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CuInSe 2 and CuIn 3 Se 5 thin films have been deposited using sodium compounds such as Na 2 Se and Na 2 S onto Corning 7059 glass substrates by the two-stage co-evaporation method. Enhanced grain growth and preferred (1 1 2) grain orientation as well as a decrease in resistivity with respect to undoped films were observed with sodium incorporation. A clear correlation between the photoluminescence spectra and the resistivity of the films was found by comparing the properties of films with and without Na incorporation. These observations suggest that compensation is reduced due to the suppression of donor-type defects by the presence of Na.

High-purity cubic GaN films have been successfully grown on a AlGaAs buffer layer by molecular beam epitaxy. A characteristic nitrided AlGaAs smooth faceted surface was observed. It was found that GaN epilayers grown on a nitrided Al 0.17 Ga 0.83 As buffer layer were epitaxial and contained only the cubic phase. No diffraction peaks associated with the hexagonal phase of GaN were observed by X-ray pole-figure measurements.

The surface morphology of several kinds of buffer layers, nitrided GaAs(1 0 0), low-temperature GaN, GaAs buffer layer, and GaAlAs buffer layer, is investigated for growing c-GaN using GaAs(1 0 0) by molecular beam epitaxy. The possibility of an epitaxial GaAlAs buffer layer for growing high-quality c-GaN on GaAs(1 0 0) is considered. A c-GaN film with a few h-GaN imperfections is successfully grown on a GaAs buffer layer, and the occurrence of h-GaN is drastically reduced by introducing an epitaxial GaAs buffer layer.

Low-resistivity ZnO films were grown by photo atomic layer deposition (photo-ALD) technique using diethylzinc (DEZ) and H2O as reactant gases. Self-limiting growth was achieved for the temperature range from 105°C to 235°C. It was found that UV light irradiation was very effective to increase the electron concentration of the films and the electron concentration of 5 × 1020 cm−3 was achieved even in undoped ZnO. Thus, the resistivity of the films grown with UV irradiation was one order of magnitude less than that grown without UV irradiation. The minimum resistivity of 6.9 × 10−4Ω cm was obtained by photo- ALD method without any intentional doping.

Compared with the activity obtained with a high-protein diet in rats, a low-protein diet doubled the activity of ornithine aminotransferase [EC 2.6.1.13] (OAT), a key enzyme for citrulline synthesis, in the small intestine. The induction of ornithine aminotransferase in the small intestine by the low-protein diet and its suppression by the high-protein diet, and the converse in the liver, were immunohistochemically verified with anti-OAT antiserum. The immunohistochemical studies revealed that ornithine aminotransferase molecules localized in the villous surface epithelia, but not in the cryptic epithelia, were most responsive to the changes in dietary conditions, these results indicating that intestinal ornithine aminotransferase may be involved in the ornithine supply to the liver, with the reversal of the enzyme reaction occurring with a low-protein diet. Reconstituted model experiments on citrulline synthesis revealed that the addition of ornithine carbamoyl-transferase and carbamoyl phosphate was essential to overcome the unfavorable equilibrium of the reverse reaction, and the further addition of glutamate dehydrogenase and ammonia resulted in a stimulating effect.

A new enzymatic assay method for ornithine carbamoyltransferase, OCT, activity was developed using ornithine:2-oxo-acid aminotransferase, OKAT, and 1-pyrroline-5-carboxylate dehydrogenase, P5CDH. The method is based on the reaction of L-ornithine formation from L-citrulline by OCT. The OCT activity was measured spectrophotometrically at 340 nm and the minimum detectable activity was about 4 units/L of serum. Results obtained by this method and by the chemical method correlated well. This method is superior to the chemical method, as it does not require boiling of the reaction mixture nor removal of interfering substances in the serum.

