Munehiko Yukioka

Filter-binding assay procedure for thyroid hormone receptors

An assay procedure for thyroid hormone receptor activity which used nitrocellulose membrane filters was developed. Receptor proteins, extracted from washed rat liver nuclei with a 0.4 M NaCl solution, were incubated with 125I-labeled thyroid hormone (T3), and filtered on the cellulose ester membranes under suction at 2 degrees C. The filters were subsequently washed with cold buffer and counted for 125I radioactivity. The method allowed an accurate estimation of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentrations. The usefulness of this filter-binding method became evident when it was compared with the conventional procedure that employs Sephadex G-25 columns. For practical application to routine assays, various filtration conditions were examined, and a standard procedure was established. Using this technique, the isolated receptors were determined to possess an apparent Kd of 1.38 X 10(-10) M and a pH optimum of T3 binding at 8.2-8.4.

Biosynthesis of gramicidin S by a cell-free system of bacillus brevis☆

Abstract Several Studies on the biosynthesis of gramicidin S by Bacillus brevis have been reported in recent years from different laboratories, such as those by Barry and Ichihara (1958) , Winnick et al . (1961) and Eikhom et al., 1963 , Eikhom et al., 1964 . However, most of these were concerned with experiments on intact cells of this organism. Kurahashi (1961) reported the formation of D -phenylalanyl- L -prolyl diketopiperazine as the starting peptide of gramicidin S synthesis by a cell-free system of B . Brevis Nagano, and Tomino and Kurahashi (1964) obtained D -phenylalanyl- L -prolyl- L -valine with the same system. In our laboratory, gramicidin S has been synthesized by a cell-free system of B . Brevis Nagano and part of this work has already been presented by Yukioka et al., 1963 , Yukioka et al., 1964 . This communication is on some features of the enzymatic formation of gramicidin S.

Common antigenicity of mouse 42°C-specific heat-shock protein with mouse hsp 105

Abstract Mammalian cells incubated at 42°C synthesize a specific heat-shock protein at 42°C (42°C-hsp) that is not induced by heat-shock at 45°C or by other stresses that induce major heat shock proteins (Hatayama et al. (1986) Biochem. Biophys. Res. Commun. 137, 957–963). Antibody raised against a heat-shock protein with molecular weight of 105,000 (hsp 105) purified from mouse FM 3A cells cross-reacted to the 42°C-hsp of the same cells. The antibody reacted only weakly to hsp 105 and 42°C-hsp of human HeLa cells. These results suggested that hsp 105 and 42°C-hsp have the same antigenic determinant, and that 42°C-hsp may have a structure similar to that of hsp 105.

Isolation of heterogeneous lymphocytes according to their cellular densities by multilayer centrifugation and detection of the separated fraction containing the immunological memory cells

Abstract 1. 1. The different stages of viable lymphoid cells are separated into several fractions by means of multilayer centrifugation using gum acacia solution as separating media. This method is based upon the difference in cellular density among the heterogeneous lymphocytes. 2. 2. It is discussed that our method which belongs to a discontinuous density gradient, is superior to other methods. 3. 3. Small, morphologically classified lymphocyte groups are revealed as consisting of a heterogeneous population as far as cellular densities and functions are concerned. 4. 4. Some of the small lymphocytes contained in the heavy fraction presumably have an immunological reaction to the antigen sensitized previously. 5. 5. Heavy small lymphocyte populations separated from various lymphocyte sources are endowed with important immunological activities.

Induction of mRNAs for heat shock proteins in livers of rats after ischemia and partial hepatectomy.

SummaryWhen the body temperature of rats is elevated to 42°C, four heat shock proteins, with the molecular weights of 70000, 71000, 85000, and 100000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), are induced in various tissues of rats (Fujio et al., J Biochem 101, 181–187, 1987). Heat shock proteins are induced by various stresses other than heat in varieties of cultured cells, so we studied whether heat shock proteins are induced in intact rats by different treatments. Analysis of the translation products of poly(A) + RNA isolated from the livers of rats recovering from ischemia of the liver showed that mRNAs for hsp 70, hsp 71, and hsp 85 were induced. These hsp-mRNAs were also induced in the livers of rats 6 h after a partial hepatectomy, and had returned to control levels 24 h after the surgery. These results suggested that heat shock proteins have not only the function of protection against various stresses but also physiological functions in the normal growth and development of animals.

