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.

Genomic cloning, structure, and regulatory elements of the 1 alpha, 25(OH)2D3 down-regulated gene for cyclic AMP-dependent protein kinase inhibitor.

The cyclic AMP-dependent protein kinase inhibitor (PKI) mRNA and protein are negatively and tissue-specifically regulated in the kidney by 1 alpha, 25(OH)2D3. A 17-kb PKI clone, isolated from a chick genomic library, revealed that the PKI gene consists of two exons separated by a 4.5-kb intron. A 411-bp upstream region (constituting 93 bp upstream and 318 bp downstream from the transcriptional start site) containing a putative negative VDRE (nVDRE) fused to the luciferase gene was used for transient transfections of primary cultures of chick kidney cells. Luciferase activity was significantly down-regulated in response to 1 alpha, 25(OH)2D3. This result suggests that the promoter region containing the putative nVDRE plays a pivotal role in the negative regulation of PKI gene transcription.

Acetylated N-terminal structures of class III alcohol dehydrogenases. Differences among the three enzyme classes.

The protein chains of mammalian alcohol dehydrogenases typically lack free alpha-amino groups. The blocked N-terminal regions of the class III type of the rat (ADH-2), human (chi chi) and horse enzymes were isolated by digestions with proteases, and characterized by mass-spectrometry supplemented with chemical analysis of the peptides and their redigestion fragments. Results were confirmed by synthesis of the corresponding peptides, followed by chromatographic comparisons of the native and synthetic products. The N-terminal regions of the three class III alcohol dehydrogenase subunits are homologous but differ from the class I and II enzymes in both the exact start position and the amino acid sequence, which suggests that different N-terminal structures are typical for each of the three classes.

Fast atom bombardment mass spectrometry and chemical analysis in determinations of acyl-blocked protein structures.

Peptide generation and fast atom bombardment mass spectrometry in combination with conventional chemical analysis was used to identify the blocking group and establish the N-terminal structure of six different proteins at the nanomole level. In this manner, the first terminal structures of three non-mammalian alcohol dehydrogenases were determined, demonstrating the presence of N-terminal acetylation in these piscine, amphibian, and avian enzymes. Similarly, two different yeast glucose-6-phosphate dehydrogenases and a minor variant of a human alcohol dehydrogenase were found to be acetylated. The exact end location of C-terminal structures was also established. Together, the analyses permit the definition of terminal regions and blocking groups, thus facilitating the delineation of remaining structures.

Morphological, biochemical, and molecular changes in endothelial cells after alpha-particle irradiation.

The response of cultured bovine aortic endothelial (BAE) cells after exposure to alpha-particle radiation from chelated 212Bi has been evaluated. The results suggest that even relatively high doses of alpha-particle radiation from 212Bi (20-72 Gy) cause only minor acute changes in the morphology of BAE cells (light and electron microscopy) under conditions of confluent monolayer growth. Significant morphological changes can be detected in cells that detach from the monolayer, though it is unclear whether these changes represent a genuine response to irradiation or reflect the causes or effects of monolayer detachment with the consequent loss of intercellular biochemical communication. After alpha-particle irradiation (20-40 Gy) angiotensin-converting-enzyme activity was not detectable in the monolayer culture medium but was significantly decreased within the cell monolayer. Neutral-elution-assay data demonstrated that DNA double-strand-break (DSB) damage occurred in these cells and that about 35% of the DSBs were repairable.

An experimental study of nerve grafting combined with silicone tubes in the rat model: Functional outcome and specificity of muscle reinnervation.

Bridging large defects in mixed nerves is still an unsolved problem in reconstructive microsurgery. Two main aspects may be distinguished: one is to obtain an appropriate substitute for the lost neural tissue, the second to direct fibers toward their previous end-organs with the highest possible specificity. In the present study, sural nerve block grafts were combined with enclosed gaps at one or both ends of the grafts. Functional outcome at the muscle level as well as the number of motor axons and their cross-sectional distribution were assessed after 3 months. The presence of a proximally placed tube was found to decrease significantly the maximal tetanic force of the tibialis anterior muscle, whereas a distally placed one tended to improve it. Morphological data from acetylcholinesterase histochemistry correlated poorly with functional results but they gave some clues about possible roles played by the chambers, according to their position relatively to the grafts. No definitive evidence for an improved regeneration by use of silicone tubes in addition to the conventional grafts could be demonstrated.

