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Biomedical subjects

B Holmquist

Publications and source records attributed to B Holmquist.

At least 91 records · Page 5Linked to original sources
PubMed1984

Cryospectrokinetic characterization of intermediates in biochemical reactions: carboxypeptidase A.

Cryospectrokinetic studies provide concurrent structural, kinetic, and chemical data on short-lived intermediates in the course of the interactions of enzymes with their substrates and of other, similar pairs of biomolecules. Subzero temperatures extend the lifetimes of these intermediates and, combined with rapid-mixing and rapid-scanning instrumentation, allow simultaneous measurement of both their physical-chemical and kinetic characteristics. For carboxypeptidase A, the spectra of a chromophoric, enzymatically functional cobalt atom at the active site signal the structure of the coordination complex during catalysis, while radiationless energy transfer between enzyme tryptophans and the fluorescent dansyl blocking group of rapidly hydrolyzed peptide and ester substrates provides the basis for measurement of the rates of formation and breakdown of intermediates. Subzero radiationless energy transfer kinetic studies of the zinc and cobalt enzymes disclose two intermediates in the hydrolysis of both peptides and esters and furnish all the rate and equilibrium constants for the reaction scheme E + S in equilibrium ES1 in equilibrium ES2----E + P. The chemical and kinetic data indicate that neither of these is an acylenzyme intermediate. Both absorption and EPR spectra of the ES2 reaction intermediates consistently demonstrate the formation of transient metal complexes, differences between the effects induced by peptides and esters, and strong similarities between those induced by all peptides on the one hand and all esters on the other. The marked alterations of the cobalt spectra likely reflect the coordination of a substrate carboxyl and/or carbonyl group to the metal at a critical step in the course of catalysis. The cryospectrokinetic approach developed here in the mechanistic study of this metalloenzyme is applicable to the examination of transients of biochemical reactions in general. It will allow molecular characterization of previously elusive intermediates and greatly magnify the range of mechanistic questions that can be answered.

Animals↗
PubMed1984

Perchloroethylene: effects on body and organ weights and plasma butyrylcholinesterase activity in mice.

The effects of continuous and intermittent inhalation of perchloroethylene (PCE) on plasma butyrylcholinesterase (BuChE) activity, organ weights, liver morphology and motor activity in mice (strain NMRI) were tested. PCE exposure increased plasma BuChE activity in a time- and concentration dependent manner in both sexes. The increase was statistically significant at 37 p.p.m. in animals continuously exposed for 30 days. BuChE increased approximately 1.5 times in females and 2.5 times in males after 120 days exposure to 150 p.p.m. After rehabilitation of animals exposed for 30 days to 150 p.p.m., BuChE levels returned to normal. Liver weight also increased in a time and concentration dependent manner. Both sexes exhibited significant liver enlargement at 9 p.p.m. The increase was about 2.3 in females and 1.9 in males after continuous exposure to 150 p.p.m. for 120 days. After rehabilitation (120 days) of animals exposed to 150 p.p.m. for 30 days, a 10% increase still remained. A decrease in body weight gain was seen in both sexes after exposure to concentrations above 75 p.p.m. Female kidney weight was slightly increased. No clear effect on spleen weight could be detected. When the same time-weighted average concentration was used, intermittent exposure for 30 days had similar effects on liver weight and BuChE activity as continuous exposure, even when exposures lasted for only one hour per day. Liver cell morphology was changed after PCE exposure. The alterations could be observed already at 9 p.p.m. but disappeared after rehabilitation.

Animals↗
PubMed1984

Pivmecillinam plus pivampicillin versus co-trimoxazole in patients undergoing transurethral prostate resection.

Patients undergoing transurethral prostate resection received a 10-day or a 20-day treatment with a combination of pivmecillinam/pivampicillin or with co-trimoxazole starting 1 day before surgery. The results were evaluated in 139 patients. Fifty-three patients had bacteriuria prior to the operation, and the bacteriological cure rate was 22 out of 25 on pivmecillinam/pivampicillin and 22 out of 28 on co-trimoxazole. Eighty-six patients had no bacteriuria pre-operatively and received treatment prophylactically. Two out of 40 patients on co-trimoxazole developed urosepsis, while pivmecillinam/pivampicillin was effective in preventing septicaemic episodes in all 46 patients treated. Tolerance was good with mild side-effects in 5 patients on pivmecillinam/pivampicillin and in 2 patients on co-trimoxazole.