Phosphorylation of HMG 17 by protein kinase NII from rat liver cell nuclei

Chromate contains a group of non&stone proteins known as the high mobility group (HMG) proteins. They consist of four main proteins: HMG 1, HMG 2, HMG 14 and HMG 17 [I]. Since limited DNase 1 digestion of chromatin causes a selective fragmentation of the active chromatins with a concomitant and preferential release of HMG proteins, it has been postulated that the actively transcribing chromatin regions are enriched in the HMG proteins relative to the inactive chromatins [2,3]. In fact, HMG 14 and HMG 17 were shown to confer a DNase I-sensitive structure which is characteristic of the active chromatin [4,5]. Similarly, micrococcal nuclease digestion rapidly releases HMG 1 and HMG 2 from the active chromatin, suggesting that these proteins are located in the nucleosomal linker regions of the active chromatin [3,6,7]. Changes in chromatin activity for RNA synthesis are strongly correlated with the alteration in the phosphorylation of nuclear chromosomal proteins (reviewed [S]). Therefore, it might be interesting to investigate the possibility of whether HMG proteins undergo a phosphorylation-dephosphorylation reaction during the changes in gene expression. In rat liver cell nucleus, there are two protein kinases which are not regulated by cyclic nucleotides. They have been designated NI and NII [9], and recently purified to homogeneity [ lo,1 11. The physiological substrates for each enzymes are, however, still unknown. We report here on the phosphorylation of isolated HMG proteins by partially purified nuclear protein kinase NII. In contrast to NI kinase, NII enzyme was

HeLa cells synthesize a specific heat shock protein upon exposure to heat shock at 42°C but not at 45°C

Abstract Upon exposure to heat shock, HeLa cells synthesize a small set of proteins having the molecular weights of 70,000, 73,000, 78,000, 85,000, 92,000, and 105,000. In addition to these proteins, we found an unusual heat shock protein induced by heat shock at 42°C, but not at 45°C. The 42°C-specific protein, the molecular weight of which was 90,000, was not produced in control cells and the induction of the protein was completely inhibited by actinomycin D. The protein was not induced by other treatments that induced most heat shock proteins. Thus, this 42°C-specific protein seems to have a peculiar induction mechanism and a specific function in the cells.

Occurrence of NI and NII type protein kinases in the nuclei from various tissues of the rat

Abstract Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were surveyed in various tissue nuclei of the rat. Enzymes were extracted from the isolated nuclei of liver, kidney, spleen, brain, heart, or testis tissue with a buffer solution containing 0.4 m NaCl, and analyzed by DEAE-Sephadex, phosphocellulose, and Bio-Gel A-1.5m column chromatographies. The chromatographic study together with characterization of the enzymes demonstrated that all the tissues contained in their cell nuclei commonly two protein kinases, the NI and NII types, and that these were exclusively found as main nuclear casein kinases. NII enzyme activity was stimulated by polyamines and strongly inhibited by heparin. By contrast, the NI enzymes were little influenced by these compounds. We interpret the present results as suggesting that NI and NII type protein kinases may be found in the cell nuclei from many tissues of rat, and have distinct functions in the cell nuclei.

Affinity labeling of the ribonucleic acid component adjacent to the peptidyl recognition center of peptidyl transferase in Escherichia coli ribosomes.

N-Iodacetylphenylalanyl-tRNA was used as an affinity label for localizing the RNA components intimately related to the peptidyl transferase activity of Escherichia coli ribosomesmthis analogue could specifically alkylate a unique nucleotide chain of 23-S RNA. The alkylation was strongly enhanced by poly(U), and was dependent on the presence of both 50- and 30-S subunits; Chloramphenicol inhibited the reaction, wheras blasticidin S stimulated it. The alkylated RNA base was found to be adenine. The nucleotide chain attacked by N-iodoacetylphenylalanyl-tRNA seemed to be localized at or near to the peptidyl recognition center of peptidyl transferase.

Enhancement of the phenylalanyl-oligonucleotide binding to the peptidyl recognition center of ribosomal peptidyltransferase and inhibition of the chloramphenicol binding to ribosomes

Abstract The binding of C-A-C-C-A(Phe) to ribosomes was critically affected by the concentration of ethanol and C-A-C-C-A(Phe) in the reaction mixture. In the presence of sparsomycin, the binding was inhibited at a low ethanol concentration, whereas it was stimulated at a moderately high ethanol concentration. At a moderately high concentration of ethanol, plots of the initial velocity of C-A-C-C-A(Phe) binding to ribosomes against C-A-C-C-A(Phe) concentration exhibited a sigmoidal saturation curve; while at low ethanol concentration, it gave a hyperbolic saturation curve. C-A-C-C-A(Phe) could not react with puromycin if a low level of C-A-C-C-A(Phe) and a low concentration of ethanol were included in the reaction mixture, while it did react with puromycin to form phenylalanyl-puromycin when a high level of C-A-C-C-A(Phe) and a high concentration of ethanol were used. In the presence of a moderately high concentration of ethanol, the binding of chloramphenicol to ribosomes was markedly inhibited by C-A-C-C-A(Phe) or C-A-C-C-A(Ac-Phe), while sparsomycin enhanced the inhibition. In the absence of ethanol, the phenylalanyl-oligonucleotides slightly affected the chloramphenicol binding to ribosomes. On the basis of these findings, it was assumed that C-A-C-C-A(Phe) could bind to the peptidyl recognition center as well as the aminoacyl recognition center of ribosomes, and that chloramphenicol could bind to the ribosomes at or very close to the peptidyl recognition center of the peptidyl transferase.