least 163 records · Page 9Linked to original sources
PubMed1985

Pancreatic damage produced by injecting excess lysine in rats.

Intraperitoneal injection of lysine (400 mg/100 g body weight) in rats caused necrosis of pancreatic acinar cells with fat necrosis and a significant increase in serum amylase and lipase. The early morphological changes in the pancreas were investigated. At 3 to 6 h, marked swelling of mitochondria was observed throughout the cytoplasm followed later by dilation of the endoplasmic reticulum and the formation of autophagic vacuoles, indicative of rapid cellular degeneration. These results suggest that transient disturbance of energy formation following mitochondrial swelling resulted in disorders of protein metabolism, with disorganization of the endoplasmic reticulum and pyknosis of the nuclei as later events.

Amylases↗
PubMed1984

Hexose phosphate binding sites of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. Interaction with N-bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate.

N-Bromoacetylethanolamine phosphate and 3-bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate have been tested in order to study the hexose phosphate binding sites of a bifunctional enzyme, fructose-6-P,2-kinase:fructose-2,6-bisphosphatase. N-Bromoacetylethanolamine phosphate is a competitive inhibitor with respect to fructose-6-P (Ki = 0.24 mM) and a noncompetitive inhibitor with ATP (Ki = 0.8 mM). The reagent inactivates fructose-6-P,2-kinase but not fructose-2,6-bisphosphatase, and the inactivation is prevented by fructose-6-P. The inactivation reaction follows pseudo first-order kinetics to completion and with increasing concentrations of N-bromoacetylethanolamine phosphate a rate saturation effect is observed. The concentration of the reagent giving the half-maximum inactivation is 2.2 mM and the apparent first order rate constant is 0.0046 s-1. The enzyme alkylated by N-bromoacetylethanolamine-P has lost over 90% of the kinase activity, retains nearly full activity of fructose-2,6-bisphosphatase, and its inhibition by fructose-6-P is not altered. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate is also a competitive inhibitor of fructose-6-P,2-kinase with respect to fructose-6-P in the forward reaction and fructose-2,6-P2 in the reverse direction. This reagent inhibits 93% of fructose-6-P,2-kinase but activates fructose-2,6-bisphosphatase 3.7-fold. 3-Bromo-1,4-dihydroxy-2-butanone 1,4-bisphosphate alters the fructose-2,6-P2 saturation kinetic curve from negative cooperativity to normal Michaelis-Menten kinetics with K0.5 of 0.8 microM. The reagent, however, has no effect on the fructose-6-P inhibition of the phosphatase. These results strongly suggest that hexose phosphate binding sites of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase are distinct and located in different regions of this bifunctional enzyme.

Adenosine Triphosphate↗
PubMed1984

Limited proteolysis and photoaffinity labeling with 8-azido-ATP of fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase.

Limited proteolysis and photoaffinity labeling of fructose-6-P,2-kinase and fructose-2,6-bisphosphatase were studied. Proteolysis by trypsin proceeds in two stages in which the first cleavage yields a product, Mr about 53,000, which has lost 90% of fructose-6-P,2-kinase, but retains nearly 80% of fructose-2,6-bisphosphatase. Further digestion of this product yields a second cleavage product, Mr about 50,000, which is completely devoid of the kinase and most of the phosphatase activities. These results indicate that fructose-6-P,2-kinase resides only in the original ("native") enzyme (Mr = 55,000), but fructose-2,6-bisphosphatase activity is present in both the native enzyme and the cleavage product(s). All three activities of fructose-6-P,2-kinase including the forward, the reverse, and ATP-ADP exchange activities are lost to the same degree by the mild proteolysis. Ki of fructose-6-P for fructose-2,6-bisphosphatase is not altered by the proteolysis. Partial protection against the proteolysis is provided by ATP, fructose-6-P, and fructose-2,6-P2. When the tryptic digestion of fructose-6-P,2-kinase:fructose-2,6-bisphosphatase was performed before and after phosphorylation of the enzyme by cAMP-dependent protein kinase, both the first and the second cleavage products contained the phosphorylation site. 8-Azido-ATP serves as a substrate for fructose-6-P,2-kinase with a Km of about 1 mM. Exposure of the enzyme-8-azido-ATP complex results in covalent incorporation (0.7 mol/mol of subunit) and 90% inactivation of fructose-6-P,2-kinase without loss of fructose 2,6-bisphosphatase. When the native and the first cleavage product of tryptic digestion were photoaffinity labeled with [alpha-32P]8-azido-ATP, the radiolabel occurred only in the native enzyme. These results provide evidence in support of, although not conclusive, the idea that the active sites of this bifunctional enzyme are different and located in two distinct sites.

