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R Sakakibara

Publications and source records attributed to R Sakakibara.

At least 145 records · Page 8Linked to original sources
PubMed1987

Electrophoretic separation of proteins on agarose gel--application for direct immunization with a gel piece containing an antigen.

An efficient method of separating proteins by electrophoresis with agarose gels containing sodium dodecyl sulfate is described. The separated proteins were then used to make antibodies. The separation of proteins in the molecular weight range 25K to 94K on 4 to 5% agarose gels showed good resolution which was comparable to that obtained by electrophoresis with polyacrylamide gels containing sodium dodecyl sulfate. The subunits of human chorionic gonadotropin were separated by the preparative agarose gel electrophoresis and the gel bands containing the subunits were used directly to raise antibodies against these proteins. The degree of solubilization of the proteins from the gel slices was found to be complete.

Antibody Formation↗
PubMed1987

Subcellular localization of intracellular forms of human chorionic gonadotropin in first trimester placenta.

To determine the subcellular sites for synthesis and processing of human chorionic gonadotropin subunits in cells, first trimester placental cells were fractionated subcellularly on sucrose density gradients. Analysis of the subcellular fractions by immunobinding techniques revealed that the rough endoplasmic reticulum-rich fraction contained only intermediates having high-mannose oligosaccharides, but the Golgi-rich fraction contained not only intermediates but also mature forms which were resistant to endoglycosidase H but sensitive to neuraminidase. These results show that human chorionic gonadotropin subunits are synthesized in the rough endoplasmic reticulum as forms containing high-mannose oligosaccharides, and their maturation occurs in the Golgi apparatus by trimming with endogenous glycosidases. They are then modified by addition of complex oligosaccharides and terminal sialic acid through glycosyltransferases.

Acetylglucosaminidase↗
PubMed1986

Intracellular molecular species of human chorionic gonadotropin from normal but non-cultured first trimester placental tissues.

Intracellular forms of human chorionic gonadotropin (hCG) were analyzed by SDS-polyacrylamide gel electrophoresis, protein blotting and immunological techniques in normal but non-cultured first trimester placentae. Placental cells were found to contain the major components of the 23K and 19K forms of the beta-subunit and the 21K form of the alpha-subunit of hCG which remained sensitive to endoglycosidase H and Con A-Sepharose 4B and small amounts of mature (urinary) subunits. An unknown molecular species of the alpha-subunit (Mr = 17K) that was not bound to Con A-Sepharose 4B was also detected. These intracellular molecular species accumulated in the placentae mainly during the first trimester. These results suggest that hCG subunits accumulate in placental cells as predominant intermediates containing high-mannose oligosaccharides.

Chorionic Gonadotropin↗
PubMed1986

Molecular cloning of rat mitochondrial glutamic oxaloacetic transaminase mRNA and regulation of its expression in regenerating liver.

cDNA clones for rat mitochondrial glutamic oxaloacetic transaminase (mGOT) have been isolated from a rat liver cDNA library. One of the clones, designated p501, contained a cDNA insert of 1.4 kilobase pairs in length and hybridized to a mRNA of 2.4 kilobases from rat liver. We measured mGOT mRNA content in a regenerating rat liver. In a regenerating rat liver, mGOT activity was increased and reached maximum (170% of control activity) at about 48 h following the operation. Using the cDNA of mGOT, it was revealed that the increase of mGOT in the regenerating rat liver depended on its mRNA content.

Animals↗
PubMed1985

Studies of the structure of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase.

