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Biomedical subjects

B M Babior

Publications and source records attributed to B M Babior.

At least 127 records · Page 7Linked to original sources
PubMed1982

Interaction of N-substituted ethanolamine analogs with ethanolamine ammonia-lyase, an adenosylcobalamin-requiring enzyme.

A number of N-substituted ethanolamine derivatives have been found to interact productively with ethanolamine ammonia-lyase, an adenosylcobalamin (AdoCbl) requiring enzyme that catalyzes the conversion of vicinal amino alcohols to oxo compounds. Inhibition, inactivation, cleavage of AdoCbl, and exchange of tritium out of [5'-3H]AdoCbl were all observed, the effects varying from analog to analog. The most striking effects were seen with azetidin-2-ol and pyrrolidin-3-ol, two aliphatic heterocycles composed of ethanolamine in which the nitrogen is tied back to the carbinol carbon by methylene bridges containing one and two carbon atoms, respectively. Incubation of azetidinol with the enzyme.AdoCbl complex resulted in extensive inactivation of the enzyme accompanied by cleavage of the cofactor; exchange of 3H from [5'-3H]AdoCbl into a nonbasic compound was also seen. Pyrrolidinol interacted with the enzyme.AdoCbl complex in a substrate-like way, causing exchange of 3H from labeled cofactor into a basic compound (perhaps the pyrrolidinol itself) together with a slight amount of C-Co bond cleavage. The interaction of the enzyme.AdoCbl complex with pyrrolidinol suggests that productively bound substrate is associated with the active site in such a way that the dihedral angle between the nitrogen and the oxygen is about 120 degrees, a configuration which maximizes bond-bond repulsions between the substituents on the adjacent carbon atoms.

Ammonia-Lyases↗
PubMed1982

Severe recurrent bacterial infections associated with defective adherence and chemotaxis in two patients with neutrophils deficient in a cell-associated glycoprotein.

We studied two patients with delayed umbilical cord detachment, recurrent bacterial infections, inability to form pus, rapidly progressive periodontitis, and persistent leukocytosis. The phagocytes of both patients were strikingly abnormal in their ability to adhere to surfaces. The adherence of polymorphonuclear leukocytes to endotoxin-coated glass coverslips, glass beads, or nylon wool was markedly reduced. Scanning electron microscopy of the few adherent polymorphonuclear leukocytes from both patients showed a failure to flatten and form fine pseudopods. In vivo polymorphonuclear leukocyte and monocyte chemotaxis assessed by skin window and skin chamber methods was dramatically impaired, and in vitro chemotaxis was severely depressed. Chemiluminescence of zymosan- but not phorbol-stimulated polymorphonuclear leukocytes was markedly reduced. Allogeneic polymorphonuclear leukocytes transfused into these patients functional normally, indicating that the defect is intrinsic to the cells and not a secondary phenomenon. A 180-kilodalton glycoprotein normally present in the particulate fraction of polymorphonuclear leukocytes was found to be completely absent in Patient 1 and present in low concentration in Patient 2. We postulate that the glycoprotein deficiency interferes with the migration of polymorphonuclear leukocytes from the bloodstream into the interstitial space and to the site of infection.

Bacterial Infections↗
PubMed1982

The enzymatic basis for O-.2 production by human neutrophils.

O-.2 production is the first step in the generation of a group of powerful microbicidal oxidants by neutrophils. The production of O-.2 is catalyzed by a membrane-bound, NADPH-preferring flavoprotein oxidase, a conclusion supported by much evidence including the discovery of a new form of chronic granulomatous disease caused by a mutation affecting that oxidase directly. Also involved in the O-.2-forming reaction is a b-type cytochrome; the role of this cytochrome is as yet undefined, though it does not appear to be on the direct route of electron transfer between NADPH and oxygen. It has been postulated that quinones too participate in the O-.2-forming reaction, but further work is necessary to define their role more fully.

Cytochrome b Group↗
PubMed1982

Studies on the interaction between GP-18-0-deficient neutrophils and vascular endothelium.

A patient whose neutrophils lack the glycoprotein gp-180 shows an increased susceptibility to bacterial infections. Neutrophils from this patient migrate abnormally both in vivo and in vitro. To examine the basis for this abnormality in migration, a study was carried out on the interaction of gp-180-deficient neutrophils with artificial surfaces and with human endothelial cell cultures. Compared with normal neutrophils. gp-180-deficient neutrophils showed decreased adhesion to cold-insoluble globulin-coated plastic surfaces, and their ability to spread on this substratum was greatly impaired. In contrast, gp-180-deficient neutrophils interacted in a normal fashion with endothelial monolayers, attaching to their surfaces and migrating between cell junctions to spread between the monolayers and the subjacent plastic. A normal interaction with endothelium in vivo was implied by the finding that the rise in the neutrophil count in response to epinephrine, an index of the marginated pool, was normal in the gp-180-deficient patient. We conclude that the abnormal function of gp-180-deficient cells is unlikely to be caused by a faulty interaction with the vascular endothelium. We postulate instead that these cells migrate poorly in vivo because of an abnormal interaction with extravascular connective tissue matrix constituents.