Adenosine Triphosphate↗
PubMed1984

Kinetic studies of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase.

The reactions catalyzed by a bifunctional enzyme, fructose-6-P,2-kinase and fructose 2,6- bisophosphatase , were studied. Fructose-6-P,2-kinase catalyzes the isotope exchange of ADP with ATP in the absence of fructose-6-P at about 3% of the rate of the overall reaction. The Km values for ATP and ADP for the reaction are 120 and 66 microM, respectively. No isotope exchange between fructose-6-P with fructose-2,6-P2 in the absence of adenine nucleotide was observed. The ADP-ATP exchange is not inhibited by the presence of 25 microM (1.6 X Km) fructose-6-P and less than 20% inhibition was observed at higher concentrations. Fructose-6-P,2-kinase catalyzes the reversal of the reaction at a rate approximately one-half that of the forward direction. The fructose 2,6-bisphosphatase reaction is not inhibited by 1 to 20 mM ADP. Plots of the reciprocal of the concentration of either substrate versus the reciprocal of the concentration of the other substrate yield intersecting lines for both forward and reverse reactions. The Michaelis constants for ATP and fructose-6-P are 150 and 16 microM, respectively, while those values for ADP and fructose-2,6-P2 are 62 and 8 microM, respectively. ADP is a competitive inhibitor with respect to ATP with an inhibition constant of 0.6 mM. All other product inhibition patterns including ADP versus fructose-6-P and fructose-2,6-P2 versus ATP and fructose-6-P are noncompetitive. Fructose-6-P,2-kinase also catalyzes the hydrolysis of ATP at about 10% of the rate of the overall reaction. There is no detectable burst of ADP formation upon incubating ATP with a stoichiometric amount of the enzyme. Similarly no burst of fructose-6-P formation is observed by reacting fructose-2,6-P2 with fructose 2,6-bisphosphatase. These results demonstrate that (a) the fructose-6-P,2-kinase reaction is consistent with a sequential mode of substrate addition rather than a "ping-pong" mechanism, (b) phosphorylenzyme , if formed, is not likely a kinetically important intermediate, and (c) no common phosphorylenzyme intermedite between fructose-6-P,2-kinase and fructose 2,6-bisphosphatase seems to exist.

Adenosine Diphosphate↗
PubMed1984

Differences in kinetic properties of phospho and dephospho forms of fructose-6-phosphate, 2-kinase and fructose 2,6-bisphosphatase.

