Two cDNA clones for rat heme oxygenase have been isolated from a rat spleen cDNA library in kgtll by immunological screening using a specific polyclonal antibody. One of these clones has an insert of 1530 nucleotides that contains the entire protein-coding region. To confrm that the isolated cDNA encodes heme oxygenase, we transfected monkey kidney cells (COS-7) with the cDNA carried in a simian virus 40 vector. The heme oxygenase was highly expressed in endoplasmic reticulum of transfected cells. The nucleotide sequence of the cloned cDNA was determined and the primary structure of heme oxygenase was deduced. Heme oxygenase is composed of 289 amino acids and has one hydrophobic segment at its carboxyl terminus, which is probably important for the insertion of heme oxygenase into endoplasmic reticulum. The cloned cDNA was used to analyze the induction of heme oxygenase in rat liver by treatment with CoCl2 or with hemin. RNA blot analysis showed that both CoCl2 and hemin increased the amount of hybridizable mRNA, suggesting that these substances may act at the transcriptional level to increase the amount of heme oxygenase.The microsomal heme oxygenase plays an essential role in physiological heme catabolism (1, 2). Heme oxygenase catalyzes the oxidative degradation of heme to biliverdin (1), which is subsequently converted to bilirubin by biliverdin reductase (3). In the rat, the activity of heme oxygenase is highest in the spleen, where senescent erythrocytes are sequestrated and destroyed (2). Other tissues such as bone marrow and liver also perform this function, especially in hemolytic states and after splenectomy (2, 4). Heme oxygenase is highly inducible by its substrate heme in kidney (2,5), liver (2, 5), and macrophages (6-8). Heme oxygenase is also induced by various other substances such as metal ions (9, 10), endotoxin (11), and bromobenzene (12).We are particularly interested in the induction of heme oxygenase by heme, because heme (ferrous protoporphyrin IX) is an essential component of hemoglobin and of other hemoproteins. Furthermore, hemin (ferric chloride protoporphyrin IX) has interesting biological properties such as stimulation ofneurite outgrowth (13), promotion ofadipocyte differentiation (14), and stimulation of globin mRNA accumulation in erythroleukemic cells (15,16). Previously, we demonstrated that hemin increased the levels of functional mRNA for heme oxygenase in cultured pig alveolar macrophages (17) and in rat liver (18), suggesting that hemin acts at the transcriptional level to increase the amount of heme oxygenase. To understand the molecular mechanisms of induction of heme oxygenase, it is essential to know the structure of the gene for heme oxygenase.In this study, we have isolated cDNA clones for rat heme oxygenase by antibody screening, and we have confirmed that our cDNA actually encodes heme oxygenase by expressing cDNA in monkey kidney cells. We determined the nucleotide sequence of the cloned cDNA and deduced the amino acid sequence of heme oxygenase. MATERIALS AN...
