Effects of polyamines on the activities of Escherichia coli ribonuclease I and II.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by E. coli ribonuclease I [ribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.23] and ribonuclease II [EC 3.1.4.1] have been studied. The degradation of poly(C) by RNase II was stimulated by spermine and spermidine, while that of poly(A) by RNase II was not affected by polyamines. Under our standard experimental conditions, the breakdown of poly(U) by RNase II was inhibited slightly by polyamines. The stimulatory effect of spermine and spermidine on the breakdown of poly(C) occurred in the absence of monovalent cations but not in the absence of divalent cations. When polyamines were used as a stimulant of RNase II, the ratio of poly(C) degradation to poly(U) degradation was greater in the presence of inhibitors such as poly(G) than in their absence. Although the breakdown of all synthetic polynucleotides by RNase I was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C)greater than poly(A)(see text)poly(U). However, the difference in degree of stimulation among polynucleotides decreased as monovalent cation concentration was increased.     

Studies on the restoration of the activities of Ribonucleases by polyamines in the presence of various ribonuclease inhibitors.

The effect of polyamines on ribonucleases in the presence of various inhibitors (poly(G), heparin, and rat liver RNase inhibitor) has been studied. Bovine pancreatic RNas A and a ribonuclease from horse submaxillary gland (RNase HS) were inhibited by the inhibitors, but RNase T1 and RNase M were not inhibited. Polyamines were found to restore the activites of RNase A and RNase HS inhibited by poly(G) or heparin but not those activities inhibited by rat liver RNase inhibitor. When poly(U) and poly(C) were used as substrates, the inhibitory effects of poly(G) and heparin were greater with poly(U) than poly(C) as a substrate. However, when poly(C) was used as a substrate in the presence of either of the above inhibitors, the restoration of RNase activity by sperimine was more efficient. In fact, a stimulatory effect was observed. From the double-reciprocal plots, it was concluded that polyamines restored the activiities of RNases by increasing the availability of the substrate and enzyme to each other. The restoration of enzyme activity by polyamines occurred through the binding of the polyamines to the inhibitor and the subsequent release of enzyme from the inhibitor.     

Coordination chemical studies on metalloenzymes. II. Kinetic behavior of various types of chelating agents towards bovine carbonic anhydrase.

In order to investigate the kinetics and mechanism of the removal of zinc ions from bovine carbonic anhydrase [EC 4.2.1.1] (BCA), several chelating agents with various stability constants were used to remove zinc from BCA. The second-order rate constants (kaap) of zinc removal from BCA were found to be in the following order; 2,6-pyridinedicarboxylic acid greater than 2-pyridinecarboxylic acid greater than 2,4-pyridinedicarboxylic acid greater than 2,3-pyridinedicarboxylic acid greater than or approximately 1,10-phenanthroline greater than or approximately 5-methyl-1,10-phenanthroline greater than 2,2'-bipyridine. With similar chelating agents the greater the stability constant, the faster was the rate of removal of zinc ions from BCA. With EDTA, trans-1,2-cyclohexanediaminetetraacetic acid, and nitrilotriacetic acid, the rate of zinc ion removal from the native enzyme was governed by the rate of spontaneous dissociation of zinc enzyme. The rate constants for the removal of zinc ions from BCA were governed by the affinity of the chelating agents for the metal ion and the conformation of the chelating agents. Based on these findings, reaction pathways for various chelating agents are proposed.     

Distribution of Sorbitol, Neutral Sugars, Free Amino Acids, Malic Acid and Some Hydrolytic Enzymes in Vacuoles of Apple Cotyledons

