Cloning and gene expression of Schistosoma mansoni protease

Schistosomes utilize proteases (termed hemoglobinases) for degradation of host globin. cDNA clones encoding Schistosoma mansoni protease were isolated by immunologically screening an expression cDNA library with antisera raised against purified hemoglobinase. Confirmation of the identities of the clones was obtained immunologically and biochemically. The bacterially produced fusion protein encoded by one clone, lambda Hb2, degraded hemoglobin in vitro. The sequence of this clone suggested that this S. mansoni protease is synthesized in a precursor form in vivo. Gene titrations indicated that S. mansoni contains multiple genes corresponding to this cDNA. The expression of these genes may be regulated during the organism's life cycle since adult, female worms contained the highest abundances of homologous mRNA and protein compared to other stages.     

Enzymatic acetylation of 10-deacetylbaccatin III to baccatin III by C-10 deacetylase from Nocardioides luteus SC 13913

Baccatin III is a polycyclic diterpene which can be used for the semi-synthesis of paclitaxel and analogs. An enzymatic process was developed for the conversion of 10-deacetylbaccatin III (10-DAB) to baccatin III without requiring protection of the 7-hydroxyl group. A C-10 deacetylase from Nocardioides luteus SC 13912 was immobilized on diethylaminoethyl cellulose (DEAE-Cellulose) and the immobilized enzyme was used in the biotransformation process. The reaction was catalyzed using vinyl acetate as acyl donor at ambient temperature and at pH 7. A reaction yield of 51% was obtained.     

Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N(2)-

[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine epsilon-aminotransferase. This enzyme catalyzed the oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required alpha-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine epsilon-amino transferase and glutamate oxidase, respectively. L-lysine epsilon-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine epsilon-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine epsilon-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine epsilon-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65-70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine epsilon-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine epsilon-aminotransferase. N-alpha-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine epsilon-aminotransferase.     

Acetylcholine release from the carotid body by hypoxia: evidence for the involvement of autoinhibitory receptors.

The purpose of the present study was to investigate whether hypoxia influences acetylcholine (ACh) release from the rabbit carotid body and, if so, to determine the mechanism(s) associated with this response. ACh is expressed in the rabbit carotid body (5.6 +/- 1.3 pmol/carotid body) as evidenced by electrochemical analysis. Immunocytochemical analysis of the primary cultures of the carotid body with antibody specific to ACh further showed that ACh-like immunoreactivity is localized to many glomus cells. The effect of hypoxia on ACh release was examined in ex vivo carotid bodies harvested from anesthetized rabbits. The basal release of ACh during normoxia ( approximately 150 Torr) averaged 5.9 +/- 0.5 fmol.min-1.carotid body-1. Lowering the Po2 to 90 and 20 Torr progressively decreased ACh release by approximately 15 and approximately 68%, respectively. ACh release returned to the basal value on reoxygenation. Simultaneous monitoring of dopamine showed a sixfold increase in dopamine release during hypoxia. Hypercapnia (21% O2 + 10% CO2) as well as high K+ (100 mM) facilitated ACh release from the carotid body, suggesting that hypoxia-induced inhibition of ACh release is not due to deterioration of the carotid body. Hypoxia had no significant effect on acetylcholinesterase activity in the medium, implying that increased hydrolysis of ACh does not account for hypoxia-induced inhibition of ACh release. In the presence of either atropine (10 microM) or domperidone (10 microM), hypoxia stimulated ACh release. These results demonstrate that glomus cells of the rabbit carotid body express ACh and that hypoxia overall inhibits ACh release via activation of muscarinic and dopaminergic autoinhibitory receptors in the carotid body.     

