Takeo Matsuzawa

Crystallization of ornithine transaminase and its properties

Abstract It is well known that ornithine-ketoacid transaminase(OKT) catalyzes the interconversion of ornithine and gluaamic-δ-semialdehyde with concomitant interconversion of α-ketoglutarate and glutamate in animal tissues. In 1963 Peraino and Pitot reported some properties of OKT using partially purified enzyme. In 1964 we reported the purification of the enzyme to a single component by ultracentrifugal and electrophoretic analysis. Strecher also reported some important kinetical properties using partially purified enzyme. Recently, we purified and crystallized the enzyme in good yiels from rat liver. This is reported in this paper with some physicochemical properties of OKT.

Retinal damage in hatched chicks induced by formoguanamine. Decrease in ornithine aminotransferase activity and vitamin B6 content

Subcutaneous injection of formoguanamine (2,4-diamino-s-triazine) induced sightlessness in newly hatched chicks within 28-32 hr. The specific activity of retinal ornithine aminotransferase, localized exclusively in the mitochondria of retinal pigment epithelium, and the retinal vitamin B6 content decreased rapidly after formoguanamine administration. An absorption spectrum of retinal extract showed reduced absorbance at 400 nm in the formoguanamine-treated chicks, but delta 1-pyrroline-5-carboxylate reductase, an enzyme in the retinal cytosol, did not change significantly. A drug-metabolizing enzyme, glutathione transferase, showed an increase in specific activity during a later phase. Histological examination of the formoguanamine-treated chicks revealed characteristic degeneration of pigment epithelium and photoreceptors, acute retinal detachment and a secondary gliosis in the outer nuclear layer. From these findings it may be concluded that formoguanamine primarily damages the physiological functions of retinal pigment epithelium and photoreceptors and results in irreversible retinal detachment in newly hatched chicks.

Amino acid synthesis from ornithine: enzymes and quantitative comparison in brain slices and detached retinas from rats and chicks.

The regional distribution of proline biosynthetic enzymes, ornithine-delta-aminotransferase and delta 1-pyrroline-5-carboxylate reductase, in the rat brain, and basic conditions for proline synthesis from ornithine in rat and chicken brain slices and chicken retinas were investigated. The cerebral regions relating to memory formation and imprinting (cortex, hypothalamus and hippocampus) exhibited a high activity of ornithine-delta-aminotransferase, while delta 1-pyrroline-5-carboxylate reductase activity was ubiquitously high. Amino acids were determined fluorometrically after separation and reaction with o-phthaldialdehyde by high-performance liquid chromatography. Proline formation was both ornithine- and 2-oxoglutarate-dependent, and the proline level was suppressed by a high-potassium medium in the brain slices but not in the detached retinas. Its main precursor in vitro seemed to be ornithine but not arginine. The retinas from formoguanamine (2,4-diamino-S-triazine)-treated chicks showed a 10-fold higher level of proline and a marked decrease in gamma-aminobutyric acid, presumably due to an impairment of the blood-retina barrier. The different response in proline level to the high-potassium medium in the brain slices and detached neuroretinas suggests that cellular distribution of the enzymes relating to ornithine and proline metabolism is different in the brain and the neuroretina.

A simple micromethod for determining human serum cortisol by high-pressure liquid chromatography using 0.1 ml serum

Abstract A simple method of high-pressure liquid chromatography for the determination of cortisol in 0.1 ml of human serum is described. Multiple steps of the previous procedures can be simplified by shaking a glass rod for effective extraction of hormones and by performing all procedures on a microscale. The recovery rate was better than that of the previous method and the coefficient of variation was 5.3%. The present method which has a limit of detection of 1–30 ng is particularly useful for the assay of serum cortisol in infantile patients.

Enzymatic assays of l-ornithine and l-Δ1-pyrroline-5-carboxylate in tissues, and ornithine-load test in human subjects

Abstract A specific enzymatic method was developed for the determinations of l -ornithine and l -Δ 1 -pyrroline-5-carboxylate using two coupling enzymes, ornithine aminotransferase and Δ 1 -pyrroline-5-carboxylate reductase. Using this method, ornithine in rat tissues and human serum could be determined at the nanomole level. The fluorometric modification of this method was used for the assay of ornithine in blood. In the ornithine-load test in human subjects, diabetic patients showed an abnormally low peak and liver cirrhotic ones gave a delayed peak in their blood ornithine response curve.