Aged↗
PubMed1983

Circular dichroism and ordered structure of bisnucleoside oligophosphates and their Zn2+ and Mg2+ complexes.

Circular dichroism, absorbance, hypochromicity, and the formation of Mg2+ and Zn2+ complexes have been measured for a series of bisnucleoside oligophosphates that contain adenosine, guanosine, and mixed guanosine/adenosine, guanosine/cytidine, and guanosine/uridine, as well as 7-methylguanosine and ribose-methylated purine nucleosides. All of the metal complex ions have stacking interactions at 2 degrees C, 10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 8.0. There is a measurable degree of base stacking for all unsubstituted purine nucleotides that differs, however, from that of bases in nucleic acids. The degree of base stacking varies with the length of oligophosphate chains and the state of methylation. The effect of 7-methylation of guanosine is interpreted as causing a switch of nucleic acid base stacking from an atypical to a typical mode, which could be important for cap function in mRNA. The Mg2+ and Zn2+ complexes give rise to characteristic circular dichroism. In all instances excepting 7-methylated bisguanosine oligophosphates, the active secondary structures are disrupted, and in this regard, Zn2+ is more effective than Mg2+. At least two sets of binding sites are involved. A single metal ion is bound tightly. Stability, in terms of equilibrium constants, increases by more than 1000-fold as a function of chain length varying from two to six phosphates. The consequences of methylation are only minor. Electrostatic attraction between metal ions and phosphates is the most likely mechanism of these phenomena as judged by the effect of high ionic strength.

Circular Dichroism↗
PubMed1983

Anion activation of angiotensin converting enzyme: dependence on nature of substrate.

Anion activation of pulmonary angiotensin converting enzyme has been examined by using 23 furanacryloyl- and 3 benzoyl-tripeptides as substrates. Chloride stimulates hydrolysis of all substrates at least 24-fold. However, the kinetic mechanism, the amount of chloride required, and the effect of pH on activation, plus the relative activating potencies of various anions, are all strongly dependent on the substrate employed. Three substrate classes have been identified. Class I substrates appear to be hydrolyzed at pH 7.5 by an ordered bireactant mechanism in which anion must bind before substrate. The apparent activation constant (KA') for Cl- ranges from 75 to 150 mM at pH 7.5, doubles at pH 9.0, and decreases to about 3 mM at pH 6.0. Class II substrates, in contrast, are hydrolyzed by a nonessential activator mechanism. The kinetically determined KA' for Cl- at pH 7.5 ranges from 2.9 to 5.0 mM and changes only slightly with pH. Class III substrates are also hydrolyzed by a nonessential kinetic mechanism but one different from that followed by class II peptides. KA' values for Cl- at pH 7.5 measured with class III substrates are 18-30 mM. Class II substrates have Arg or Lys at the ultimate or penultimate position. The features distinguishing class I and III peptides are less clear, although all class III substrates identified have penultimate alanine residues. Possible explanations for this substrate dependence are offered.

Acetates↗
PubMed1983

One hundred fold increased activity of Aeromonas aminopeptidase by sequential substitutions with Ni(II) or Cu(II) followed by zinc.

Full substitution of Cu(II) or Ni(II) for the two g-atom zinc in Aeromonas aminopeptidase hyperactivates the enzyme 6.5 and 25 fold respectively. Even greater enhancements of activity can be achieved with mixed metal substitutions. Thus, apoenzyme reactivated by first adding one g-atom zinc followed by one g-atom of either Cu(II) or Ni(II) is 15 and 22 times more active than the native enzyme. Reversing the order, i.e. by first adding either one g-atom Cu(II) or Ni(II) followed by one g-atom zinc, activates the enzyme nearly 100 fold. The order of metal addition is critical and suggests the existence of two non-identical metal sites, each with a different function.