Escherichia coli TUH12191, which is resistant to piperacillin, cefazolin, cefotiam, ceftizoxime, cefuzonam, and aztreonam but is susceptible to cefoxitin, latamoxef, flomoxef, and imipenem, was isolated from the urine of a patient treated with -lactam antibiotics. The -lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed -lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cephaloridine, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by -lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to -lactams, streptomycin, spectinomycin, sulfamethoxazole, and trimethoprim was transferred by conjugational transfer from E. coli TUH12191 to E. coli ML4903, and the transferred plasmid was about 58 kbp, belonging to incompatibility group M. The cefotaxime resistance gene for Toho-1 was subcloned from the 58-kbp plasmid by transformation of E. coli MV1184. KTG). Toho-1 was about 83% homologous to the -lactamase mediated by the chromosome of K. oxytoca D488 and the -lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated -lactamase Toho-1 produced by E. coli TUH12191 is similar to -lactamases produced by K. oxytoca D488, K. oxytoca E23004, and E. coli MEN-1 rather than to mutants of TEM or SHV enzymes. Toho-1 has shown the highest degree of similarity to K. oxytoca class A -lactamase. Detailed comparison of Toho-1 with other -lactamases implied that replacement of Asn-276 by Arg with the concomitant substitution of Thr for Arg-244 is an important mutation in the extension of the substrate specificity.Expanded-spectrum cephalosporins have chemical structures which confer stability to many -lactamases from gram-negative bacteria. However, many members of the family Enterobacteriaceae other than Escherichia coli developed resistance to the expanded-spectrum cephems (40). The primary mechanism of this resistance was demonstrated to be excessive production of a chromosomal -lactamase (AmpC) (23). However, bacteria that show resistance mediated by other -lactamases appeared in 1984 (8). Species of the Enterobacteriaceae such as Klebsiella pneumoniae, Klebsiella oxytoca, and E. coli acquired resistance against expanded-spectrum cephem antibiotics by producing extended-spectrum -lactamase. The extended spectrum of the -lactamase was often acquired by the variation of -lactamase genes on transmissible plasmids (43,44). Under the influence of antimicrobial agents, bacteria producing primarily TEM-type or SHV-type -lactamases developed point mutations in structural genes which served to extend the substrate specificity of the enzymes (44). These TEM-type and SHV-type -lactamases show about 65% amino acid sequence homology, with isoelectric points of 5.5 to 6.3 and 7.0 to 8.2, respectively (8, 9).
1. Thromomodulin decreased by 20-30% the Michaelis constant of two tripeptidyl p-nitroanilide substrates of thrombin. Thrombomodulin increased the rate ofinactivation of thrombin by two peptidyl chloromethane inhibitors by a similar amount. This effect appeared to be due to a decrease in the dissociation constants of the inhibitors. 2. An improved method for the separation of fibrinopeptides A and B by h.p.l.c. was developed, and this method was used to study the effect of thrombomodulin on the thrombin-catalysed cleavage of fibrinogen. In this reaction, thrombomodulin was a competitive inhibitor with respect to the Aa-chain of fibrinogen. The release of fibrinopeptide B was also inhibited by thrombomodulin. Analysis of the inhibition caused by thrombomodulin with respect to fibrinopeptides A and B yielded the same dissociation constant for the thrombin-thrombomodulin complex. 3. In the presence of thrombomodulin, the rate of inactivation of thrombin by antithrombin III was stimulated 4-fold. This stimulation showed saturation kinetics with respect to thrombomodulin. 4. Thrombomodulin was found to compete with hirudin for a binding site on thrombin. As a result of this competition, hirudin became a slow-binding inhibitor of thrombin at high thrombomodulin concentrations. 5. Estimates of the dissociation constant for thrombomodulin were obtained in several of the above experiments, and the weighted mean value was 0.7 nm.
p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.
The effects of structural properties and their changes during cellulose hydrolysis on the enzymatic hydrolysis rate have been studied from the reaction mechanism point of view. Important findings are the following: (1) The crystallinity index (CrI) of partially crystalline cellulose increases as the hydrolysis reaction proceeds, and a significant slowing down of the reaction rate during the enzymatic hydrolysis is, in large part, attributable to this structural change of cellulose substrate. (2) The crystallinity of completely disordered cellulose, like phosphoric-acid-treated cellulose, does not change significantly, and a relatively high hydrolysis rate is maintained during hydrolysis. (3) The specific surface area (SSA) of partially crystalline cellulose decreases significantly during enzymatic hydrolysis while the change in SSA of regenerated cellulose is found to be negligible. (4) The value of degree of polymerization (DP) of highly ordered crystalline cellulose remains practically constant whereas the change in DP of disordered regenerated cellulose is found to be very significant. (5) Combination of these structural effects as well as cellulase adsorption, product inhibition, and cellulase deactivation all have important influence on the rate of cellulase reaction during cellulose hydrolysis. More experimental evidence for a two-phase model, which is based on degradation of cellulose by simultaneous actions of cellulase complex on the crystalline and amorphous phases, has been obtained. Based on experimental results from this study and other results accumulated, the mode of cellulase action and a possible reaction mechanism are proposed.
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