Membrane-bound DD-carboxypeptidase and transpeptidase activities from Bacillus megaterium KM at pH 7. General properties, substrate specificity and inhibition by beta-lactam antibiotics.
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1. The membranes from Bacillus megaterium KM contained a DD-carboxypeptidase with optimum activity under the following conditions: pH 7; ionic strength, 1.3 M; temperature, 40 degrees C and below 20 degrees C. It did not require any divalent cation, but was inactivated by Cu2+ and Hg2+. It was stimulated by 2-mercaptoethanol and low concentrations of p-chloromercuribenzoate. 2. The membrane preparation also catalyzed a simple transpeptidation reaction using as carboxyl acceptors D-alanine or glycine. 3. The conditions for optimum activity, temperature-inactivation, temperature-dependence of the activity, carboxyl donor specificity, sensitivity to beta-lactam antibiotics, and insensitivity to potential peptide inhibitors of both enzyme activities, was identical. The DD-carboxypeptidase showed inhibition by D-alanine and Ac2-L-Lys-D-Ala. 4. The inhibition by beta-lactam antibiotic was reversible for both enzymic activities and the time-dependence for their recovery was identical. 5. The DD-carboxypeptidase was very sensitive to changes in the configuration and size of the side-chains of the C-terminal dipeptide of the substrate. Amino acid residues at the C-terminus that precluded the peptide from being a DD-carboxypeptidase substrate were not acceptors in the transpeptidation reaction. Dipeptides were not acceptors for the 'model transpeptidase'. 6. It is suggested that both activities are catalysed by the same enzyme molecule, whose physiological role is not the formation of peptide crosslinks during peptidoglycan biosynthesis. (+info)
Studies on the uptake, metabolism, and release of endogenous and exogenous chemicals by use of the isolated perfused lung.
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The isolated perfused lung is a valuable tool for studying many lung functions. The kinds of information one can obtain from the isolated perfused lung are illustrated by examples from our studies on the uptake, accumulation, and metabolism of endogenous and exogenous chemicals. (+info)
Incorporation of [14C]glucose from UDP[14C]glucose into a phosphoglycolipid by cell-free particulate systems of a moderately halophilic gram-negative bacterium, Pseudomonas halosaccharolytica ATCC 29423.
(19/6763)
A glucosyl group from uridine diphosphate [U-14C]glucose is incorporated into a phosphoglycolipid, probably a glucosylphosphatidylglycerol, by a disrupted membrane enzyme preparation from a gram-negative, moderately halophilic bacterium, Pseudomonas halosaccharolytica ATCC 29423. The conversion of [14C]phosphatidylglycerol into phosphoglycolipid by the particulate preparation was also enhanced in the presence of non-labelled UDP-glucose. A chemical degradation study of labelled phosphoglycolipid showed the bulk of the radioactivity from UDP[U-14C]glucose to be associated with the glucose moiety, which also appeared to be attached to the hydroxyl group of a second glycerol. (+info)
Bystander killing of tumour cells by antibody-targeted enzymatic activation of a glucuronide prodrug.
(20/6763)
RHI-betaG-PEG, formed by linking poly(ethylene glycol)-modified beta-glucuronidase to Mab RH1, was employed to examine bystander killing of antigen-negative N1S1 rat hepatoma cells by activation of a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at antigen-positive AS-30D rat hepatoma cells. Sequential treatment of cells with 10 microg ml(-1) RH1-betaG-PEG and 20 microM BHAMG was not toxic to N1S1 cells but killed 99% of AS-30D cells. Over 98% of N1S1 cells, however, were killed in mixed populations containing as few as 2% AS-30D cells after identical treatment, demonstrating an in vitro bystander effect. Subcutaneous injection of AS-30D and N1S1 cells in BALB/c nu/nu mice produced solid tumours containing both cells. Uptake of radiolabelled RH1-betaG-PEG in solid AS-30D and mixed AS-30D/N1S1 tumours was 11.6 and 9.3 times greater than a control antibody conjugate 120 h after i.v. injection. Intravenous treatment with RH1-betaG-PEG and BHAMG cured seven of seven nude mice bearing solid s.c. AS-30D tumours and significantly delayed, compared with control conjugate and prodrug treatment, the growth of mixed N1S1/AS-30D tumours with one cure, showing that targeted activation of BHAMG kills bystander tumour cells in vivo. (+info)
A novel strategy affords high-yield coupling of antibody to extremities of liposomal surface-grafted PEG chains.
