Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.     

Immunocytochemical localization of 2,4-dienoyl-CoA reductase in the liver of normal and di-(2-ethylhexyl)phthalate-fed rats

Summary Localization of 2,4-dienoyl-CoA reductase (DCR) in rat liver was studied using immunoenzyme and immunogold techniques. The animals were fed on a laboratory diet with or without 2% di-(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for two weeks. For light microscopy (LM), semithin Epon sections were stained by immunoenzyme technique after removal of the epoxy resin. For electron microscopy (EM), ultrathin Lowicryl K4M sections were stained by the protein A-gold technique. By LM, in untreated rats reaction deposits showing the antigenic sites for DCR were present in the cytoplasmic granules. Hepatocytes, epithelial cells of interlobular bile duct, and sinus-lining cells contained these granules. After administration of DEHP, the cytoplasmic granules stained similarly. The staining intensity of the heaptocytes increased markedly, but that of the other cells decreased. The sinus-lining cells became mostly negative. By EM, gold particles indicating the antigenic sites for DCR were present in both the mitochondria and peroxisomes of hepatocytes of untreated rats. In the other cells, the gold label was confined to the mitochondria. After administration of DEHP, labelling intensity of the hepatocyte mitochondria increased markedly, but that of the peroxisomes conversely decreased. Quantitative analysis of labelling density showed that the mitochondrial DCR increased to about three times that in the untreated rat, but the peroxisomal DCR decreased to 1/6. The results show that in the rat liver, DCR exists in both, mitochondria and peroxisomes. DEHP can induce mitochondrial DCR, but not peroxisomal DCR.     

Studies on prolyl endopeptidase from shakashimeji (Lyophyllum cinerascens): purification and enzymatic properties

High prolyl endopeptidase (post-proline cleaving enzyme) [EC 3.4.21.26] activity was detected in fruit bodies of shakashimeji (Lyophyllum cinerascens), tsukuritake (mushroom: Agaricus bisporus), hirohachichitake (Lactarius hygrophoroides), and yaburebenitake (Russula lepida) which belong to the genus Basidiomycetes. Cell-free extract of shakashimeji showed high activities of proline iminopeptidase and arylamidase as well as prolyl endopeptidase. The prolyl endopeptidase was purified from the extract of shakashimeji by sequential chromatographies on DEAE-Toyopearl, DEAE-Sephadex and hydroxyapatite, and high-performance liquid chromatography with a DEAE-5PW column. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 6.8 as checked with Z-Gly-Pro-beta-naphthylamide as a substrate and was stable in the range of pH 5.8-7.4. The isoelectric point of the enzyme was 5.2 and the molecular weight was estimated to be 76,000 by gel filtration on Sephadex G-150 and by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme was a monomer. The enzyme was completely inhibited by diisopropyl fluorophosphate (DFP), Z-Gly-Pro-CH2Cl, and Z-Pro-prolinal, while it was not inhibited by p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), or metal chelators. It was estimated that at least five subsites were concerned with the enzyme-substrate binding. Among them, the S1, S2, and S1' sites showed high stereospecificity, as in mammalian, microbial, and plant enzymes. The enzyme hydrolyzed TRH at the carboxyl side of the proline residue. The mushroom enzyme, that was sensitive to DFP, Z-Pro-prolinal, and Z-Gly-Pro-CH2Cl, but not to PCMB, were quite similar in characteristics to the Flavobacterium enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inducing effects of clofibric acid on 1-acylglycerophosphorylcholine acyltransferase in kidney and intestinal mucosa of rats

Administration of p-chlorophenoxyisobutyric acid (clofibric acid) markedly increased the activity of microsomal 1-acylglycerophosphorylcholine (1-acyl-GPC) acyltransferase in kidney, intestinal mucosa and liver, but not in brain, heart, lung, spleen, testis or skeletal muscle. In both kidney and liver, a marked dose-dependent increase in the activities of both microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation was observed. In the rats treated with clofibric acid at a relatively low dose, the increase in the activity of 1-acyl-GPC acyltransferase in kidney was more marked than that in liver. The extent of the relative increase in the activity of 1-acyl-GPC acyltransferase to the activity of peroxisomal beta-oxidation in kidney was more marked than that in liver. The increased activity of 1-acyl-GPC acyltransferase in both kidney and liver lasted throughout the 8-week treatment period of rat with clofibric acid.     

Effect of polyamines on in vitro reconstitution of ribosomal subunits

The effect of polyamines on in vitro reconstitution of Escherichia coli 30S and 50S ribosomal subunits has been studied. Spermidine stimulated the reconstitution of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits at least 1.6-fold. The reconstitution of 30S particles from normal 16S rRNA and total proteins of 30S subunits exhibited only slight spermidine stimulation. However, the optimal Mg2+ concentration of the reconstitution was decreased from 20 mM to 16 mM in the presence of 3 mM spermidine. In the absence of spermidine the assembly of 30S particles from normal 16S rRNA was more rapid than the assembly from 16S rRNA lacking the methyl groups on two neighboring adenines. The reconstitution of 50S particles from 23S and 5S rRNA and total proteins of 50S subunits was not influenced greatly by spermidine. Gel electrophoresis results, from reconstitution experiments of 30S particles from 16S rRNA lacking the methyl groups on two neighboring adenines and total proteins of 30S subunits, showed that the assembly of S1 and S9 proteins to 23S core particles was stimulated by spermidine during reconstitution. The relationship of polyamine effects on in vitro ribosome assembly from its constituents to in vivo ribosome assembly is discussed. The reconstitution of Bacillus subtilis 30S particles from 16S rRNA and total proteins of 30S subunits was also stimulated approximately 1.3-fold by 3 mM spermidine.     

