Search PubMed⌕ Search

Biomedical subjects

B Holmquist

Publications and source records attributed to B Holmquist.

At least 109 records · Page 6Linked to original sources
PubMed1981

Trichloroethylene: effects on body and organ weights in mice, rats and gerbils.

The influence of continuous inhalation of 150 ppm trichloroethylene (TCE) on body, liver, spleen, and kidney weights in rats, mice, and mongolian gerbils was tested. An age dependent decrease in body weight gain was observed in female rats exposed to TCE. All 3 species showed liver enlargement caused by the exposure. The effect was much more pronounced in mice, in which the increase was 60--80%, than in rats and gerbils where it was only 20--30%. After the end of the TCE-exposure the liver weights of the mice decreased rapidly. After 5 days of rehabilitation the weight was only 10--20% higher than that of the controls. This difference persisted for at least 25 days. The spleen weight appeared unaffected or somewhat smaller in TCE-exposed animals of all species. An increased kidney weight (15%) was observe din TCE-exposed gerbils. This effect was less pronounced in mice and rats. Effects on the liver have earlier been seen only after exposure to concentrations much higher than that used in the present study. This difference in results is proposed to be due to the different schedules used for the exposure.

Animals↗
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

Structural and electronic mimics of the active site of cobalt(II)-substituted zinc metalloenzymes.

Complexes of cobalt(II) and zinc(II) which involve monodentate coordination of two alkyl carboxylate and two imidazole ligands in a slightly distorted tetrahedral fashion have visible and magnetic circular dichroism spectra remarkably similar to the cobalt(II)-substituted proteolytic enzymes thermolysin and carboxypeptidase A. Single crystal x-ray structure determinations on [Co(C2H5COO)2Im2], Im = imidazole, and its zinc counterpart reveal only minor structural differences between the cobalt and zinc species. Electron paramagnetic resonance spectra of cobalt(II) doped into zinc(II) complexes with known structures demonstrate the extreme sensitivity of the g-values to minor structural differences.

Binding Sites↗
PubMed1979

Metal-coordinating substrate analogs as inhibitors of metalloenzymes.

A group of active-site metal coordinating inhibitors of zinc proteases (carboxypeptidase A, thermolysin, Bacillus cereus neutral protease, and angiotensin-converting enzyme) have been synthesized and their properties investigated. Their general structures are R-SH and R-NH-PO2(O phi)H, where-S- or -O- serve as metal ligands and R refers to an amino acid or peptide group designed to interact with substrate recognition sites. These inhibitors can be extremely potent; thus, N-(2-mercaptoacetyl)-D-phenylalanine, e.g., inhibits carboxypeptidase A with a Kiapp of 2.2 x 10(-7) M. The spectral response of cobalt(II)-substituted thermolysin or carboxypeptidase A to the sulfur-containing inhibitors signals the direct interaction of the mercaptan with the metal. An S leads to Co(II) charge transfer band is generated near 340 nm and is detected by absorption, circular dichroism, and magnetic circular dichroism. The cobalt(II) spectra indicate both inner sphere coordination with sulfur and 4-coordination in the enzyme-inhibitor complex. Thus, the metal undergoes a simple substitution reaction, the inhibitor most likely displacing water at the fourth coordination site.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1977

Characterization of the "microprotease" from Bacillus cereus. A zinc neutral endoprotease.

The neutral protease isolated from Bacillus cereus (BRL-70) has been purified by affinity chromatography and characterized. The enzyme exhibits a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has a molecular weight of 34 000 by ultracentrifugation, and contains one enzymatically essential zinc atom per 34 000 g. These data together with the amino acid composition, response to metal substitution, chemical modification, and substrate specificity all indicate that this protease is monomeric and is a typical bacterial neutral metalloprotease.

Amino Acids↗
PubMed1976

Superactivation of neutral proteases: acylation with N-hydroxysuccinimide esters.