The complete amino acid sequence of human liver cytosolic alanine aminotransferase (GPT) (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. Isolated peptides were analyzed with a protein sequencer or with a plasma desorption time of flight mass spectrometer and placed in the sequence on the basis of their molecular mass and homology to the sequence of rat GPT. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The Mr of the subunit was calculated to be 54,479, which was in good agreement with a Mr of 55,000 estimated by SDS-PAGE, and also indicated that the active enzyme with a Mr of 114,000 was a homodimer composed of two identical subunits. The amino acid sequence is highly homologous to that of rat GPT (87.9% identity) recently determined [Ishiguro, M., Suzuki, M., Takio, K., Matsuzawa, T., & Titani, K. (1991) Biochemistry 30, 6048-6053]. All of the crucial amino acid residues are conserved in human GPT, which seem to be hydrogen bonding to pyridoxal 5'-phosphate in rat GPT by the sequence homology to other alpha-aminotransferases with known tertiary structures.

The complete amino acid sequence of rat kidney ornithine aminotransferase [EC 2.6.1.13] is presented. The 404-residue sequence was determined by analysis of peptides generated by digestion of the S-carboxyamidomethylated protein with CNBr, Achromobacter protease I, arginylendopeptidase, or Staphylococcus aureus V8 protease. Mueckler and Pitot have reported the amino acid sequence of the rat liver enzyme (440 residues) as predicted from the nucleotide sequence of the cDNA [Mueckler, M.M. & Pitot, H.C. (1985) J. Biol. Chem. 260, 12993-12997]. The amino acid sequence of the rat kidney enzyme presented herein coincides with residue 36 (Gly) through 440 (Phe) of the predicted precursor protein, indicating that the liver and kidney enzymes are identical, and that the enzyme is processed at the amino-terminal region after translation.

Background: Serum ornithine carbamoyltransferase is a diagnostic marker of hepatic disorders due to its localization in periportal mitochondria. Methods: We have developed a new method for the determination of serum ornithine carbamoyltransferase. It is based on the reverse reaction of ornithine carbamoyltransferase, using ornithine-ketoacid aminotransferase, ∆1-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase, which together convert citrulline through ornithine to glutamate. The glutamate is then quantitatively measured using glutamate oxidase and Trinder's reagent. Results: The results obtained by this method agreed well with those obtained using the diacetylmonoxime method as a gold standard [correlation coefficient (r) = 0·973 P1-pyrroline-5-carboxylate) were eliminated by the initial futile reaction. The new method appears to be more accurate at low levels of ornithine carbamoyltransferase activity than the diacetylmonoxime method. Conclusions: Here we report a new method for serum ornithine carbamoyl-transferase assay which might be useful for clinical diagnosis of hepatic disorders, including hepatic cancer.

Native human cytosolic alanine aminotransferase (EC 2.6.1.2) was found to be a homodimer consisting of two 55 kDa subunits. The human enzyme was more cationic and more susceptible to heat inactivation and heavy metal-inhibition than its rat homologue. Isoelectric focussing separated three human isoforms and four rat isoforms of cALT that differed in their isoelectric points. Two subtypes of the enzyme which differed in apparent molecular weight on sodium dodecylsulfate polyacrylamide gel electrophoresis were detected in rat, the liver type and the muscular type. This microheterogeneity was not found for human cytosolic alanine aminotransferase. Immunohistochemical studies revealed that the rat liver enzyme was exclusively localized in periportal hepatocytes, consistent with its role in gluconeogenesis. In human hepatocytes the plasma membrane was intensely stained, implicating an intracellular localization near the plasma membrane.

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.

Molecular dynamics simulation of molten LiBr was performed at 1000 K and 1500 K with the Born-Mayer-Huggins type potential functions. The temperature effects on the g ( r ) functions, the coordination numbers, the angular distribution functions, the diffusion coefficients, the velocity autocorrelation functions, and the power spectra are examined.

CuIn3Se5 films have been deposited with Na2S on Coning 7059 glass substrates by the two-stage co-evaporation method. Stoichiometric CuIn3Se5 films with p-type conduction with the resistivities of 106 Ω·cm range were obtained by our sodium control technique. Enhanced grain growth and preferred (112) orientation were also clearly observed with sodium incorporation; the same effect that has been reported for chalcopyrite CuInSe2 thin films. The effects of sodium in these films have been investigated by comparing the electrical and optical properties of CuIn3Se5 films with and without the Na incorporation. A correlation between the photoluminescence spectra and the resistivity or conduction type of the CuIn3Se5 films was found, suggesting a reduction in compensation due to the suppression of donor-type defects by the presence of Na.