A mathematical model for regeneration rate and initial delay following surgical repair of peripheral nerves.

A mathematical model is presented by which the regeneration rate and initial delay for peripheral nerve regeneration can be calculated from sensory pinch test data following surgical repair of peripheral nerves. The model is based on the assumption that experimental variations in regeneration distances between animals is due to the initial delay period--the time period before the regenerating fibers cross the suture line whereas the rate of regeneration is constant. This model which accounts for all observed data including 'regenerating failures' showed that nerve fibers in the rat sciatic nerve repaired with a fresh nerve graft regenerated at a rate of 1.5 mm/day after an initial delay of 3.6 days.

Predegeneration enhances regeneration into acellular nerve grafts.

In the present study, we determined the regeneration rate and the initial delay in rat sciatic nerve grafts first made hypercellular by predegeneration then acellular by freeze-thawing. 7-day predegenerated nerve pieces from the distal nerve stump on the right side were made acellular by repeated freeze-thawing and inserted as grafts into a 10-mm long freshly created defect on the left contralateral side. Freshly made (no predegeneration period) acellular nerve grafts were used as controls. Both types of grafts supported outgrowth of regenerating axons as demonstrated by the sensory pinch test. However, the predegenerated acellular nerve grafts had a significantly shorter initial delay period (2.7 days) as compared with freshly made acellular nerve grafts (9.5 days). The initial delay period for predegenerated acellular nerve grafts was similar to that for fresh cellular nerve grafts but significantly longer than that for predegenerated cellular nerve grafts [24]. The rate of regeneration appeared independent of the type of grafts used. We suggest that modifications of the basal lamina and/or factors produced during the predegeneration period by non-neuronal cells survive the freeze-thawing cycle and account for the decrease in the initial delay period.

Pre-degenerated nerve grafts enhance regeneration by shortening the initial delay period.

In the present study we tested how nerve grafts with different pre-degeneration periods (1-28 days) influenced the early regenerative response in the rat sciatic nerve. The sciatic nerve on the right side was crushed and after 1-28 days of pre-degeneration, a 10 mm segment was used as an autologous nerve graft and transposed to a freshly made 10 mm long nerve defect on the left side. The regeneration distance was measured by the sensory pinch test 2-10 days after nerve repair. A newly developed mathematical model was used to calculate regeneration rates and initial delay periods from the measured regeneration distances. Pre-degenerated nerve grafts improved nerve regeneration by decreasing the initial delay period as compared to fresh nerve grafts without affecting the regeneration rate. Only one day of pre-degeneration was sufficient to reduce the initial delay period from 3.6 days to 1.7 days. The maximal effect on the initial delay period was achieved after 3 days of pre-degeneration. The initial delay period at later pre-degeneration intervals (7-14 days) was about 1 day. The effect persisted for at least 28 days of pre-degeneration. The regeneration rate was 1.5 mm/day for fresh nerve grafts and between 1.8-2.1 mm/day for pre-degenerated grafts. The results suggest that the effects of pre-degeneration are not only due to the increased cell proliferation in the graft, but that also trophic and/or inflammatory mechanisms may be of importance. Grafts pre-degenerated by crush may have clinical implications since they are easy to perform if an elective nerve grafting procedure is planned.

Identification of the carboxypeptidase responsible for the post-synthetic modification of creatine kinase in human serum.

The enzyme responsible for the post-translational modification of creatine kinase-MM isoenzyme was purified from human plasma. The enzymatic activity of this enzyme (modifying protein) on the synthetic substrates hippuryl-L-arginine, hippuryl-L-lysine, 3-(2-furylacryloyl)-L-arginine and 3-(2-furylacryloyl)-L-alanyl-L-lysine and the ratio of activities on these substrates are in good agreement with the enzymatic activity of the human serum carboxypeptidase N. The effect of metal ions, chelating agents, proteolytic inhibitors and carboxypeptidase N inhibitor could not differentiate the modifying protein from human serum carboxypeptidase N. Affinity chromatography on Concanavalin-A-Sepharose demonstrated the glycoprotein nature of the modifying protein. The difference in molecular weight observed between modifying protein and carboxypeptidase N can be explained by known instability characteristics and the influence of proteolytic enzymes during purification. Double immunodiffusion analysis with purified antiserum to human carboxypeptidase N confirmed the identity of the modifying protein and carboxypeptidase N.