Aeromonas↗
PubMed1983

Human serum procarboxypeptidase A.

Zymogen activation is an important biochemical control process and has important physiological and pathological implications. We have simultaneously measured both procarboxypeptidase A, the enzyme precursor, and carboxypeptidase A, its active product, in serum by using an affinity resin and the synthetic peptide substrate N-(2-furanacryloyl)-L-phenylalanyl-L-phenylalanine. Serum procarboxypeptidase A is activated by trypsin, chymotrypsin, plasmin, subtilisin, or urokinase but not by thrombin or enteropeptidase. The molecular weight of the precursor is approximately 5000-10 000 greater than that of the active product. Both enzyme and precursor increase in serum in the course of pancreatic inflammation, but the degree of activation can vary up to 2000-fold, independent of the amount of precursor present. The existence of this pancreatic proteolytic precursor in serum opens new avenues for the investigation of zymogen activation and its regulation.

Carboxypeptidases↗
PubMed1983

Spectral properties of cobalt carboxypeptidase A. Interaction of the metal atom with anions.

At pH greater than 7 the absorption and magnetic circular dichroic spectra of cobalt carboxypeptidase A are insensitive to anions [Latt, S. A., & Vallee, B. L. (1971) Biochemistry 10, 4263-4270], but at pH less than 6 chloride and other anions perturb them in a manner specific for each anion. Lowering of the pH apparently facilitates the entry of an anion into the metal coordination sphere, suggesting that an acidic group normally stabilizes a metal-coordinated water molecule against displacement. The lack of sensitivity to anions at pHs between 7 and 9--when the enzyme is maximally active--and its evident abolition upon protonation of an active-site group are consistent with this interpretation. Selective modification of cobalt carboxypeptidase at Glu-270 using a carbodiimide affinity reagent generates sensitivity to anions at pH 7 very similar to that of the unmodified enzyme at pH approximately 5. This suggests that the group stabilizing the metal-coordinated water is the catalytically essential carboxylate of Glu-270. These and related results provide evidence for a mechanistically important interaction of Glu-270 with a metal-bound water molecule.

Binding Sites↗
PubMed1983

Substrate specificity and kinetic characteristics of angiotensin converting enzyme.

Furanacryloyl-Phe-Gly-Gly has been shown to be a convenient substrate for angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). A detailed kinetic analysis of the hydrolysis of this substrate indicates normal Michaelis-Menten behavior with kcat = 19000 min-1 and KM = 3.0 x 10(-4) M determined at pH 7.5, 25 degrees C. The enzyme is inhibited by phosphate and activated by chloride; maximal activity is observed with 300 mM NaCl. In the absence of added zinc, activity is lost rapidly below pH 7.5 due to spontaneous dissociation of the metal, but in the presence of zinc, the enzyme remains fully active to about pH 6. The pH-rate profile indicates two groups on the enzyme with apparent pK values of 5.6 and 8.4. The substrate specificity of the enzyme has been examined in terms of the fundamental specificity quantity kcat/KM as well as the separate constants by using a series of furanacryloyl-tripeptides. The activity toward furanacryloyl-Phe-Gly-Gly has been compared with that toward the physiological substrates angiotensin I and bradykinin.

Acrylates↗
PubMed1983

Effects of continuous trichloroethylene inhalation on different strains of mice.

Seven different strains of mice (wild, C57BL, DBA, B6CBA, A/sn, NZB and NMRI) were continuously exposed to 150 ppm trichloroethylene (TCE) during 30 days. After exposure all strains showed large increases in liver weight, while changes in kidney and spleen weights were small. Plasma butyrylcholinesterase (BuChE) activity increased in males of all strains and in females of strains A/sn and NZB. The increase in the females was less than that of the corresponding males. We concluded that there are moderate, but in some cases, significant differences in TCE sensitivity between different mouse strains. The differences observed between sexes emphasizes the importance of specifying sex as well as strain when reporting on toxic effects.

Animals↗
PubMed1983

Trichloroethylene: further studies of the effects on body and organ weights and plasma butyrylcholinesterase activity in mice.

