Pyridoxal phosphate-dependent reactions in water pools.

Pyridoxal 5'-phosphate, its Schiff base with L-alanine, and cytosolic aspartate aminotransferase were dissolved in isooctane solutions containing reverse micelles of the surfactant di-2-ethylhexylsodium sulfosuccinate and water. The physico-chemical properties of these compounds in the new environment have been studied.

Metabolism of straight saturated medium chain length (C9 to C12) dicarboxylic acids.

A method utilizing thin-layer chromatography, high performance liquid chromatography, and mass spectrometry was developed for the quantification of C9, C10, C11, and C12 dicarboxylic acids in serum, urine, and feces of human volunteers and rats after oral administration of the acids. The method allowed good resolution and measurement of the dicarboxylic acids at nanogram levels. In humans, excretion was independent of the dosage; about 60% of C9, 17% of C10, 5% of C11, and 1% of C12 were excreted in the urine during the first 12 hours after administration. The concentration of the acids in serum peaked between 2 and 3 hours. Excretion was also independent of dosage in rats. About 2.5% of C, 2.1% of C10, 1.8% of C11, and 1.6% of C12 were excreted in the urine over a period of 5 days. The serum concentration and the urinary excretion of the diacids reached a maximum at the second day after the oral dose. In both humans and rats, the dicarboxylic acids found in serum and urine were 2, 4, or 6 carbon atoms shorter than the corresponding administered diacid. This indicates that there was beta-oxidation of the ingested diacids to some extent. The administration of [1,9-14C]azeliac acid and of [10,11-3H]dodecandioic acid confirmed the occurrence of beta-oxidation, and led to elucidation of the fate of the ingested diacids that were not excreted as such in the urine.

X-ray absorption near edge structure (XANES) determination of calcium sites of troponin C and parvalbumin.

Using synchrotron radiation at the Frascati storage ring ADONE, the X-ray Absorption Near Edge Structure (XANES) has been applied to determine homologies and modifications of the local structure of the calcium binding sites of troponin C. In all four calcium binding sites, Ca2+ appears to be co-ordinated to carboxyl and carbonyl groups in a characteristic configuration. No structural difference has been found between high and low-affinity sites. A distortion of the Ca2+ site geometry by binding of Mg2+ has been observed. The XANES of parvalbumin has been measured and found to be different from troponin C. A tentative identification of the characteristic XANES spectra of the two different Ca2+ sites in this protein is reported.

Non-invasive near infrared spectroscopy of brain in fluorocarbon exchange-transfused rats.

Near infrared spectra (700-930 nm) were recorded from the head of anesthetized rats before and after blood substitution with a fluorocarbon emulsion. A large band, centered at 850 nm and characteristic of oxidized cytochrome a, a3, was evidenced in hemoglobin-free living animals. Variations in the amplitude of this absorption band were observed under different respiratory conditions.

Circular-dichroism study of the interaction of aspartate-aminotransferase isoenzymes with a coenzyme analog.

The interaction between a coenzyme derivative, 4'-N-(2,4-dinitro-5-fluorophenyl)-pyridoxamine 5'-phosphate, and the apoenzyme of cytoplasmic and mitochondrial aspartate aminotransferase, was studied by circular dichroism. The specific complexes initially formed were characterized by their circular dichroic spectra. The spectra indicate that the complex is very probably the same for the two isoenzymes. In contrast the spectra recorded during further reaction, in agreement with previous results, monitor different reaction paths and characterize the irreversible labeling at the active site of the cytoplasmic enzyme and regeneration of pyridoxal 5'-phosphate in the mitochondrial enzyme. By following circular dichroic changes in the mitochondrial enzyme, initial kinetic characterization of the cleavage of 4'-N-(2,4-dinitro-5-fluorophenyl)-pyridoxamine 5'-phosphate to form pyridoxal 5'-phosphate at the active site, is provided.

Rat brain monitoring by near-infrared spectroscopy: an assessment of possible clinical significance.

Near-infrared 700-950 absorbance of some chromophores has been measured in different biological tissues, in particular rat and rabbit brain, to assess value of this noninvasive technique for monitoring of the regional functional state. Experiments carried out under different induced circulatory conditions confirmed the possibility of obtaining information on local blood content and hemoglobin oxygenation level as well as an indication of redox state of cytochrome c oxidase, the enzymatic complex that catalyzes about 95% of cellular oxygen consumption. The near IR spectral data were correlated with the result of independent simultaneous measurements of circulatory function: blood pressure, local blood flow, and local blood volume.

Kinetics of coupled reactions catalyzed by aspartate aminotransferase and glutamate dehydrogenase.

Liver mitochondrial aspartate aminotransferase and glutamate dehydrogenase catalyze following sequence of reactions: see formula in text. In the presence of a slight excess of dehydrogenase, the time course of NADPH oxidation resulting from the overall reaction goes through a lag phase and reaches a linear phase. The slopes of the linear part of this curve is a linear function of transaminase concentration. At high concentration (approximately or equal to 10 microM) of both enzymes the lag phase, as observed after rapid mixing of the two enzymes in a Durrum stopped-flow spectrophotometer, is shorter, than that predicted from the kinetic parameters determined for the separate reactions catalyzed by each enzyme.