Bacterial Infections↗
PubMed1982

Expression of a chronic granulomatous disease-like defect by fluoride-exhausted neutrophils.

Neutrophils incubated with 20 mM F- express a respiratory burst without degranulating or performing phagocytosis. After 60 min of F- treatment, the burst is exhausted and cannot be restarted. Neutrophils so treated have a microbicidal defect similar to that of chronic granulomatous disease (CGD): they kill Str. mitis at a nearly normal rate, but show a marked impairment in the destruction of S. aureus. They differ from CGD neutrophils in that they also display a defect in motility. This defect, however, is not so severe as to seriously impair their ability to kill bacteria by mechanisms that are independent of endogenously generated microbicidal oxidants.

Blood Bactericidal Activity↗
PubMed1981

The O2--producing enzyme of human neutrophils. Further properties.

The enzyme responsible for the respiratory burst in human neutrophils is a membrane-bound oxidase that catalyzes the reduction of oxygen to O2- at the expense of a reduced pyridine nucleotide. We describe further properties of the solubilized oxidase. The rates of O2- production and NADPH consumption are consistent with the stoichiometry: 2 O2 + NADPH leads to 2 O2- + NADP The enzyme is highly specific for oxygen, failing to reduce several artificial electron acceptors including ferricyanide. FAD, an essential cofactor, binds tightly to the enzyme, as indicated by a Km of 61 nM. A requirement for a free --SH group is suggested by a 2-fold increase in activity in the presence of low concentrations of dithiothreitol; the higher dithiothreitol concentrations lead to inhibition. The solubilized enzyme is highly unstable, losing one-half its activity after 1 h at room temperature. Loss of activity is accelerated 2- to 3-fold by salts and EDTA. The substrate analog 2',5'-ADP is similar to other salts in its effect on the inactivation rate. ATP, on the other hand, causes loss of activity in seconds, raising the possibility that ATP is a physiological regulator of the catalytic activity of the enzyme.

Cations, Divalent↗
PubMed1981

Arrangement of the respiratory burst oxidase in the plasma membrane of the neutrophil.

Evidence was obtained regarding the way the O-2-forming NADPH oxidase of human neutrophils is arranged within the plasma membrane. O-2 production by particles from zymosan-activated human neutrophils rose two- to threefold when the particles were assayed in the presence of appropriate concentrations of Triton X-100. The portion of activity revealed by the detergent was not affected by treating the particles with trypsin or with p-chloromercuribenzene sulfonate, a nonpenetrating sulfhydryl reagent, but the activity detectable in the absence of detergent was abolished by these treatments. O-2 production by phagocytic vesicles was not augmented by detergent, and was almost entirely eliminated by tryptic digestion of the vesicles regardless of whether or not detergent was present during the assay. These results suggest that the O-2-forming oxidase is embedded in the plasma membrane with a portion extending into the cytoplasm and the rest buried in the lipid bilayer. It is proposed that the pyridine nucleotide-binding site is located on the cytoplasmic extension and the oxygen binding site is on the intramembranous portion of the enzyme.

4-Chloromercuribenzenesulfonate↗
PubMed1981

The third component of complement: covalent attachment of a radioactive sugar to the labile binding site of C3 via the alternative pathway.

A complement- (C) fixing particle consisting of agarose beads to which 5-thioglucose was attached by a --S--S-- bond (agarose-thioglucose) was employed to investigate the mechanism of attachment of C3 to surfaces. When whole serum containing [125I] C3 was incubated with agarose-thioglucose, labeled C3b was taken up in a form that was not removed by 2 M NaCl but was released by 10 mM dithiothreitol. Deposition of DTT-releasable C3b was dependent upon the alternative pathway of C activation. Gel electrophoresis of DTT-releasable C3b from similar experiments performed with unlabeled serum and agarose-[3H]thioglucose showed that the liberated C3b contained a molecule of radioactive thioglucose attached to the alpha'-chain by a covalent bond that was stable to mercaptoethanol. We propose that the thioglucose-alpha' chain bond was formed during the course of C activation by a reaction between the "labile binding site" of newly released C3b and the (then) particle-bound sugar. This formulation implies that the reaction by which C3b attaches to 5-thioglucose in this system is the reaction responsible for opsonization by C3b, and that the C3b-linked sugar represents a marker for the labile binding site. Incubation of the particle-bound C3b in serum resulted in the cleavage of the covalently linked alpha'-chain to several smaller polypeptides, the major cleavage product having a m.w. of 70,000.

Binding Sites↗
PubMed1980

The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Studies with isopropanolamine, a true substrate.

