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C Nakai

Publications and source records attributed to C Nakai.

At least 19 recordsLinked to original sources
PubMed1992

Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI.

The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein. The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves. Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C. Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins.

Base Sequence↗
PubMed1992

A hybrid ribonuclease H. A novel RNA cleaving enzyme with sequence-specific recognition.

A hybrid enzyme which site-specifically hydrolyzes RNA was created by covalently linking an oligodeoxyribonucleotide to Escherichia coli ribonuclease HI, an enzyme which specifically cleaves RNA moiety of DNA/RNA hybrids. A cysteine residue was substituted for Glu135 by site-directed mutagenesis in the mutant enzyme, in which all 3 free cysteine residues were replaced by alanine (Kanaya, S., Kimura, S., Katsuda, C., and Ikehara, M. (1990) Biochem. J. 271, 59-66), and coupled with a maleimide group, which is attached to the 5' terminus of the nonadeoxyribonucleotide (5'-GTCATCTCC-3') with a flexible tether. The resulting hybrid enzyme, d9-C135/RNase H, cleaved the phosphodiester bond between the fifth and sixth residues of the complementary nonaribonucleotide, without addition of the oligodeoxyribonucleotide. The nonaribonucleotide is cleaved by the wild-type or unmodified mutant enzyme only when the complementary oligodeoxyribonucleotide is present. When the kinetic parameters of the hybrid enzyme for the hydrolysis of the nonaribonucleotide were compared with those of the unmodified mutant enzyme for the hydrolysis of the nonanucleotide duplex, the hybrid enzyme exhibited a 7- and 4-fold decreases in the Km and kcat values, respectively, indicating that it performs multiple turnovers and has a sufficiently high hydrolytic activity. Hybrid ribonucleases H with various oligodeoxyribonucleotides in size and sequence, therefore, might be used as excellent tools for structural and functional studies of RNA.

Autoradiography↗
PubMed1991

COOH terminus of membrane IgM is essential for an antigen-specific induction of some but not all early activation events in mature B cells.

Transfectants of mature B cell lines that bind phosphorylcholine were made in order to understand the role of the COOH terminus of the mu chain of membrane IgM (mIgM) in generation of antigen-specific signals. A chimeric receptor (I-A alpha tail) was constructed by replacing 40 amino acids from the mu COOH terminus with that of major histocompatibility complex class II I-A alpha chain. The effect of wild-type and chimeric tails were studied on representative immediate-early antigen-specific signals. The I-A alpha tail hybrid, but not the wild-type receptor, was defective in antigen-driven Ca2+ mobilization, although it could effectively endocytose ligand-receptor complexes. Signal(s) transduced through the wild-type receptor led to transient induction of selected immediate-early gene messages (Egr-1, c-fos, Jun) above basal levels. However, the signal(s) generated after crosslinking of the I-A alpha tail receptor either showed no effect (c-fos) or actually repressed basal level expression of Egr-1 and Jun. Thus, we have established that receptor-mediated endocytosis can be distinguished from other early events associated with B cell activation, based on their differential dependence upon the structural fidelity of the COOH-terminal sequence of mIgM.

Amino Acid Sequence↗
PubMed1991

[Three cases of psittacosis].

Case 1 was a 55 year old female whose primary complaint was pyrexia. Anamnesis consisted of a fever at a 39 degrees C level beginning on September 15, 1987 with headache and muscle pain. The patient was examined by a nearby physician. Despite taking medication, there was no break in the fever. The patient was examined and admitted to this institution on September 18. Case 2 was a 32 year old female whose primary complaint was pyrexia and an abnormal sensation in the pharynx. Anamnesis consisted of a fever at a 39 degrees C level beginning on February 13, 1989 and the manifestation of a cough beginning on February 17. Although the fever broke on February 20, as the abnormal sensation in the pharynx persisted, the patient was examined and admitted to this institution on February 21. Case 3 was a 42 year old male whose primary complaint was pyrexia. Anamnesis consisted of a fever of 38 degrees C, cough and a sensation of pressure in the right precordia beginning on November 18, 1989. The patient was examined and admitted to this institution on November 21. Chest X-rays revealed the presence of atypical pneumonia in all three cases. As a significant rise in antibodies to the Chlamydia of parrot fever was observed in the cases at the time of admittance as well as after admittance, treatment was begun by administration of Minocycline.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Three isozymes of catechol 1,2-dioxygenase (pyrocatechase), alpha alpha, alpha beta, and beta beta, from Pseudomonas arvilla C-1.