Crystal structure of a murine glutathione S-transferase in complex with a glutathione conjugate of 4-hydroxynon-2-enal in one subunit and glutathione in the other: evidence of signaling across the dimer interface.

mGSTA4-4, a murine glutathione S-transferase (GST) exhibiting high activity in conjugating the lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) with glutathione (GSH), was crystallized in complex with the GSH conjugate of 4-HNE (GS-Hna). The structure has been solved at 2.6 A resolution, which reveals that the active site of one subunit of the dimeric enzyme binds GS-Hna, whereas the other binds GSH. A marked asymmetry between the two subunits is evident. Most noticeable are the differences in the conformation of arginine residues 69 and 15. In all GST structures published previously, the guanidino groups of R69 residues from both subunits stack at the dimer interface and are related by a (pseudo-) 2-fold axis. In the present structure of mGSTA4-4, however, the two R69 side chains point in opposite directions, although their guanidino groups remain in contact. In the subunit with bound GSH, R69 also interacts with R15, and the guanidino group of R15 points away from the active site, whereas in the subunit that binds GS-Hna, R15 pivots into the active site, which breaks its interaction with R69. According to our previous results [Nanduri et al. (1997) Arch. Biochem. Biophys. 335, 305-310], the availability of R15 in the active site assists the conjugation of 4-HNE with GSH. We propose a model for the catalytic mechanism of mGSTA4-4 in conjugating 4-HNE with GSH-i.e., the guanidino group of R15 is available in the active site of only one subunit at any given time and the stacked pair of R69 residues act as a switch that couples the concerted movement of the two R15 side chains. The alternate occupancy of 4-HNE in the two subunits has been confirmed by our kinetic analysis that shows the negative cooperativity of mGSTA4-4 for 4-HNE. Disruption of the signaling between the subunits by mutating the R69 residues released the negative cooperativity with 4-HNE.     

Peripheral chemoreceptors in health and disease.

Peripheral chemoreceptors (carotid and aortic bodies) detect changes in arterial blood oxygen and initiate reflexes that are important for maintaining homeostasis during hypoxemia. This mini-review summarizes the importance of peripheral chemoreceptor reflexes in various physiological and pathophysiological conditions. Carotid bodies are important for eliciting hypoxic ventilatory stimulation in humans and in experimental animals. In the absence of carotid bodies, compensatory upregulation of aortic bodies as well as other chemoreceptors contributes to the hypoxic ventilatory response. Peripheral chemoreceptors are critical for ventilatory acclimatization at high altitude. They also contribute in part to the exercise-induced hyperventilation, especially with submaximal and heavy exercise. During pregnancy, hypoxic ventilatory sensitivity increases, perhaps due to the actions of estrogen and progesterone on chemoreceptors. Augmented peripheral chemoreceptors have been implicated in early stages of recurrent apneas, congestive heart failure, and certain forms of hypertension. It is likely that chemoreceptors tend to maintain oxygen homeostasis and act as a defense mechanism to prevent the progression of the morbidity associated with these diseases. Experimental models of recurrent apneas, congestive heart failure, and hypertension offer excellent opportunities to unravel the cellular mechanisms associated with altered chemoreceptor function.     

The role of arginine residues in interleukin 1 receptor binding.

Interleukin 1 (IL-1) is a family of polypeptide cytokines that plays an essential role in modulating immune and inflammatory responses. IL-1 activity is mediated by either of two distinct proteins, IL-1 alpha or IL-1 beta, both of which bind to the same receptor found on T-lymphocytes, fibroblasts and endothelial cells (Type 1 receptor). The effect of specific chemical modification of recombinant IL-1 alpha and IL-1 beta on receptor binding was examined. Modification of the proteins with phenylglyoxal, an arginine-specific reagent, resulted in the loss of Type 1 IL-1 receptor binding activity. The stoichiometry of this modification revealed that a single arginine in either IL-1 alpha or IL-1 beta is responsible for the loss of activity. Cyanogen bromide cleavage of phenylglyoxal modified IL-1 alpha and IL-1 beta, followed by sequencing of the peptides, revealed that arginine-12 in IL-1 alpha and arginine-4 in IL-1 beta, which occupy the same topology in the respective crystallographic structures, are the target of phenylglyoxal. These results suggest that an arginine residue plays an important role in ligand-receptor interaction.