Fructose-6-P,2-kinase:fructose 2,6-bisphosphatase has been purified to homogeneity. The ratio of the activities of fructose-6-P,2-kinase to fructose 2,6-bisphosphatase is 1.2. The enzyme ("native") contains 0.2 mol of phosphate/mol of subunit, and it is fully phosphorylated to 0.96 mol of phosphate/mol of subunit by cAMP-dependent protein kinase. Kinetic behavior of the native and phosphorylated forms of these enzymes was investigated. Both native and phosphofructose-6-P,2-kinase show sigmoidal kinetics with respect to fructose-6-P with an apparent K0.5 of 15 microM and 50 microM, respectively. The Hill coefficients are also increased from 1.3 to 2 by phosphorylation. The initial velocity patterns with respect to ATP follows Michaelis-Menten kinetics but the K0.5 of the phosphoenzyme (0.5 mM) is higher than that of the native enzyme (0.25 mM). The native fructose 2,6-bisphosphatase shows a biphasic saturation curve with respect to fructose-2,6-P2 which appears to be negatively cooperative. The phosphofructose 2,6-bisphosphatase, however, exhibits no cooperativity, and the apparent K0.5 for the substrate is 0.5 microM. Both forms of the phosphatase show the same Vmax. Based on these results possible allosteric regulation of fructose-6-P, 2-kinase and fructose 2,6-bisphosphatase in a reciprocal manner in vivo is discussed.

Adenosine Triphosphate↗
PubMed1984

Immunohistochemical appearance of calmodulin in the developing brain: a comparison with neuron specific enolase.

Calmodulin is a small, acidic, calcium-binding protein thought to regulate many cellular functions. In the brain of the adult mouse, calmodulin was found immunohistochemically to localize mainly in the neurons. In the developing brain, the immunoreactivity to anti-calmodulin antibody appeared early in the cells in the low brain stem but late in the cerebral cortex, hippocampus, and cerebellum, except for the deep cerebellar nuclei. The cells in the major proliferative layer present during early development, such as the matrix cells in the cerebral cortex and the cells in the external granular layer in the cerebellum, did not show the immunoreactivity. In the cerebral cortex, the migrating cells and the cells in the cortical plate were also negative while the deep cortical cells, which had probably settled in their final position, became positive. The comparison of these results with the immunohistochemical appearance of neuron specific enolase, a characteristic protein in the brain, suggested that calmodulin appeared with some maturation of the neurons as neuron specific enolase.

Animals↗
PubMed1983

Significance of phosphorylation of phosphofructokinase.

In order to understand the effect of phosphorylation on phosphofructokinase, the allosteric kinetic behavior, ligand binding at various pHs, and pH-dependent cold inactivation of phosphofructokinase phosphorylated to different extents were studied. A subtilisin-digested phosphofructokinase from which a COOH-terminal peptide containing a phosphorylation site has been cleaved (Riquelme, P. T., and Kemp, R. G. (1980) J. Biol. Chem. 255, 4367-4371) was also included in these studies in order to investigate the possible role of this region of the molecule. Allosteric kinetics and direct binding experiments have shown that increasing phosphorylation of phosphofructokinase results in increased sensitivity to ATP inhibition and stronger binding of ATP to the inhibitory site of the enzyme. Ths subtilisin-cleaved phosphofructokinase is the least sensitive to the inhibition and shows the weakest binding of ATP. The opposite effect is observed with the binding isotherms of fructose-6-P. There is no difference in the binding of fructose-2,6-P2 among these enzymes. Binding of ATP to the inhibitory site of these enzymes as determined by fluorescence quenching (Pettigrew, D. W., and Frieden, C. (1979) J. Biol. Chem. 254, 1887-1895) is affected by pH; the binding is greatly enhanced at lower pH. Moreover, there is little difference in the binding among the modified enzymes at pH 8, but at lower pHs the binding to the phosphorylated enzyme is much more enhanced than the dephosphoenzyme. A pH-dependent cold inactivation study has shown that the phosphorylation of the enzyme causes an increase in the pK value for the inactivation, and the extent of the pK shift depends upon the degree of phosphorylation. Based on these results, a model originally proposed by Frieden et al. (Frieden, C., Gilbert, H. R., and Bock, P. E. (1976) J. Biol. Chem. 251, 5644-5647) can be applied to explain a possible role for the phosphorylation and the peptide portion of phosphofructokinase in its complex allosteric kinetic behavior.

Allosteric Regulation↗
PubMed1983

A binding study of the interaction of beta-D-fructose 2,6-bisphosphate with phosphofructokinase and fructose-1,6-bisphosphatase.