Thyrotropin-releasing hormone and insulin in chemically induced pancreatic islet cell tumors in rats

Thyrotropin-releasing hormone (TRH) and insulin were measured by radioimmunoassay in acetic-acid extracts of 19 pancreatic islet cell tumors induced by streptozotocin and nicotinamide in rats. In addition, gel filtration properties of TRH-immunoreactivity and immunoreactive insulin (IRI) were examined in 5 and 14 tumors, respectively. TRH was demonstrated in 10 of 19 tumors, with a mean of 166 +/- 47 (SEM) pg/mg wet weight, whereas the concentration was less than 3 pg/mg wet weight in the other tumors. In contrast, all tumors contained IRI, with a mean of 11.0 +/- 1.6 micrograms/mg wet weight. Ten tumors in which TRH was demonstrated contained more IRI than those in which TRH was not detected (13.1 +/- 1.8 vs 6.5 +/- 1.7 micrograms/mg wet weight, P less than 0.02). After gel filtration, all TRH immunoreactivity was eluted at the same place as synthetic TRH in the 5 tumors. In addition, gel filtration elutes showed essentially the same pattern of IRI in the 14 tumors, with 3 peaks. The predominant IRI peak comigrated with marker insulin (95.7 +/- 0.8%), another prominent peak occurred coincident with proinsulin standard (3.3 +/- 0.5%), a third peak was present in the void volume (0.28 +/- 0.04%). These distributions of IRI were similar to those in extracts of normal pancreases. The present studies demonstrate TRH immunoreactivity in pancreatic islet cell tumors induced by streptozotocin and nicotinamide in rats. Chemically induced insulinomas can serve as a model for insulin storage which is analogous to islet B cells.     

The complete nucleotide sequence of the group II RNA coliphage GA

The complete nucleotide sequence of the RNA coliphage GA, a group II phage, is presented. The entire genome comprises 3466 bases. Three large open reading frames were identified, which correspond to the maturation protein gene (390 amino acids), the coat protein gene (129 amino acids) and the replicase beta-subunit protein gene (531 amino acids). In addition, untranslated regions occur at the 5' (135 bases) and 3' (122 bases) ends of the molecule. Two intercistronic untranslated regions occur between the cistrons for the maturation and coat proteins, and between the coat and beta-subunit proteins. We have compared the nucleotide sequence of GA RNA with the published sequence of MS2 RNA, and show that they are related. The comparative structures of two important regulatory regions are presented; the coat protein binding site which is involved in translational repression of the replicase beta-subunit protein gene, and a hairpin in a region proximal to the lysis protein gene.     

Benzodiazepines and their metabolites: relationship between binding affinity to the benzodiazepine receptor and pharmacological activity

Experiments were carried out to study the relationship between binding affinity to the benzodiazepine receptor and pharmacological activity, especially anti-anxiety activity, of clinically useful benzodiazepines. In the in vitro experiments, fludiazepam showed the highest affinity to the benzodiazepine receptor with 4 times more potency than that of diazepam, which paralleled the in vivo activity. Diazepam and nimetazepam also bound with high affinities as expected from their in vivo activities. On the contrary, medazepam and cloxazolam showed extremely low affinities and oxazolam showed no affinity, although they showed moderate in vivo activity. However, their metabolites were found to have both high affinity and in vivo activities. These results strongly suggest that in the case of medazepam, cloxazolam and oxazolam, their metabolites may bind to receptor sites in the brain and then elicit pharmacological action. This conclusion was supported by the fact that a good correlation between the binding affinity and the anti-anxiety activity of the tested compounds was observed.     

Changes in the activity of enzymes involved in purine "salvage" and nucleic acid degradation during the growth of Catharanthus roseus cells in suspension culture

Changes during growth in the activity of several enzymes involved in purine "salvage", adenine phosphoribosyltransferase (EC 2.4.2.7), guanine phosphoribosyl-transferase (EC 2.4.2.8), hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20), the enzymes which catalyze the conversion of nucleoside monophosphate to triphosphate, nucleoside monophosphate kinase (EC 2.7.4.4) and nucleoside diphosphate kinase (EC 2.7.4.6), and several degradation enzymes, deoxyribonucleae(s), ribonuclease(s). phosphatase(s), nucleosidase (EC 3.2.2.1), 3'-nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were examined in cells of Catharanthus roseus (L.) G. Don cultured in suspension. In addition, the incorporation of [8-¹⁴C] adenine, [8-¹⁴C] adenine, [8-¹⁴C]hypoxanthine. [8-¹⁴C] adenosine and [8-¹⁴C]inosine into nucleotides and nucleic acids was also determined using intact cells.
The activities of all purine "salvage" enzymes examined and those of nucleoside monophosphate and diphosphate kinases increased rapidly during the lag phase and decreased during the following cell division and cell expansion phases. The rate of incorporation of adenine, guanine, hypoxanthine, and adenosine into nucleotides and nucleic acids was higher in the lag phase cells than during the following three phases. The highest rate of [8-¹⁴C]inosine incorporation was observed in the stationary phase cells. The activity of all degradation enzymes examined decreased when the stationary phase cells were transferred to a new medium.
These results indicated that the increased activity of purine "salvage" enzymes observed in the lag phase cells may contribute to an active purine "salvage" which is required to initiate a subsequent cell division.