CuInSe2 films with Cu/In ratios of m=0.83–0.99 have been deposited on glass substrates by the co-evaporation method using a bi-layer process. The effects of sodium in these films have been investigated by comparing the electrical and optical properties of the films with and without the Na2Se incorporation. Two donor-acceptor pair emissions, [D, A]α at 0.89–0.94 eV and [D, A]β at 0.84–0.87 eV, were typically observed in the photoluminescence spectra. The relative intensity of [D, A]α to [D, A]β was found to depend strongly on both the Cu/In ratio and Na incorporation. For the films with m=0.94, [D, A]α was predominantly observed regardless of Na incorporation. For m≤0.90, [D, A]β became dominant for the films without Na, while [D, A]α remained as the dominant emission for the films with Na. A correlation between the prominence of [D, A]α and resistivities below 2×103 Ω· cm was found, suggesting a reduced compensation by suppressed Se vacancy donors due to the presence of Na.

The cDNA encoding ornithine aminotransferase (EC 2.6.1.13; OAT) was isolated from a human kidney cDNA library. The isolated cDNA contained the entire protein coding region and partial 3′- and 5′-untranslated regions. The nucleotide sequences of human kidney OAT cDNA were absolutely homologous with those of human liver OAT cDNA, and human kidney and liver OAT fused completely against the antibody to human kidney OAT in an Ouchterlony double diffusion test. These findings settled the controversy as to which characteristics of liver and kidney OAT isozymes are different. An N-terminal sequence analysis of purified mature human kidney OAT clarified that the leader peptide was cleaved between Gln-35 and Gly-36.

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.

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.

Δ1-pyrroline-5-carboxylate reductase ( l -proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) from Baker's yeast has been purified and characterized. Purification to an apparently homogenous protein was effected by using ‘reagent-grade’ water containing dithiothreitol and by maintaining a constant pH 7.5, because of the instability of the enzyme protein. The enzyme was purified approximately 200-fold from the crude extract of baker's yeast, and it is a negatively-charged protein with a molecular weight of 125 000, containing an active SH group which participates in binding with NAD(P)H. The Km value for dl -pyrroline-5-carboxylate is 0.8 · 10−4 M; for NADH, it is 4.8 · 10−5 M; and for NADPH, it is 5.6 · 10−5 M. These Km values are much smaller than those of enzymes from other sources. The purified enzyme is free of contaminating enzymes which might interfere with its use in assays. The enzyme has been applied successfully to the assays of l Δ 1 - pyrroline -5- carboxylate and l -ornithine in tissue, and in vivo levels of these amino acids in rat liver are reported.

Bovine retinal pyrroline-5-carboxylate reductase (L-proline: NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) was purified 180-fold from crude extract, using affinity chromatography with 2',5'-ADP Sepharose 4B. The purified enzyme utilized either NADH or NADPH as the coenzyme. The enzyme activity was inhibited with ATP, and activated with potassium and phosphate ions, suggesting that it may be regulated by the energy level and the redox state in the retina. The distribution of this enzyme in bovine lens suggests a developmental trail of the enzyme in the lens.

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.

We found heterogenous ornithine oxoacid aminotransferase ( L -ornithine: 2-oxo-acid aminotransferase, EC 2.6.1.13) in rat ascites hepatoma AH 130 cells. Compared with enzymes from normal rat tissues, this heterogenous enzyme showed larger K m values for 2-oxoglutarate, a different elution-profile upon affinity chromatography with 2-oxoglutarate, more anionic mobility upon polyacrylamide gel electrophoresis, and a clearly different salting-out property upon ammonium sulfate fractionation. Similar heterogeneity of this aminotransferase was found in human cancer cells.

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.