Vacuoles of apple cotyledons (Malus pumila Mill. var. domesticaSchneid.) were obtained by purification with Ficoll densitygradient centrifugation after the protoplasts were lysed byboth osmotic shock and the addition of EDTA. High levels ofacid protease and carboxypeptidase activity were detected inthe vacuoles along with acid phosphatase, phosphodiesteraseand ATPase. The distribution of sorbitol and other sugars inthe vacuoles, the protoplast and extracellular free space wasdetermined. About 45, 60 and 90% of the sorbitol, glucose andsucrose, respectively, contained in whole tissue were foundin the extracellular free space, and 54% of the sorbitol inthe protoplast was detected in the vacuole. The sorbitol inthe vacuoles and the extravacuole of the protoplast was about80 and 70%, respectively, of the total sugar content of thecell. Arginine was the most abundant free amino acid in theprotoplast, and about 90% of it was contained in the vacuole.More than 80% of the total amino acids and 50% of the proteinwere also located in the vacuole, as well as most of the malate.The amounts of the total sugars, total amino acids, proteinand malate in the vacuoles were to 250, 400, 48 and 9 µg/10⁶cells, respectively. The results suggest that the vacuoles ofapple cotyledons contain a large pool of amino acids and proteinsrather than sugars, and have a close connection with proteinbody degradation. ¹ This paper is contribution A-l37 of the Fruit Tree ResearchStation. (Received January 20, 1982; Accepted May 18, 1982)     

Role of an intrachain disulfide bond in the conformation and stability of ovalbumin

Ovalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the non-reactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6.8 degrees C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation.     

Identification of lysyl residues at the AMP-binding site of biodegradative threonine deaminase from Escherichia coli.

The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP. To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2-PL). In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110 microM. The former value is comparable to the Kd value of the enzyme for AMP. In the presence of AMP, the Schiff base formation was greatly reduced. Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same. These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme. To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I. Reverse-phase HPLC was used to isolate two labeled peptides from the digest. Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled. We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme.     

Suppression by IL-2 of IgE production by B cells stimulated by IL-4.

IgE production was obtained from B cells of BALB/c or nude mice when these cells were cultured with IL-4 plus LPS. IL-2 added to these cultures at the start (day 0), 1 or 2 days later completely suppressed the production of IgE. The production of IgG1 was also inhibited, but only if IL-2 was added on day 0. The production of other isotypes (IgM, IgG2a, IgG2b) was only slightly decreased by addition of IL-2. No suppression of IgE or IgG1 production was observed if monoclonal anti-IL-2 was added, whereas anti-IFN-gamma had no effect on the suppression of the production of these isotypes. The expression of CD23 on the third day of culture on B cells stimulated with LPS and IL-4 was markedly decreased when IL-2 was added to the cultures on day 0. Addition of monoclonal anti-IL-2 suppressed all effects produced by IL-2, whereas addition of anti-IFN-gamma had no effect. These results show that the suppression by IL-2, at least for the first signaling processes, are different from the suppression produced by IFN-gamma.     

Inhibition of cholesterol absorption and synthesis in rats by sesamin

The effects of sesamin, a lignan from sesame oil, on various aspects of cholesterol metabolism were examined in rats maintained on various dietary regimens. When given at a dietary level of 0.5% for 4 weeks, sesamin reduced the concentration of serum and liver cholesterol significantly irrespective of the presence or absence of cholesterol in the diet, except for one experiment in which the purified diet free of cholesterol was given. On feeding sesamin, there was a decrease in lymphatic absorption of cholesterol accompanying an increase in fecal excretion of neutral, but not acidic, steroids, particularly when the cholesterol-enriched diet was given. Sesamin inhibited micellar solubility of cholesterol, but not bile acids, whereas it neither bound taurocholate nor affected the absorption of fatty acids. Only a marginal proportion (ca. 0.15%) of sesamin administered intragastrically was recovered in the lymph. There was a significant reduction in the activity of liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase after feeding sesamin, although the activity of hepatic cholesterol 7 alpha-hydroxylase, drug metabolizing enzymes, and alcohol dehydrogenase remained uninfluenced. Although the weight and phospholipid concentration of the liver increased unequivocally on feeding sesamin, the histological examination by microscopy showed no abnormality, and the activity of serum GOT and GPT remained unchanged. Since sesamin lowered both serum and liver cholesterol levels by inhibiting absorption and synthesis of cholesterol simultaneously, it deserves further study as a possible hypocholesterolemic agent of natural origin.     

Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase.

A cDNA clone encoding transglutaminase was isolated from a bovine-endothelial-cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial-cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea-pig liver, human and rat keratinocyte transglutaminases, and the human blood-coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern-blot analysis of mRNA from retinoid-treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels.     

Photoreversible antigen-antibody reactions

A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K = 5 x 10(7) M-1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light.