An application of high performance liquid chromatography to analysis of lipids in archaeological samples.

Five samples from three different types of 1500-year-old Mediterranean amphorae, as well as from a contemporary oil lamp found in the same deposit, were analyzed for the presence of lipid residues. Each sample of finely ground amphorae powder weighed 1-2 g. The abundance of interfering secondary products makes thin-layer chromatography (TLC) an essential step of the procedure. The fractionation of the extract into its various lipid components by means of TLC was followed by quantitative recovery of the triglyceride (TG) and free fatty acid (FFA) fractions from the plates and by the measurement of their components by high performance liquid chromatography (HPLC) after esterification. The minimum detectable level is 1 ng. The amphorae samples revealed a more abundant FFA fraction than a TG fraction, which is the reverse of what we know about the composition of fresh oil. Despite the considerable age of the amphorae and their preservation under non-ideal conditions, the lipid residues have retained certain identifiable characteristics that enable one to make valid suggestions as to the type of commodity originally transported in the amphorae. The results of these experiments yield important information that enables the biochemist to observe an aging process irreproducible in the laboratory and provides the archaeologist with previously unavailable information about trade 15 centuries ago.

Evidence for the presence of dipeptidyl carboxypeptidase and its inhibitors in inflammatory synovial fluids.

Evidence is reported for the presence of dipeptidyl carboxypeptidase (peptidyldipeptide hydrolase, EC 3.4.15.1) and of inhibitor(s) of this enzyme in synovial fluids from patients with rheumatoid arthritis and gout.

ATP-dependent exonuclease V from Micrococcus luteus. The enzyme-DNA complex, the processive mechanism, and the role of ATP.

Some kinetic predictions of the proposed processive mechanism for the hydrolysis of DNA by the ATP-dependent enzyme exonuclease V have been checked. The method is to trap enzyme molecules not attached to radioactive DNA substrate with an excess of nonradioactive DNA, so that enzyme molecules attached to the radioactive substrate contribute to the liberation of radioactive products only until they dissociate from it. The experiments show that enzyme molecules remain attached to a T7 double-stranded DNA molecule, while hydrolysing it, for about 2 min under our conditions, in agreement with the predictions of the processive mechanism. However, the mechanism of degradation of single-stranded DNA is not processive. Formation of an enzyme-DNA complex is largely dependent on the presence of ATP. This formation does not appear to be synchronous. ATP analogs do not stimulate formation of, nor stabilize, the enzyme-DNA complex. EDTA causes dissociation of enzyme molecules from the DNA complex.

Light and GTP effects on the turbidity of frog visual membrane suspensions.

The time course of turbidity changes of frog visual membranes, dependent on osmotic shocks, on light and on nucleotide substrates or effectors of enzyme activities, were measured as absorption changes in a rapid mixing stopped-flow spectrophotometer. As a result of studies on different preparations, it is concluded that light can cause both rapid (within 50 msec) and slow (within 90 sec) changes in the turbidity of visual membranes, not associated with permeability changes, and that they are affected by GTP or its analog guanyl-5'-yl imidodiphosphate; however, the light and GTP effects are lost when a water soluble fraction containing the light-sensitive enzyme cGMP-phosphodiesterase, is removed from the rod outer segments membranes. It is suggested that the fast light and GTP-sensitive response is related to the activation of cGMP-phosphodiesterase.

An improved viscosimetric assay for vertebrate collagenase activity.

An improved viscosimetric assay for vertebrate collagenase acitivity is described. The assay is carried out at 35 degrees C in the presence of 1 M glucose to prevent fibril formation. The decrease in viscosity is linear with ime and proportional to enzyme concentration.

Collagenase activity in human synovial fluids from joint diseases of diverse etiology.

The content of collagenase in latent as well as in apparent active form has been determined in synovial fluids from 21 patients with rheumatoid arthritis, 16 with non-rheumatoid inflammatory joint diseases, and 15 with degenerative joint disease. Collagenase activity was measured before and after activation of the latent enzyme by NaSCN treatment. Before activation, collagenase activity was present in the synovial fluids from 1 case of rheumatoid arthritis, 3 cases of degenerative joint disease, and 1 case of Behçet's syndrome. An excess of inhibitor was present in inactive synovial fluids. The incidence of collagenolytic activity markedly increased after treatment with NaSCN. When NaSCN-dependent collagenolytic activity was present, its value was of the same order of magnitude in all patients regardless of disease type. The diagnostic value of the finding of collagenase activity in synovial fluid, and the physiological meaning of the enzyme with reference to joint diseases, are discussed.

Catalytic activity of aspartate aminotransferase in the crystal. Equilibrium and kinetic analysis.

Natural substrates and analogs rapidly diffuse through crystals of pig heart mitochondrial aspartate aminotransferase and react at the active sites causing spectral changes that can be measured by single-crystal microspectrophotometry. Dissociation constants for natural substrates and rate constants of transamination for slowly reacting substrates have been determined. A comparison between the data obtained in the crystal and in solution shows that the crystalline enzyme is catalytically competent and that events occurring in the crystal essentially parallel those occurring in solution, even though minor differences have been detected.