Effects of organic solvents on motor activity in mice.

Groups of male mice were exposed via inhalation to methylene chloride, perchloroethylene, toluene, trichloroethylene or 1,1,1-trichloroethane. The exposures were started at 2300 h. Generation of vapor was stopped after 1 h. Motor activity of the animals during the exposures was measured with a Doppler radar. Several concentrations of each solvent were tested. Concentrations could be found for all solvents at which they initially increased the motor activity. When the generation of vapor was terminated and the concentration started to decline, a new phase of changes in motor activity was induced. At this phase, motor activity was in most cases influence in the opposite direction to that at the beginning of the exposure. Trichloroethylene concentrations could be found which gave no increase in activity at the start of exposure but a prominent decrease at termination. The lowest concentration at which effects could be seen was different for the different solvents. Perchloroethylene was more and 1,1,1-trichloroethane less potent than the other solvents in inducing motor activity. The time pattern of the motor activity alterations was specific for each solvent. Both the concentration and the rate of the concentration increase were responsible for the effects on motor activity. The differences between the solvents probably reflect differences in their site of action, their distribution and their biotransformation.

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.

Effects of mechanism-based reversible inhibitors on the metal environment of cobalt(II)carboxypeptidase A: an electronic spectral study.

Electronic absorption, circular dichroic (CD), and magnetic circular dichroic (MCD) spectra have been determined for complexes of cobalt(II)-substituted carboxypeptidase A and five reversible inhibitors. Three of the inhibitors, N-(1-carboxy-5-butyloxycarbonylaminopentyl)-L-phenylalanine, (I); (R,S)-2-benzyl-4-oxobutanoic acid, (III); and 2-benzyl-4-oxo-5,5,5-trifluoropentanoic acid, (IV) are mechanism-based inhibitors. Another, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine, (II), is a tight binding, slowly hydrolyzed substrate. The fifth, phosphoramidon, (V), is a mechanism-based inhibitor of thermolysin, and may also bind to carboxypeptidase in a mechanism-based mode. The absorption and CD spectra of the enzyme-inhibitor complexes all differ from the spectrum of the free enzyme and from each other. The MCD spectra indicate that the tetrahedral coordination geometry of cobalt, which is distorted in the free enzyme, is also distorted in the inhibitor complexes, although to various degrees. The complexes of I and III are spectrally similar despite being structurally dissimilar, and that of IV, whose structure resembles III, is spectrally distinct, indicating that I and III, but not IV, may perturb the metal in nearly the same way. The absorption spectrum of IV is identical to that, at high pH, of Co(II)carboxypeptidase in which Glu-270 has been modified by a carbodiimide reagent, possibly pointing to a common perturbation of this residue. The absorption and CD spectra of II are similar to those of the catalytic intermediate that precedes the rate-limiting step in peptide hydrolysis [D. S. Auld, A. Galdes, K. F. Geoghegan, B. Holmquist, R. Martinelli, and B. L. Vallee, Proc. Natl. Acad. Sci. USA 81, 4675-4681 (1984)]. Since II is a substrate, the steady-state bound species that it generates may therefore be a true productive intermediate rather than a nonproductive mimic of an intermediate. The spectra of the complexes with II and V differ considerably despite structural similarities. The negative CD ellipticity of the free enzyme is reversed in sign in the presence of V, a phenomenon previously observed with complexes of Co(II)carboxypeptidase and dipeptides. This resemblance may result from a similar interaction of cobalt with the phosphoramidate group of phosphoramidon and the N-terminal amine of dipeptides. The spectra of reversible, mechanism-based inhibitors permit general structural predictions about true intermediates but require caution when used for assigning precise conformation and ligands of bound catalytic species.

An angiotensin converting enzyme inhibitor is a tight-binding slow substrate of carboxypeptidase A.