The binding of beta-D-fructose 2,6-bisphosphate to rabbit muscle phosphofructokinase and rabbit liver fructose-1,6-bisphosphatase was studied using the column centrifugation procedure (Penefsky, H. S., (1977) J. Biol. Chem. 252, 2891-2899). Phosphofructokinase binds 1 mol of fructose 2,6-bisphosphate/mol of protomer (Mr = 80,000). The Scatchard plots of the binding of fructose 2,6-bisphosphate to phosphofructokinase are nonlinear in the presence of three different buffer systems and appear to exhibit negative cooperativity. Fructose 1,6-bisphosphate and glucose 1,6-bisphosphate inhibit the binding of fructose-2,6-P2 with Ki values of 15 and 280 microM, respectively. Sedoheptulose 1,7-bisphosphate, ATP, and high concentrations of phosphate also inhibit the binding. Other metabolites including fructose-6-P, AMP, and citrate show little effect. Fructose-1,6-bisphosphatase binds 1 mol of fructose 2,6-bisphosphate/mol of subunit (Mr = 35,000) with an affinity constant of 1.5 X 10(6) M-1. Fructose 1,6-bisphosphate, fructose-6-P, and phosphate are competitive inhibitors with Ki values of 4, 2.7, and 230 microM, respectively. Sedoheptulose 1,7-bisphosphate (1 mM) inhibits approximately 50% of the binding of fructose 1,6-bisphosphate to fructose bisphosphatase, but AMP has no effect. Mn2+, Co2+, and a high concentration of Mg2+ inhibit the binding. Thus, we may conclude that fructose 2,6-bisphosphate binds to phosphofructokinase at the same allosteric site for fructose 1,6-bisphosphate while it binds to the catalytic site of fructose-1,6-bisphosphatase.

Animals↗
PubMed1983

Inhibition of adenylate cyclase by a membrane protein from rat cerebrum.

The inhibitor protein of adenylate cyclase was partially purified from detergent-extract of rat brain. The inhibition occurred without lag phase. Calmodulin, GTP, guanyl 5'-yl imidodiphosphate (Gpp (NH) p) and forskolin did not change the inhibition, but activities stimulated by NaF or Mn2+ were more resistant for the inhibition. The inhibitor might include an essential sulfhydryl group.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Activation of adenylate cyclase by forskolin in rat brain and testis.

Detergent-dispersed adenylate cyclase from rat cerebrum was detected in two components, one sensitive to Ca2+ and calmodulin and another sensitive to fluoride or guanyl-5'-yl imidodiphosphate (Gpp(NH)p). The enzyme activity of both components was markedly augmented by forskolin assayed in the presence or absence of other enzyme activators (e.g., NaF, Gpp(NH)p, calmodulin). The catalytic subunit fraction in which G/F protein was totally lacking was also activated by forskolin. During 1-35 days of postnatal development, the basal adenylate cyclase activities in either cerebrum and cerebellum particulate preparations progressively increased. While the fluoride sensitivity of the cerebrum and cerebellum enzyme increased during postnatal development, the responsiveness to forskolin remained unaltered. There was no enhancement of soluble adenylate cyclase (from rat testis) by forskolin under the assay conditions in which there was a marked stimulatory action on the particulate enzyme. The results seen with the solubilized enzyme, with either Lubrol PX or cholate, indicate that the effects of forskolin on the cyclase do not require either G/F protein or calmodulin and the results of our study of brain enzymes support this view. Data on soluble testis cyclase (a poor or absent response to forskolin by this enzyme) imply that it lacks a protein (other than the catalytic unit) which could confer greater stimulation. The present results do not rule out an alternative explanation that forskolin stimulates adenylate cyclase by a direct interaction with the catalytic subunit, if the catalytic proteins do differ widely in various species of cells and their response to this diterpene.

Adenylyl Cyclases↗
PubMed1983

Immunohistochemical localization of calmodulin in mouse brain.

