The reaction mechanism of bovine kidney biliverdin reductase.

The steady-state kinetics of biliverdin reductase can be studied in detail at pH 9 as under these conditions the Km for biliverdin is high enough to obtain reliable measurements of the initial rate in the absence of any biliverdin binding proteins. The initial rate kinetics and the product-inhibition studies are consistent with an ordered sequential mechanism provided the biliverdin concentration was below 20 microM. Above this concentration significant flux occurs through a substrate inhibition pathway involving an enzyme-NAD(P)-biliverdin complex. Chloride is shown to cause a significant activation of the enzyme under certain conditions and this is shown to result from an inability of biliverdin to bind to an enzyme-NAD-chloride complex.

Molecular structure at 1.8 A of mouse liver class pi glutathione S-transferase complexed with S-(p-nitrobenzyl)glutathione and other inhibitors.

The three-dimensional crystal structure of pi class glutathione S-transferase YfYf from mouse liver complexed with the inhibitor S-(p-nitrobenzyl)glutathione has been determined at 1.8 A resolution by X-ray diffraction. In addition two complexes with glutathione sulphonic acid and S-hexylglutathione have been determined at resolutions of 1.9 and 2.2 A, respectively. The high resolution of the S-(p-nitrobenzyl)glutathione complex allows a detailed analysis of the active site including the hydrophobic (H-) subsite. The nitrobenzyl moiety occupies a hydrophobic pocket with its aromatic ring sandwiched between Phe8 and the hydroxyl group of Tyr108. An insertion of two residues Gly41 and Leu42, with respect to the pig enzyme, splits helix alpha B into an alpha-helix and a 3(10) helix. Water bridges between carbonyl oxygen atoms of the alpha-helix at its C terminus and the amide NH groups of the 3(10) helix at its N terminus provide structural continuity between these two secondary elements. Tyr7 appears to be the only residue close to the sulphur atom of glutathione, while three conserved water molecules lie in the surrounding area in all complexes. The enzyme mechanism is discussed on the basis of the structural analysis.

A normal population study of human salivary insulin-like growth factor 1 (IGF 1) concentrations from birth through puberty.

Insulin-like growth factor 1 (IGF 1) concentrations in mixed saliva samples, collected from a normal population (n = 327, ranging in age from birth to adolescence), were determined by RIA. Salivary IGF 1 concentrations remained steady over a 24-h period when collected at basal rates, but were diminished in saliva samples collected at a maximally stimulated flow rate. A similar pattern was observed for males and females, when IGF 1 levels in saliva were plotted as a function of age. The pattern was that of low levels in early childhood, rising with age, peaking in puberty and falling again in late adolescence. Salivary IGF 1 measurement differed from plasma measurement in three ways: 1) salivary IGF 1 concentrations (70 +/- 50 pM) were 100- to 200-fold less than plasma IGF 1 levels; 2) salivary IGF 1 levels in age-matched male and female samples were not different outside of pubertal influences; 3) salivary IGF 1 levels in neonates were highly variable with concentrations ranging up to pubertal concentrations. The study provides salivary IGF 1 reference data for a pediatric population.

Salivary insulin-like growth factor-I originates from local synthesis.

Insulin-like growth factor-I (IGF-I) is a GH-dependent growth factor found in its highest concentrations in plasma. It is also measurable in saliva. The origins of salivary IGF-I concentrations were studied. Intracardial administration of Sprague-Dawley rats with 125I-labelled IGF-I and subsequent analysis of plasma and saliva samples by exclusion gel chromatography and SDS-PAGE, followed by autoradiography, demonstrated the apparent inability of IGF-I to cross from the plasma pool through to saliva. 125I-Labelled IGF-I was not chromatographed immediately before injection, resulting in administration of free iodide along with the iodinated peptide. This free iodide was demonstrable in saliva, indicating that movement of substances from plasma to saliva was measurable using the levels of 125I activity administered. Free iodide in saliva was not contributed to by 125I-labelled IGF-I degradation since 125I-labelled IGF-I was shown to be stable in saliva over 24 h. These data indicated that IGF-I in saliva is produced locally. Identification of a 4.7 kb IGF-I mRNA transcript in rat parotid salivary gland was consistent with IGF-I synthesis within that tissue.

The nature of the inhibition of rat liver monoamine oxidase types A and B by the acetylenic inhibitors clorgyline, l-deprenyl and pargyline.