Disease of Ornithine-Proline Pathway: A △1-pyrroline-5-carboxylate Reductase Deficiency in the Retina of Retinal Degeneration Mice

The discovery of a genetic defect in ornithine oxoacid aminotransferase (L-ornithine: 2-oxoacid aminotransferase, EC 2.6.1.13) in gyrate atrophy of the choroid and retina1,2,3, prompted us to study the ornithine metabolism of the eye4. We soon found high 361 activities of ornithine oxoacid aminotransferase and △1-pyrroline-5-carboxylate reductase (L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2) (P5C reductase) in the retina and retinal pigment epithelium4,5. Gyrate atrophy of the choroid and retina is a disease related to the category of choroideremia and retinitis pigmentosa (primary, hereditary, pigmentary retinopathy), these are much frequent cause of the human hereditary blindness, however the biochemical pathogenesis of these diseases remains to be solved. In this paper we describe an intracellular proline synthetic pathway from ornithine uncovered in the bovine cornea and retinal outer layers(including the choroid) which presumably participates in the biosynthesis of proline-rich proteins such as collagen and glycoproteins, and the characteristic postnatal changes and the deficiency of this pathway in C3H retinal degeneration mice.

Enzymes metabolizing ornithine-proline pathway in the rovine eye

We studied bovine ocular tissues biochemically for the following enzyme activities related to ornithine metabolism: arginase, Δ1-pyrroline-5-carboxylate reductase, Δ1-pyrroline-5-carboxylate dehydrogenase and proline oxidase. Arginase activity was high in the retina and uvea; Δ1-pyrroline-5-carboxylate reductase activity was high in the lens, cornea and retina; Δ1-pyrroline-5-carboxylate dehydrogenase activity was high in the uvea but lower in the retina; and proline oxidase activity was negligible in all ocular tissues. It is possible that ornithine in the retina may be supplied from arginine, or from the uptake of ornithine itself, and converted into proline by the cooperative action of ornithine-ketoacid-transaminase and Δ1-pyrroline-5-carboxylate reductase.

Ornithine metabolism in relation to stimulation of urea cycle, induced by high protein diet☆

Abstract Approximate rates of some in vivo ornithine metabolisms in rats were calculated by pulse-labeling data, on the assumption that hepatic metabolite levels are constant during a given 3-min period. The rate of ornithine catabolism was 70–110 nmol/min/g liver; that of urea output was 3–6 μmol/min/g liver; the rotary rate of the “ornithine flux” was 10–12 rpm. A change from a 25 to a 70% casein diet resulted in a 1.5-fold augmentation in the rate of ornithine catabolism and a 1.6-fold increase in the rate of urea output; however, the rate of the “ornithine flux” remained nearly constant These findings suggest that stimulation of the urea cycle is accompanied not by acceleration of the cycle rotation, but rather by increased mass of the metabolite flux.

Colorimetric assays for serum alanine transaminase and lactic dehydrogenase using diazonium zinc salt

Abstract Rapid and accurate methods for the determination of alanine transaminase and lactic dehydrogenase in serum and tissue using the diazonium salt and the coupling enzymes have been developed. Several factors affecting the coupling reactions were studied in detail and are discussed. By the present methods it was possible to measure the ranges of 0–150 m-I.U. in alanine transaminase assay and 0–1500 m-I.U. in lactic dehydrogenase assay.

Ornithine oxoacid aminotransferase found in AH 130 ascites hepatoma cells

We found heterogenous ornithine oxoacid aminotransferase (L-ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.1.3) in rat ascites hepatoma AH 130 cells. Compared with enzymes from normal rat tissues, this heterogenous enzyme showed larger Km values for 2-oxoglutarate, a different elution-profile upon affinity chromatography with 2-oxoglutarate, more anionic mobility upon polyacrylamide gell electrophoresis, and a clearly different salting-out property upon ammonium sulfate fractionation. Similar heterogeneity of this aminotransferase was found in human cancer cells.