Calmodulin is a well-known calcium-binding protein which is ubiquitous in the plant and animal kingdoms and regulates many cellular processes. In this paper, the distribution of calmodulin in mouse brain was studied immunohistochemically using specific anti-calmodulin IgG which was raised in the rabbit by immunization with native calmodulin. Immunoreactive staining was observed in the cells in almost all areas of the brain, but its intensity varied. Some areas were stained heavily even in the presence of high concentration of NaCl. These differed from those stained immunohistochemically with antibody against other calcium-binding proteins, parvalbumin or vitamin D-dependent calcium-binding protein.

Animals↗
PubMed1983

Stimulus repetition and an amplitude increase of the occipital late positive component in the human visual evoked potential.

VEPs to number and checkerboard stimuli were measured from F3, F4, P3, P4, Cz, O1 and O2 electrode loci. A trial was composed of 8 successive stimuli: a warning stimulus (WS) and 7 task stimuli. The task stimuli were a hexad composed of 6 identical stimuli (intratrial stimulus positions 1-6) and a single stimulus (position 7). A subject's task was either naming the numbers or pattern matching the checkerboards in the hexad and in position 7. Centrofrontal N140s and parietal N180s to the number and checkerboard stimuli did not change in amplitude across stimulus positions 1-7. Parietal and centrofrontal P350s were large in amplitude at positions 1 and 7 and decreased at positions 2-6. Parietal P270s and centrofrontal P200s to the number and checkerboard stimuli behaved in the same way as the P350s with respect to the stimulus positions. Occipital N180s to the number and checkerboard stimuli were maximum in amplitude at position 1, decreased with stimulus repetition (positions 2-6) and increased at position 7. Occipital P270s to the number and checkerboard stimuli were almost the same in amplitude at position 1. The P270 to the number stimulus increased in amplitude with stimulus repetition (positions 2-6) and decreased at position 7. The P270 to the checkerboard stimulus was the same in amplitude at positions 1-3 increased at positions 4-6 and slightly decreased at position 7. These P270 amplitude changes were interpreted as due to the amplitude changes of the overlapping occipital N250 which reflected task-specific perceptual activities.

Adolescent↗
PubMed1983

Ontogeny of calmodulin and calmodulin-dependent adenylate cyclase in rat brain.

The development of calmodulin, calmodulin-dependent adenylate cyclase and beta-adrenergic receptors was studied in the rat brain. Membrane-bound calmodulin detected was approximately 40-50% of the total calmodulin throughout the postnatal development of either in the cerebrum or cerebellum. No significant difference was found between the quantitative patterns of the membrane-bound and cytosolic calmodulin during the entire period of postnatal development in either of these tissues. Both the cytosolic and membrane-bound calmodulin were present in low concentrations in the immature brain after birth. Their contents rapidly increased during the second postnatal week. Subsequently, the cytosolic calmodulin content remained constant, but showed a considerable decrease in the particulate fraction after day 14. Basal adenylate cyclase activity in the rat cerebrum slowly increased up to the second postnatal week and decreased after day 14. The responsiveness to calmodulin of this enzyme remained unaltered during postnatal development, whereas fluoride and guanine nucleotide sensitivities increased in the same period. The maximum number of (-)-[3H]dihydroalprenolol binding site sharply increased during day 9-14 in the rat cerebrum, although the dissociation constant Kd of the binding site was not affected by age. The results in the latter study suggest that calmodulin-dependent adenylate cyclase may be already present in the earlier postnatal ages of the rat brain, while the beta-adrenergic receptor and guanine nucleotide regulatory unit, both of which are required for a hormone-sensitive adenylate cyclase, may sharply increase in the second postnatal week.

Adenylyl Cyclases↗
PubMed1983

Human NADH-cytochrome b5 reductases: comparison among those of erythrocyte membrane, erythrocyte cytosol, and liver microsomes.