The kinetics of inhibition of rat liver mitochondrial monoamine oxidase by clorgyline, l-deprenyl and pargyline are consistent with a mechanism whereby a reversible interaction between the inhibitor and the enzyme active site under conditions of thermodynamic equilibrium is followed by a time-dependent formation of the covalently-bound enzyme-inhibitor adduct. The Ki value for the reversible interaction between clorgyline and monoamine oxidase A is about 1000 times lower than that towards the B-form of the enzyme, and this difference is sufficient to account for most, but not all, of the selectivity of the inhibition caused by this compound. The Ki value of the monoamine oxidase B selective inhibitor l-deprenyl towards that form of the enzyme is only about 40-fold lower than that towards the A-form. However, in this case, the rate of formation of the irreversible adduct is considerably faster for the B-form than for the A-form and this makes a major contribution to the selectivity of this compound. Pargyline shows a Ki value towards monoamine oxidase B that is only 8 times lower than that towards the A-form and in this case the rates of formation of the enzyme-inhibitor adducts are similar. The significance of these results are discussed in terms of the selective inhibition of monoamine oxidase.

On the possible role of biliverdin stimulation of cyclic AMP levels as a trigger for liver regeneration in the rat.

Contrary to a recent report by Okazaki et al. (Biochem. biophys. Res. Commun., 1978, 81, 512-520) we show that high concentrations of biliverdin (100 mg/kg, i.p.) slightly reduce the mitotic rate in rat liver. Adenylate cyclase and both the "high Km" and "low Km" phosphodiesterase of rat liver plasma membranes are inhibited by high concentrations of biliverdin. Biliverdin has no effect on cyclic AMP production in isolated hepatocytes. An intracellular binding protein (glutathione-S-transferase B) is shown to bind biliverdin providing a mechanism for maintaining a low free intracellular concentration of the tetrapyrrole analogous to that of albumin in plasma. In conclusion, these results are not consistent with a role for biliverdin in stimulating liver regeneration in the rat via a mechanism involving elevated cyclic AMP levels.

5-Hydroxytryptamine is a substrate for both species of monoamine oxidase in beef heart mitochondria.

The activity of beef heart mitochondrial monoamine oxidase towards 5-hydroxytryptamine (5-HT) is inhibited by the selective inhibitors clorgyline, PCO [5-phenyl-3-(N-cyclopropyl)-ethylamine-1,2,4-oxidiazole] and Deprenyl with a biphasic dependence on the inhibitor concentration. The activities towards tyramine, dopamine and tryptamine were also inhibited in a biphasic manner, but the apparent proportions of the two enzyme species active on dopamine and tryptamine depended on the inhibitor used. Phenethylamine oxidation was inhibited in a monophasic manner suggesting that only a single enzyme species was responsible for the oxidation of this substrate. The biphasic response of 5-HT oxidation to inhibition by clorgyline persisted when functionally competent mitochondria were used and was unaffected by the soluble amine oxidase inhibitors semicarbazine and aminoguanidine. These results indicate that the behaviour of the beef heart enzyme towards selective inhibitors is considerably different from that of any preparations previously studied and suggest that the classification of monoamine oxidase activites into A and B types may be only of limited usefulness.

Haem degradation in animals and plants.

Two enzyme systems have evolved for the reduction of linear tetrapyrroles: one family, found in plants, algae and cyanobacteria, uses ferredoxin and catalyses the reduction of the terminal pyrrole rings (A and D) and one of the vinyl side chains to form various light-harvesting and light-sensing chromophores. The other group (biliverdin reductases A and B) utilize NAD(P)H and catalyse reduction at C10 (hydride addition) to form the 'bile' pigments bilirubin-IX alpha and bilirubin-IX.

Enzymes: nature's nanomachines. Royal Irish Academy Medal Lecture.

The development of enzyme kinetics, protein crystallography and NMR studies allows enzyme-catalysed reactions to be described in terms of mechanistic chemistry, albeit applied to relatively enormous molecules. These nanomachines, which so inspired Drexler's "Engines of Creation," have been working in biological systems for over three billion years and represent a useful knowledge base for our further understanding of mechanistic biology. They also provide a tantalizing glimpse into what may be the basis for novel technologies with industrial applications for the twenty-first century.

Studies on the glutathione S-transferase activity associated with rat liver mitochondria.

The major proportion of rat liver glutathione S-transferase is cytosolic. Carefully washed mitochondria contain 0.25-0.47% of the cytosolic activity. Subfractionation of washed mitochondria using digitonin treatment revealed that glutathione S-transferase release did not parallel that of any of the mitochondrial marker enzymes. Glutathione S-transferase release paralleled that of lactate dehydrogenase, suggesting that these 'mitochondrial' activities are due to loosely bound cytoplasmic forms.