1-Pyrroline-5-carboxylate dehydrogenase was purified and crystallized from Bacillus sphaericus. The crystalline preparation gave a single band on polyacrylamide slab gel electrophoresis. The molecular weight of the enzyme was determined to be about 100,000 by gel filtration. The enzyme consists of two subunits which are identical in molecular weight (50,000), as judged on SDS slab gel electrophoresis. The enzyme shows an optimum pH of 6.5 to 7.0. Its activity was 8.1 times higher with NADP+ than with NAD +, and the enzyme was stabilized by NADP+. The apparent Km values for l-l-pyrroline-5-carboxylate, NADP+ and NAD+ are 4.2 × 10–5m (with NADP+), 9.5 × 10~6m and 2.5 × IO-3 m, respectively. The enzyme reaction is irreversible. A simple method for the determination of l-ornithine involving ornithine ?-aminotransferase and 1- pyrroline-5-carboxylate dehydrogenase from B. sphaericus was developed. A linear relationship was found between the absorbance at 340 nm and the amount of l-ornithine (50 ~ 400 nmol), an...

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.

A recent report from this laboratory ( Segal, Abraham,and Schatz, 1968 ) described some in vitro stability studies with rat liver alanine aminotransferase in which it was found that L-proline protected the enzyme against heat denaturation better than any other amino acid tested. It was of interest to us to pursue this observation in order to decide whether the interaction was at the active site, and if so whether a complex was formed with the pyridoxal moiety. The results allow us to conclude that L-proline does interact with the prosthetic group and to speculate about the nature of the complex and its relationship to normal intermediates in the transamination reaction.

The half-life of alanine aminotransferase in rat muscle is about 20 days, compared with that of the same enzyme in liver of about three days. The thermal denaturation rate of the enzyme in , vitro gives a calculated half-life of about 400 days at body temperature. A liver lysosomal preparation effectively inactivated the enzyme under conditions in which it was otherwise quite stable. From our results we conclude that turnover of alanine aminotransferase in , vivo is a reflection of an active (enzymatic) process which does not depend on prior thermal denaturation.

Rat liver alanine aminotransferase has been crystallized and has a specific activity of 500 to 550 units (micromoles per min) per mg. The enzyme contains two pyridoxal phosphate groups and 23 to 30 titratable sulfhydryl equivalents per molecule (114,000 daltons). The amino acid composition has been determined and includes 34 half-cystine residues per molecule. Reaction of the first seven or eight sulfhydryl groups with p-mercuribenzoate or 5,5'-dithiobis-(2-nitrobenzoic acid) led to no loss of activity, after which there was a loss of activity proportional to the extent of reaction of the next 16 equivalents with 5,5'-dithiobis-(2-nitrobenzoic acid). Resolution and partial reconstitution of the apoenzyme has been achieved. The spectrum of the pyridoxal form of the enzyme is pH dependent, with an acid peak at 430 mµ, a basic peak at 335 mµ, and an isosbestic point at 380 mµ.

A homogeneous preparation of Δ1-pyrroline-5-carboxylate reductase ( l -proline: NAD (P)+ 5-oxidoreductase, EC 1.5.1.2) was obtained from baker's yeast by an affinity chromatography, using 5′ AMP-Sepharose 4B. After the 1st DEAE-Sephadex column chromatography, the enzyme absorbed on 5′ AMP-Sepharose column was eluted with 1 mM ATP. The final preparation was homogeneous on polyacrylamide gel electrophoresis and purified 3500-fold from the crude extract. This purified enzyme was very useful as a coupling enzyme for the assays of ornithine and pyrroline-5-carboxylate in tissues, and for the rate assay of ornithine aminotransferase activity.

We examined four cases of Japanese patients with gyrate atrophy of the choroid and retina. All cases had hyperornithinemia and deficiency of ornithine ketoacid transaminase (OKT). Two types of disease in responsiveness to vitamin B6, the responsive and non-responsive, were found. The responsive type showed the in vivo reduction of serum ornithine level after oral vitamin B6 and the OKT activity increased by high concentration of pyridoxal phosphate.

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.

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.