Three isozymes of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas arvilla C-1 were separated using DEAE-Toyopearl chromatography. The specific activities of each isozyme were similar to one another. The molecular weights of isozymes 1, 2, and 3 were estimated to be approximately 67,000, 64,000, and 59,000, respectively, from gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isozymes 1 and 3 gave a single protein band, corresponding to Mr = 32,000 and 30,000, respectively, and isozyme 2 gave two bands corresponding to Mr = 32,000 and 30,000. These results indicated that isozymes 1 and 3 were homodimers, while isozyme 2 was a heterodimer. The NH2-terminal sequences up to 20 residues of these three isozymes confirmed that isozymes 1, 2, and 3 consisted of beta beta, alpha beta, and alpha alpha, respectively, based on our previous data (Nakai, C., Kagamiyama, H., Saeki, Y., and Nozaki, M. (1979) Arch. Biochem. Biophys. 195, 12-22). Properties of these isozymes such as absorption spectrum, iron content, substrate specificity, and kinetic constants were similar to one another. Subunit exchange between the different isozymes and dissociation of the isozymes into subunits was not observed under nondenaturing conditions. Available evidence indicates that these isozymes exist naturally in the bacterium and were not due to artifacts caused by purification.

Amino Acid Sequence↗
PubMed1989

Immunoglobulin receptor signaling depends on the carboxyl terminus but not the heavy-chain class.

To examine the isotypic and structural requirements involved in signaling through the immunoglobulin (Ig) receptor on B lymphocytes, we established a panel of T15 idiotype-positive transfectants that expressed wild-type IgM, wild-type IgD, or hybrid IgM molecules. Growth inhibition of the transfected lymphoma cells in response to anti-idiotype antibodies was used to measure signaling. Hybrid IgM molecules were constructed so that the membrane-spanning region of the mu heavy chain was replaced by that of delta, gamma 2b, or alpha heavy chains or that of the I-Ab class II (Ia) alpha chain. All transfectants that expressed IgM or hybrid IgM molecules with membrane-spanning regions from another Ig isotype underwent signaling in response to anti-idiotype antibodies, whereas the IgM-Ia hybrid transfectants did not. Transfectants that expressed wild-type IgD molecules also underwent signaling, although this response was particularly sensitive to serum concentrations. These results imply that signaling occurs in a similar manner through heavy-chain receptors of any isotype and suggest that conserved amino acid sequences in the transmembrane regions are important in this process.

Amino Acid Sequence↗
PubMed1988

Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1.

Catechol 1,2-dioxygenase (pyrocatechase) has been purified to homogeneity from Pseudomonas putida mt-2. Most properties of this enzyme, such as the absorption spectrum, iron content, pH stability, pH optimum, substrate specificity, Km values, and amino acid composition, were similar to those of catechol 1,2-dioxygenase obtained from Pseudomonas arvilla C-1 [Y. Kojima et al. (1967) J. Biol. Chem. 242, 3270-3278]. These two catechol 1,2-dioxygenases were also found, from the results of Ouchterlony double diffusion, to share several antigenic determinants. The molecular weight of the putida enzyme was estimated to be 66,000 and 64,000 by sedimentation equilibrium analysis and Sephadex G-200 gel filtration, respectively. The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to Mr 32,000. The NH2-terminal sequence, which started with threonine, was determined up to 30 residues by Edman degradation. During the degradation, a single amino acid was released at each step. The NH2-terminal sequence up to 20 residues was identical to that of the beta subunit of the arvilla enzyme, with one exception at step 16, at which arginine was observed instead of glutamine. The COOH-terminal residue was deduced to be arginine on carboxypeptidase A and B digestions and on hydrazinolysis. These results indicate that the putida enzyme consists of two identical subunits, in contrast to the arvilla enzyme which consists of two nonidentical subunits, alpha and beta [C. Nakai et al. (1979) Arch. Biochem. Biophys. 195, 12-22], although these two enzymes have very similar properties.

Amino Acid Sequence↗
PubMed1977

Protein inhibitors of phosphorylase phosphatase and cyclic AMP-dependent protein kinase from rabbit skeleta muscle.