A series N-hydroxysuccinimide esters of acylamino acids previously shown to acylate and thereby increase the activity of thermolysin by several orders of magnitude (Blumberg, S., and Vallee, B. L. (1975), Biochemistry 14, 2410) has been used to modify the related neutral proteases from Bacillus subtilis, Bacillus megaterium, and Aeromonas proteolytica. Each of these enzymes is activated to a level characteristic of the particular protein and the particular acyl group incorpporated when monitored with the substrate furylacryloyl-Gly-Leu-NH2. Thus, for the modification of B. megaterium, B. subtilis, and A. proteolytica proteases with Ac-Trp-ONSu, kcat/Km increases 11-, 2.5-, and 18-fold whereas those of the Ac-Phe(4-DnpNH)-ONSu modified enzymes before and after deacylation with hydroxylamine indicate that from 1 to 2 residues are modified. The rate of removal of the Ac-Phe(4-DnpNH) label by 0.1 M hydroxylamine correlates directly with that of the return of native enzymatic activity, at a rate comparable with the rate of deacylation of O-acyltyrosine models. The competitive inhibitors Zn2+ and beta-phenyl-propionyl-Phe do not prevent activation indicating that modification occurs at a site(s) distinct from that at which inhibitors bind. The degree of activation depends also on the substrate employed, generally being greater for substrates which the native enzymes hydrolyze slowly. These data are interpreted to indicate the modification of a residue near the active site, but which serves as a subsite for substrate interaction.

Aeromonas↗
PubMed1976

Esterase activity of zinc neutral proteases.

The hydrolysis of a series of depsipeptides demonstrates that the zinc neutral endopeptidases of bacteria are active esterases. Esters such as BzGly-OPhe-Ala, BzGly-OLeu-Ala, and FA-Gly-OLeu-NH2 are hydrolyzed at rates three- to eightfold slower than are their exact peptide analogues, when hydrolyzed by thermolysin, Bacillus subtilis neutral protease and the neutral protease from Aeromonas proteolytica. Ester hydrolysis by zinc neutral proteases follows the characteristic preference for hydrophobic amino acids adjacent to the site of cleavage, discerned from the hydrolysis of peptide substrates. Removal of zinc from thermolysin abolishes the esterase activity of the native enzyme. Among the metals examined, only Co2+ and Zn2+ restore esterase activity to any significant extent, Co2+ restoring 50% and Zn2+ 100% of the native thermolysin activity. The hydrolysis of esters and peptides by thermolysin does not differ with respect to either the binding or catalytic steps. Substrate specificity, pH-rate profiles, inhibitor, and deuterium isotope effects are identical for both types of substrates.

Binding Sites↗
PubMed1975

Magnetic circular dichroic spectra of cobalt(II) substituted metalloenzymes.

The magnetic circular dichroic (MCD) spectra of cobalt(II) sugstituted metalloenzymes have been studied and compared to a series of four-, five-, and six-coordinate cobalt(II) model complexes previously examined (T. A. Kaden et al. (1974), Inorg. Chem. 13, 2582). The MCD spectra of cobalt substituted carboxypeptidase A, procarboxypeptidase ta, and thermolysin are consistent with earlier deductions of tetrahedral coordination from absorption spectra and also with X-ray structure analysis. Inhibitors fail to alter their MCD spectra significantly. The MCD spectra of cobalt alkaline phosphatase and carbonic anhydrase are more complex and their pH dependence and alteration by inhibitors are discussed in terms of known cobalt(II) models.

Acetazolamide↗
PubMed1975

Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes.

The visible fluorescence of terbium(III) when bound to a calcium binding site of thermolysin is greatly enhanced with an excitation maximum at 280 nm but substitution of cobalt(II) for zinc at the active site decreases the intensity by 89.5%. Treatment with N-bromosuccinimide quenches enzyme tryptophan and Tb(III) fluorescence to a similar extent and suggests the operation of tryptophan vector Tb(III) vector Co(II) energy relay system in the enzyme. Dipoledipole radiationless energy transfer between the Tb(III) donor and the Co(II) acceptor can account for this quenching. The inherent characteristics of the metal pair limits the value of the orientation factor, K2, of the Förster equation, thereby reducing uncertainties in distance measurements by energy transfer compared with other systems. A quantum yield of 0.51 yields a value of R0, the distance for 50% energy transfer, of 19.6 A, and a distance, R, between Tb(III) and Co(II) of 13.7 A, a value identical to that measured for the distance between the active site zinc atom and calcium atom number 1 by x-ray analysis in native thermolysin crystals. The limits of confidence of this measurement are discussed. Energy transfer between two different metal atom sites of a protein provides a new class of probes to measure intramolecular distances of biological macromolecules in solution.

Binding Sites↗