The effects of hypoxia and a metabolic inhibitor, sodium cyanide (NaCN), on the spontaneous action potential and membrane current systems were studied in small preparations (0.2 x 0.2 x 0.1 mm) of rabbit atrioventricular node. When the PO2 of the superfusate was lowered from 500 to less than 20 mm Hg, all the preparations initially showed reductions in the spontaneous firing frequency (due to the decreased rate of diastolic depolarization) and maximal rate of depolarization, which were followed by a cessation of automatic activity with subsequent membrane hyperpolarization to -57 +/- 3 mV (n = 4). Voltage clamp experiments using double microelectrode techniques revealed that hypoxia reduced the slow inward current by 20 to 80% without affecting its inactivation kinetics. The delayed rectifying potassium current tail was also reduced or abolished by severe hypoxia, whereas the background outward current was greatly increased. The hyperpolarization-activated inward current was decreased by hypoxia. Similar changes in the spontaneous action potential and membrane currents were obtained on adding 0.1 to 0.5 mM NaCN to the superfusate under conditions of normal PO2. Hypoxia-induced suppression of automaticity was reversed completely by 5 mM glucose, and partially by 40 mM valine. These results suggest that (1) hypoxia impairs atrioventricular nodal conduction by reducing the slow inward current and the delayed rectifying potassium current; (2) hypoxia depresses automaticity by increasing the background outward current and, to some extent, by reducing the slow inward current; and (3) glucose and valine contribute to the generation of high-energy phosphates in cytoplasm and mitochondria, respectively, and protect the atrioventricular node from hypoxia.

Reports on hereditary deficiencies of the urea cycle enzyme, e.g. hyperammonemia with homocitrullinuria1 and ornithine carbamoyl transferase(OCT) deficiency2, led us to the view that ornithine transport into the mitochondrial matrix represents an important factor controlling citrulline synthesis and plays a role in the regulation of carbamoyl phosphate synthesis3. We have long been interested in whether ornithine is derived from arginine or proline, i.e., whether ornithine, produced from arginine in the cytosol, is transported into the mitochondria, or whether ornithine is derived from proline via Δ1-pyrroline-5-carboxylate(P5C) in the mitochondrial matrix. Once the urea cycle is activated, ornithine is continuously transported into the mitochondrial matrix from cytosol and released from mitochondria into the cytosol(“ornithine flux”); this transport system is composed of a citrulline-exchange transporter and an H+ antiporter4,5. We now present some in vitro mitochondrial studies on ornithine transport and citrulline formation from proline, and an analysis of in vivo arginine and ornithine metabolisms. This investigation was undertaken to elucidate the characteristics of “orni-thine flux” and its relation to dietary stimulation of urea cycle.

FUJIMURA, Y., MATSUZAWA, T., KAWAMURA, M., TADA, K. and MIZUNO, K. Mass Screening of Urea Cycle Diseases: A New Mass Screening Method of Hyperornithinemia by Using Two Coupling Enzymes. Tohoku J, exp. Med., 1983, 141 (3), 257-261 - We devised a new microfluorometric mass screening method for determining ornithine in only one blood disc of 3mm in diameter (blood of 2-3μl) by use of two coupling enzymes, ornithine aminotransferase and Δ'-pyrroline-5-carboxylate reductase. We could apply this method to some cases of gyrate atrophy of the choroid and retina. Our method can be applied in range of 0 to 12-16 nmoles or about 54.8mg% or about 4mM ornithine in blood. Ornithine values by our method were well correlated with those by an amino acid analyser.

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.

Publisher Summary This chapter discusses the preparation of porphobilinogen (PBG). PBG is a normal intermediate in the biosynthesis of heme. By using both δ-aminolevulinic acid and the enzyme δ- aminolevulinic acid dehydratase, an enzymatic method has been developed for the preparation of porphobilinogen with good yield. Enzyme is stored as a precipitate at -10°. A solution of 10–20 mg protein per milliliter in 0.134 M Na 2 HPO 4 -KH 2 PO 4 buffer (pH 6.8) is prepared when needed. PBG isolated can be identified by using different methods. By paper chromatography, a biosynthetic PBG runs as a single spot having the same R f as that for the markers. A solution of biosynthetic PBG when mixed with an equal volume of Ehrlich's reagent gives maxima at 555 m μ and 525 m μ. PBG can be enzymatically converted into uroporphyrinogens by means of the deaminase–isomerase enzymatic complex.

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.