Ethanolamine ammonia-lyase catalyzes the adenosylcobalamin (AdoCbl)-dependent deamination of 1-amino-3-propanol (isopropanolamine) to acetone and NH3. During the course of the reaction a hydrogen is transferred from the carbinol carbon of the substrate to one of the methyl carbons of the product, the cofactor serving as intermediate hydrogen carrier. This result, together with previous results obtained with this and other AdoCbl-requiring enzymes, suggests that an initial step in the catalytic mechanism is the abstraction of a hydrogen atom from the substrate to produce the 1-amino-2-hydroxyprop-2-yl radical. This is a bulky species which for steric reasons is unlikely to alkylate cob(II)alamin. Thus, an organocobalamin formed by a reaction between the substrate radical and cob(II)alanin is probably not involved in the mechanism of deamination of isopropanolamine by ethanolamine ammonia-lyase.

Ammonia-Lyases↗
PubMed1980

Deamination of 2-aminopropanol by ethanolamine ammonia-lyase, an AdoCbl-requiring enzyme. Kinetics and isotope effects for the R and S enantiomers of the substrate.

Both (R)-2-aminopropanol and (S)-2-aminopropanol are substrates for ethanolamine ammonia-lyase, reacting to yield propionaldehyde and ammonia via a common enzyme-bound intermediate. (S)-Propanolamine shows a lower Km and a higher Vmax than the R enantiomer, indicating that the former is a substantially better substrate than the latter. From the isotope effects and other considerations, it appears that the rate-limiting step for (S)-propanolamine is the same as that for ethanolamine, namely, the transfer of hydrogen from the cofactor to the product. In contrast, isotope effects seen with (R)-propanolamine indicate that there are two rate-limiting steps, both located in the segment of the reaction mechanism which is unique to the R enantiomer. One of these is the transfer of hydrogen from the substrate to the cofactor. The other is a subsequent step whose rate is similar to that of the first hydrogen transfer step but which does not involve the rupture of a carbon-hydrogen bond. It is speculated that this second rate-limiting step may have to do with the migration of the amino group.

Ammonia-Lyases↗
PubMed1980

An inherited abnormality of neutrophil adhesion. Its genetic transmission and its association with a missing protein.

Neutrophils from a five-year-old boy with recurrent bacterial infections failed to spread on surfaces, leading to a severe defect in chemotaxis and a mild impairment in phagocytosis. Failure to spread was also seen in a fraction of the neutrophils from the patient's mother and sister, but cells from his father and brother were normal. Gel electrophoresis revealed that a protein with a molecular weight of 110,000 daltons (designated gp 110) present in the particulate fraction of normal neutrophils was absent from the patient's cells, and that its levels were below normal in cells from his mother and sister but normal in neutrophils from his father and brother. These findings suggest that gp 110 is necessary for the spreading of neutrophils onto surfaces, that the functional abnormality in the patient's cells is caused by its absence, and that deficiency of gp 110 is an X-linked congenital disease.

Blood Proteins↗
PubMed1980

The mechanisms of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence that carbon-cobalt bond cleavage is driven in part by conformational alterations of the corrin ring.

Previous work has shown that the interaction between ethanolamine ammonia-lyase (ethanolamine ammonia-lyase, EC 4.3.1.7) and adenosylcobalamin weakens the C-Co bond of the cofactor with respect to homolytic cleavage. To obtain information concerning the mechanism by which this is accomplished, a study was conducted in which optical and circular dichroism spectroscopy were used to explore the interaction between ethanoloamine ammonia-lyase and a series of adenosylcobalamin analogs composed of an adenyl residue attached to the cobalt atom of cobalamin by a methylene chain whose length varies from 2 to 6 carbons. These studies indicated that the binding of a cobalamin to the active site activates forces which tend to alter the conformation of the enzyme, and with it that of the corrin ring, but that these conformational changes are blocked by bulky Co-beta substituents which restrict corrin ring flexibility. We postulate that at least one element of the force which weakens the C-Co bond of the enzyme-bound cofactor is the relief of conformational strain which occurs when C-Co bond cleavage, by releasing the interfering adenosyl group, permits the enzyme and the corrin ring to assume the energetically favored conformation.

Ammonia-Lyases↗
PubMed1980

Incorporation of glucosamine by activated human neutrophils. A myeloperoxidase-mediated process.

Zymosan-activated neutrophils were found to incorporate large amounts of [3H]glucosamine into TCA-precipitable material as compared with resting cells. The burst of glucosamine incorporation began 2 min after zymosan exposure and lasted 3 to 5 min, after which the incorporation rate returned to that of resting cells. Studies with cells from patients with chronic granulomatous disease and hereditary myeloperoxidase deficiency as well as experiments with inhibitors indicated that glucosamine incorporation required both the respiratory burst and the myeloperoxidase system. SDS-polyacrylamide gel electrophoresis of [3H]glucosamine-containing TCA precipitates from zymosan-activated cells revealed radioactivity migrating throughout the length of the gel. The radioactivity in precipitates from resting cells or cells activated in the presence of a small amount of a myeloperoxidase inhibitor was found in a peak migrating close to the tracking dye. These results indicate that zymosan-activated neutrophils are able to incorporate glucosamine into protein by a process dependent on H2O2 and myeloperoxidase. The biosynthetic significance of this phenomenon is not certain, but it most likely represents the reaction of amino sugar with macromolecular degradation products formed by the action of the myeloperoxidase system on cellular and particulate constituents.

Glucosamine↗