Carboxypeptidase A-catalyzed hydrolysis of peptides and depsipeptides is competitively inhibited by N-(1-carboxy-5-t-butyloxycarbonylaminopentyl)-L-phenylalanine (Boc-CA-Phe, Ki = 1.3 microM) and the angiotensin converting enzyme inhibitor, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine (Z-CA-Gly-Phe, Ki = 4.5 microM). The latter compound is actually a slow substrate of carboxypeptidase. Indirect observation of inhibitor binding by stopped-flow measurement of radiationless energy transfer between carboxypeptidase tryptophans and dansylated substrates reveals slow binding for both compounds. The visible absorption spectrum of the complex of cobalt(II)-substituted carboxypeptidase and Z-CA-Gly-Phe, which differs from the corresponding spectrum of the Boc-CA-Phe complex, is remarkable in its resemblance to the spectrum of the complex between Co(II)carboxypeptidase and a transient intermediate previously observed during hydrolysis of peptide substrates. The spectrum slowly changes to that of the free enzyme indicating hydrolysis. Chromatographic quantitation of substrate and products confirms that carboxypeptidase converts Z-CA-Gly-Phe to Z-CA-Gly and L-Phe with an apparent kcat of 0.02 s-1. Absorption spectroscopy indicates that the Z-CA-Gly-Phe-Co(II)carboxypeptidase spectrum is not that of bound products. Moreover, spectral titrations indicate that the products (both with spectral Ki values of about 3 mM), as well as D-Phe, compete for the same site on the enzyme.

Thioamide substrate probes of metal-substrate interactions in carboxypeptidase A catalysis.

Three thioamide peptides in which the oxygen atom of the scissile peptide bond is replaced by sulfur (denoted by (= S)) were synthesized and found to be good, convenient substrates for carboxypeptidase A. The thioamide bond absorbs strongly in the ultraviolet region, and enzymatic hydrolysis is monitored easily using a continuously recording spectrophotometric assay. The reaction follows Michaelis-Menten kinetics with kcat values of 68, 9.0, and 3.7 sec-1 and Km values of 0.83, 0.81, and 0.53 mM for Z-Glu-Phe(= S)-Phe, Z-Gly-Ala(= S)-Phe, and Z-Phe(= S)-Phe, respectively. Activities of the thioamides and their oxygen amide analogs were determined with a series of metal-substituted carboxypeptidases. The Cd(II), Mn(II), Co(II), and Ni(II) enzymes exhibit 30%-35%, 60%-85%, 150%-190%, and 40%-55% of the Zn(II) enzyme activity with the amide substrates; this compares with 240%-970%, 0%-15%, 340%-840%, and 30%-140% of the Zn(II) activity, respectively, with the thioamides. The activity of the Cu(II) and Hg(II) enzymes is less than 3% toward all substrates. Cadmium, a thiophilic metal, yields an enzyme which is exceedingly active with the thioamides; the kcat/Km values are 2.4-9.7-fold higher than with Zn(II) carboxypeptidase. In contrast, Mn(II), which has a relatively low affinity for sulfur, yields an enzyme with correspondingly low activity toward the thioamides. The results are consistent with a mechanism for peptide bond hydrolysis in which the metal atom interacts with the substrate carbonyl atom during catalysis.

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.

Pitfalls in the interpretation of long-term inhalation experiments.

Age- and weight-matched groups of mice were enclosed in airtight chambers and exposed to clean, filtered air for 1 month. At the end of the exposure period, body, liver and spleen weights and plasma butyrylcholinesterase (BuChE) activity were measured. More than twice as many significant differences in these parameters occurred compared with the expected results if only random differences existed between the groups. Thus, isolation of animal groups for extended periods of time in inhalation experiments alone may lead to differences in various biological parameters. When testing the effects of unknown substances such differences may be mistaken for reactions to the test agent, which actually may have no effect.

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%.

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.

Analysis of film coating thickness and surface area of pharmaceutical pellets using fluorescence microscopy and image analysis.

A method is presented which enables geometrical characterisation of pharmaceutical pellets and their film coating. It provides a high level of details on the single pellet level. Image analysis was used to determine the coating thickness (h) applied on the pellets and the surface area (A) of the pellet cores. Different definitions of A and h are evaluated. Hierarchical analysis of variance was used to resolve different sources contributing to the total variance. The variance within pellets and the variance between pellets were found as significant sources of variation. Special emphasis was put on evaluation of A/h due to its influence on the release rate of an active drug substance from the pellet core. The pellet images were thus used to predict variations in the release rate using a mathematical model as a link between the image data and the release rate. General aspects of image analysis are discussed. The method would be useful in calibration of near infrared spectra to h in process analytical chemistry.