The effects of continuous and intermittent inhalation of trichloroethylene (TCE) were studied in male and female mice. Plasma butyrylcholinesterase (BuChE) activity, body, liver, kidney and spleen weights were measured. The liver was studied histologically and motor activity measured with doppler radar. Continuous TCE-exposure (37-300 p.p.m.) increased plasma BuChE activity in the males in a time and concentration dependent manner. After 30 days at 37 p.p.m. the increase was about 25%. Exposure to 300 p.p.m. for 30 days increased the activity three times. BuChE activity in females was only slightly influenced even at 300 p.p.m. Liver weight was increased in a time and concentration dependent manner in both sexes. In animals continuously exposed for 30 days to 300 p.p.m., liver weight was roughly twice that of the air-exposed controls. Morphological changes were observed in the liver of TCE-exposed animals. Above 150 p.p.m. kidney weight in both sexes was significantly increased. This effect was more pronounced in the males than in the females. Spleen weight was not influenced by the exposure. Body weight increase was slightly lower in exposed animals. Plasma BuChE activity and liver weight returned to normal when exposure was terminated. Intermittent exposure to short pulses of high concentration of TCE had roughly the same effect on BuChE, body and organ weights as continuous exposure to the same time-weighted average. Motor activity was affected by the intermittent exposure schedules. At 900 p.p.m. decrease in activity was observed. At 3600 p.p.m. motor activity was considerably increased.

Animals↗
PubMed1982

Sensitivity of Mongolian gerbils to trichloroethylene exposure during neonatal growth.

Continuous exposure of young mongolian gerbils to trichloroethylene (230 ppm) was started at different times during the first month of life. The onset of exposure was accompanied by an increased number of deaths among the pups and by an immediate reduction in growth rate. The proportion of dead pups was greatest when exposure was started at birth, and decreased rapidly with increasing age at onset of exposure. Growth rate was partially restored approx. 1 week after the onset of exposure. After weaning the effect of the exposure on growth decreased, and the variation in the effect among different litters was considerably reduced. The enhanced sensitivity of the pups to trichloroethylene (TCE) exposure is believed to be due to a disturbance in the mother-offspring relationship.

Animals↗
PubMed1982

Effects of trichloroethylene inhalation on acid phosphatase in rodent brain.

Rats, mice and gerbils were continuously exposed to 150 ppm trichloroethylene (TCE) for 30 days. In all three species, there was a marked increase in liver weight. In mice the weight increased more (86%) than in rats and gerbils (20%). After exposure the activity of acid phosphatase, a lysosomal marker enzyme, was tested in different brain areas, using a system which had a limit of detection of +/- 10-15%. In most areas no significant influence was found. However, in the brain stem of mice and gerbils the phosphatase activity increased by approx. 10%.

Acid Phosphatase↗
PubMed1981

Spectral studies of cobalt (II)- and Nickel (II)-metallothionein.

The zinc and cadmium of native rabbit metallothionein-1 were replaced stoichiometrically with either cobalt (II) or nickel (II). The electronic, magnetic circular dichroic (MCD), and electron spin resonance spectra of Co (II)-metallothionein reflect distorted tetrahedral coordination of the cobalt atoms. Both the d-d and charge-transfer spectral regions closely resemble those of simple cobalt-tetrathiolate complexes, implying that their coordination chemistry is analogous. Ni (II) complex ions and Ni (II)-metallothionein similarly exhibit analogous MCD bands in the d-d region. The circular dichroic bands associated with ligand-metal charge-transfer transitions in the non-d-d region of Co (II)- and Ni (II)-metallothionein afford additional evidence for the similarity in tetrahedral microsymmetry of the two metal derivatives. The known ratio of 20 thiolate ligands to 7 metal ions, in conjunction with the spectral evidence for tetrathiolate coordination in metallothionein, represents good evidence that these metal thiolates are organized in clusters.

Animals↗
PubMed1981

An angiographic study of erection in the dog.

Erections were produced in dogs by stimulating the pudendal, the pelvic and the hypogastric nerves either singly or in combination and angiographic studies were then performed. During an erection an increase in blood flow to the erectile tissue could be demonstrated together with an impaired venous return.

Angiography↗