A heat-and acid-stable protein inhibitor of phosphorylase phosphatase is present in a highly purified preparation of protein inhibitor of cyclic AMP-dependent protein kinase from rabbit skeletal muscle. Although these two inhibitors have strikingly similar properties to each other, such as sensitivity to trypsin and behavior on gel permeation chromatography, they can be separated by polyacrylamide disc gel electrophoresis. This indicates that the phosphatase-inhibitory and kinase-inhibitory activities reside with different protein species. The inhibition of both the enzymes is not altered by incubating the inhibitor preparation with a general phosphoprotein phosphatase, with phosvitin kinase, or with cyclic AMP-dependent protein kinase. Inhibition of phosphorylase phosphatase is of a non-competitive type supporting the idea that the phosphatase inhibitor is not an alternative substrate for the enzyme. Inhibition of phosphatase activity is selective in that it does no occur when phosphorylated histone or phosphorylated protamine are used as substrates.

Animals↗
PubMed1977

Inhibition of rabbit skeletal muscle phosphorylase phosphatase by spermine.

The effect of three naturally occurring polyamines (putrescine, spermidine, and spermine) on the activity of rabbit skeletal muscle phosphorylase phosphatase was investigated. Only spermine significantly inhibited the enzyme. The mode of inhibition (ki value of 0.3 mM) of the phosphatase by spermine appears to be different from that caused by divalent metal ions or by other organic cations, such as arginine and lysine esters, since it is noncompetitive with respect to the substrate, phosphorylase a.

Animals↗
PubMed1975

Effects of magnesium on the kinetic properties of bovine heart glycogen synthase D.

Highly purified glycogen synthase D, free of synthase kinase and phosphatase activities, was prepared from bovine heart. The enzyme had no activity without glucose 6-phosphate. Kinetic studies of this enzyme at various concentrations of UDP-glucose demonstrated that there was no cooperativity with respect to the substrate at any concentration of glucose-6-P with or without Mg2+. Glucose 6-phosphate increased the maximum velocity (Vmax) of the enzyme, but had very little or no effect on the Michaelis constant for UDP-glucose (Km equals 0.33 mM). Free Mg2+ gave a high Vmax at all glucose 6-phosphate concentrations without affecting the Km for the substrate. The double reciprocal plots of reaction rates versus glucose 6-phosphate concentration were biphasic and were interpreted as evidence for two kinetic forms, each with a glucose 6-phosphate binding site of different affinity (A1/2 values equals 0.31 and 1.1 mM). High Mg2+ concentrations nearly abolished the biphasic kinetic behavior of glucose 6-phosphate, suggesting that the Vmax of both enzyme forms was the same at saturating concentration of Mg2+ and glucose 6-phosphate and that magnesium ion might have no effect on the binding of glucose 6-phosphate or on the state of the equilibrium between two forms. Plots of reaction velocity versus Mg2+ concentration showed no cooperativity of Mg2+ activation in the presence or absence of glucose 6-phosphate. Both kinetic forms of glycogen synthase D had the same affinity for Mg2+ (A1/2 is approximately equal to 4 mM). Studies on the inhibition of the enzyme by Pi, ATP, and UTP were carried out with assays specific for the form of synthase with A1/2 for glucose 6-phosphate equals 0.31 mM (high affinity form) and the form with A1/2 for glucose 6-phosphate equals 1.1 mM (LOW AFFINITY FORM) BY ASSAYING WITH AND WITHOUT 5.0 MM free Mg2+, respectively. Both forms of synthase exhibited positive cooperativity with respect to UDP-glucose when inhibited by UTP, but not with Pi or ATP. Thus, each form of the enzyme had more than one UDP-glucose site, and these sites showed cooperativity only in the presence of a uridine nucleotide inhibitor. In the absence of Mg2+ (low affinity form), the inhibitors, Pi, ATP, and UTP, all induced positive cooperativity with respect to glucose 6-phosphate binding to this enzyme. The positive cooperativity induced by ATP was obliterated by adding free Mg2+ (high affinity form), but that induced by other inhibitors was affected slightly or not at all by the cation. These results indicate that each of the enzyme forms (high or low affinity forms) has more than one glucose 6-phosphate site and that these may function in a cooperative manner. The preceding findings are interpreted in relation to the importance of Mg2+ in the regulation of glycogen synthase D activity as well as the regulation of glycogen synthase phosphatase activity in heart.

Adenosine Triphosphate↗