NADH-cytochrome b5 reductases purified from human red cell membranes and cytosol were compared with those prepared from human liver microsomes. Minimal molecular weights of the membrane and the cytosol enzymes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were 36,000 and 32,000 daltons, respectively, which are comparable to those of the detergent-solubilized reductase (dfp) and the protease-solubilized one (tfp) of liver microsomes, respectively. All the enzymes contained FAD and had essentially the same turnover numbers and apparent Km values for NADH and protease-solubilized cytochrome b5. The membrane enzyme and liver dfp reduced cytochrome c in the presence of detergent-solubilized cytochrome b5 70-80 times faster than in the presence of trypsin-solubilized cytochrome b5, whereas the cytosol enzyme and liver tfp showed essentially the same low activities with both preparations of cytochrome b5. SDS-PAGE mapping of the limited proteolytic products of the reductases obtained by digestion with staphylococcal protease or a-chymotrypsin showed essentially the same patterns of peptides between the red cell membrane enzyme and liver dfp and between the red cell cytosol enzyme and liver tfp. These results suggest that the NADH-cytochrome b5 reductase of human red cell membranes is identical with that of liver microsomes and that the enzyme of red cell cytosol is a proteolytic product of the membrane enzyme.

Cytochrome Reductases↗
PubMed1983

Production of antibodies to calmodulin in rabbits and enzyme immunoassays for calmodulin and anti-calmodulin.

The production of anti-calmodulin antibodies in rabbit was examined with the use of performic acid-oxidized calmodulin from bovine brain or an emulsion of native calmodulin and methylated bovine serum albumin as the antigen. The antibodies in rabbit sera were determined serially by means of a sensitive enzyme immunoassay method that uses a mini-column of goat (anti-rabbit IgG) IgG-coupled Sepharose 4B for separation. The antibodies could be produced in rabbits with either antigen. Although the native calmodulin with methylated bovine serum albumin seemed to be a better antigen, the titer of antiserum was not raised to levels detectable in the double immunodiffusion test. Monospecific antibodies were purified from the antiserum, and a competitive enzyme immunoassay method for the assay of calmodulin was developed with the use of the column-separation technique employed in the assay of anti-calmodulin. The method could determine from 10 ng to 10 micrograms (0.6 pmol to 0.6 nmol) of rat calmodulin and gave no cross-reaction with S-100 protein. Calmodulin contents in rat brain determined by the present method were consistent with those measured by the bioassay using calmodulin-deficient phosphodiesterase.

Animals↗
PubMed1983

Immunogenic dialyzable factor derived from a ribosomal fraction of Salmonella typhimurium. II. Isolation and characterization of the protective moiety in the dialyzable factor.

An immunogenic dialyzable factor was obtained by dialysis of the freeze-thawed ribosomal fraction derived from a smooth virulent strain (LT2) of Salmonella typhimurium. Ion exchange chromatography of the dialyzable factor on Dowex 1-X2 (Cl- form) demonstrated the presence of four peaks and the fourth peak eluted with 0.4 M NaCl in 0.005 N HCl was found to be necessary for protection. This effective peak was not obtained by chromatography of nonprotective dialyzable factors such as an RNase digest. Dowex chromatography of the dialyzable factors isolated from rough mutants of strain LT2 revealed that the dialyzable factor of strain SL1004 whose live vaccine is capable of inducing protective immunity contained fairly large amounts of peak IV. DEAE-cellulose for two-dimensional thin layer chromatography was used to identify the composition of the dialyzable factor and peak IV. Eight spots were located under ultraviolet light and seven spots were characterized by their absorption ratios. In peak IV, four nucleotides were located and identified by comparison with a map of the original dialyzable factor. The data show that the effective components of the dialyzable factor are mixed nucleotides and may be unique to ribonucleic acids of strains of S. typhimurium in which live vaccines are capable of affording mouse protection.

Bacterial Vaccines↗