(21/6763)
Several methodologies for the preparation of polyethylene glycol-grafted immunoliposomes have been developed by attaching antibodies to the terminus of the polymer. Unilamellar liposomes were prepared containing a combination of a functionalized polyethylene glycol(3400) and an inert polyethylene glycol(2000) phosphatidylethanolamine derivate up to 5 mol%. The greater length of the functionalized polyethylene glycol derivate did not alter the liposomal sterical stability or the remote loading of doxorubicin. Anti-CD34 immunoliposomes were prepared by the reaction of maleimide-derivatized My10 antibody with generated thiol groups at the periphery of the liposomes and efficiencies of nearly 100% were obtained. The greater accessibility of the reactive group makes this strategy more efficient than others described. The immunoliposomes prepared bound specifically to CD34+ cells. (+info)
Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure.
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The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase. (+info)
Targeted delivery and improved therapeutic potential of catalase by chemical modification: combination with superoxide dismutase derivatives.
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Four types of bovine liver catalase (CAT) derivatives, succinylated (Suc-CAT), galactosylated (Gal-CAT), mannosylated (Man-CAT), and polyethylene glycol conjugate (PEG-CAT), were synthesized and their pharmacokinetics and therapeutic potential in a hepatic ischemia/reperfusion injury model were studied in mice. About 90% of the CAT enzymatic activity was retained after chemical modification. Biodistribution studies showed that 111indium (111In)-Gal-CAT accumulated selectively in the liver parenchymal cells as 111In-CAT, whereas an increased amount of 111In-Suc-CAT and 111In-Man-CAT was delivered to liver nonparenchymal cells. 111In-PEG-CAT exhibited prolonged retention in plasma. Pharmacokinetic analysis revealed that the hepatic uptake clearances of 111In-Suc-CAT, 111In-Gal-CAT, and 111In-Man-CAT were much greater than that of 111In-CAT, whereas that of 111In-PEG-CAT was very small. In the ischemia/reperfusion injury model, in which hepatic injury was induced by occlusion of the portal vein for 30 min followed by 1 h reperfusion, the elevation of plasma glutamic pyruvic transaminase and glutamic oxaloacetic transaminase levels was slightly inhibited by treatment with native CAT or Gal-CAT. PEG-CAT was less potent. In contrast, Suc-CAT and Man-CAT effectively suppressed the increase in plasma glutamic pyruvic transaminase and glutamic oxaloacetic transaminase. Coinjection of mannosylated superoxide dismutase marginally improved the inhibitory effects of CAT derivatives. These results demonstrate that targeted CAT delivery to liver nonparenchymal cells via chemical modification is a promising approach to prevent hepatic injuries caused by reactive oxygen species. The potential usefulness of combining of CAT and superoxide dismutase derivatives is also demonstrated. (+info)
Influenza hemagglutinin mediated fusion of membranes containing poly(ethylene-glycol) grafted lipids: new insights into the fusion mechanism.
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The influence of a hydrophilic layer covering the membrane on influenza hemagglutinin (HA) mediated fusion was investigated using membranes containing poly(ethylene-glycol) grafted phosphatidylethanolamine (PEG-2000-PE). Steric inhibition of HA-membrane interactions by these lipids affected virus fusion (half-maximal inhibition at 0.8 mol% for lipids with 114 ethylene glycol residues, or at 3.2 mol% for 45 residues (PEG-2000-PE), concentrations at which the PEG moieties adopt a random coil structure). Reconstituted viral membranes containing 3 mol% PEG-2000-PE retained 40% of their fusion activity. Therefore, efficient fusion is possible with membranes completely covered by a hydrophilic layer of several nanometers, and fusogenic virosomes containing PEG-PE are feasible. (+info)