Some physicochemical properties of a homogeneous preparation of a bifunctional enzyme, fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase, were determined. The molecular weight of the enzyme is 101 000 as determined by high-speed sedimentation equilibrium. The molecular weight of dissociated enzyme is 55 000 in 6 M guanidinium chloride by sedimentation equilibrium and in sodium dodecyl sulfate by polyacrylamide gel electrophoresis. A value of 4.7 was observed for the isoelectric point. Tryptic peptide maps and high-performance liquid chromatography of the trypsin-digested enzyme revealed approximately 60 peptides. Amino acid analysis of the enzyme shows that it contains 27 lysine and 36 arginine residues per 55 000 daltons. No free N-terminal amino acid residue was detectable, suggesting that it is blocked. Hydrolysis of the enzyme by carboxypeptidases A and B releases tyrosine followed by histidine and arginine, indicating that the amino acid sequence at the carboxyl terminus is probably -Arg-His-Tyr. Tryptic digestion of [32P]phosphofructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase yields a 32P-labeled peptide detected by tryptic peptide mapping and high-performance liquid chromatography. Thermolysin digestion of CNBr-cleaved 32P-enzyme also yields a single 32P-peptide. These results indicate that fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase is a dimer of 55 000 daltons and the subunits are very similar, if not identical.

Amino Acids↗
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↗
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

Differences in the allosteric properties of pure low and high phosphate forms of phosphofructokinase from rat liver.

Low phosphate and high phosphate forms of phosphofructokinase (Furuya, E., and Uyeda, K. (1980) J. Biol. Chem. 255, 11656-11659) from rat liver were purified to homogeneity and various properties were compared. The specific activities of these enzymes and their electrophoretic mobilities on polyacrylamide in sodium dodecyl sulfate are the same. A limited tryptic digestion yields products with no change in the enzyme activity but with a reduction in the molecular weight of about 2000. Both low and high phosphate enzymes can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, and approximately twice as much [32P]phosphate is incorporated into the low phosphate than the high phosphate enzyme. A comparison of their allosteric kinetic properties reveal that the high phosphate enzyme is much more sensitive to inhibition by ATP and citrate and shows a higher K0.5 for fructose 6-phosphate than the low phosphate enzyme, and the difference in the K0.5 values becomes greater at lower pH values. Furthermore, the high phosphate phosphofructokinase is less sensitive to activation by AMP and fructose 2,6-bisphosphate. Moreover, when the low phosphate enzyme is phosphorylated by protein kinase, the resulting phosphorylated enzyme exhibits a higher K0.5 for fructose 2,6-bisphosphate than does the untreated enzyme. These results demonstrate that the phosphorylation affects the allosteric kinetic properties of the enzyme and results in a less active form of phosphofructokinase.

Adenosine Monophosphate↗
PubMed1983

Precursor of mitochondrial glutamic oxaloacetic transaminase isozyme exists as a dimer.

Mitochondrial glutamic oxaloacetic transaminase is synthesized in the cytoplasm as a larger precursor and then imported into mitochondria in association with its processing to mature form. The precursor corresponded to about 0.05-0.1% of the total proteins synthesized on membrane-free polysomes. Gel permeation chromatography showed that the molecular weight of the precursor was about 100,000 daltons, which was slightly larger than that of mature enzyme (homodimer: 45,000 x 2). On the other hand, sodium dodecylsulfate treated precursor had the molecular size (about 50,000 daltons) slightly larger than that of the subunit of mature one by the gel permeation chromatography. These results suggest that the precursor of mitochondrial glutamic oxaloacetic transaminase exists as a dimer.

Animals↗
PubMed1981

The complete amino acid sequence of mitochondrial glutamic oxaloacetic transaminase from rat liver.

The complete amino acid sequence of mitochondrial glutamic oxaloacetic transaminase from rat liver was determined. Sequence analyses were performed on the 12 cyanogen bromide peptides isolated after cleavage with cyanogen bromide. The amino acid sequences of all these cyanogen bromide peptides were determined by a combination of tryptic digestion, carboxypeptidase digestion and manual Edman degradation. The large peptides were hydrolyzed with trypsin after maleylation or treatment with 1,2-cyclohexanedione. These cyanogen bromide peptides were aligned by homology with the corresponding cyanogen bromide peptides from pig heart isozyme. The polypeptide chain has 401 amino acid residues and a calculated molecular weight of 44,358. Comparison showed that 24 of the 401 residues of mitochondrial glutamic oxaloacetic transaminase from rat liver are different from those of pig heart isozyme and that homology between the two isozymes is 94%.

Amino Acid Sequence↗