Organic Acid Metabolism in Cellular Organelles of Vigna cylindrica (Mitorisasage) Cotyledons

In starchy cotyledons of Vigna cylindrica (L.) Skeels (Mitorisasage)during seed germination, the enzymes of the glyoxylate cyclewere located in the matrix of mitochondria. Glyoxysomes wereabsent. The glyoxylate cycle in the mitochondria supplies organicacids to the tricarboxylic acid cycle. In mitochondria, isocitratelyase activity was much higher than malate synthase activity.Part of the glyoxylate thus produced in mitochondria may benonenzymatically converted to formate by H₂O₂ and the formatethen converted to CO₂ by peroxidase or by formic dehydrogenase.The activity of superoxide dismutase, which supplies H₂O₂, washigher in mitochondria than in peroxisomes. The remaining glyoxylatein mitochondria is possibly converted to glycine by alanine-glyoxylateaminotransferase or transported to peroxisomes which lackedisocitrate lyase activity but had high malate synthase activity.In peroxisomes, glyoxylate may be also produced from urate,as is suggested by the fairly high activities of uricase, allantoinaseand allantoicase. Judging from the enzyme distribution, Vignaperoxisomes should be capable of producing malate, oxalacetate,citrate, isocitrate and a-ketoglutarate. ¹Present address: Department of Horticulture, College of Agricultureand Animal Science, Yeugnam University, Gyeongsan 632, Korea. (Received May 27, 1987; Accepted October 7, 1987)     

Identity of alanine:glyoxylate aminotransferase with alanine:2-oxoglutarate aminotransferase in rat liver cytosol

Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a pI of 5.2 and an Mr of approx. 110,000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The pI 6.0 enzyme with an Mr of approx. 220,000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the pI 8.0 enzyme with an Mr of approx. 80,000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine: glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase.     

Alanine aminotransferase from Cucurbita moschata cotyledons

Alanine aminotransferase increased in pumpkin cotyledons duringgermination with the greatest increase occurring in green cotyledons.The enzyme was found in the soluble fraction and was inhibitedby NH₂OH and p-chloromercuribenzoate. Pyridoxal phosphate andglutathione or dithiothreitol overcame die respective inhibition.Dialysis of the enzyme reduced enzyme activity but the activitywas restored by the addition of pyridoxal phosphate. Alanineaminotransferase was proposed to play a major role in the synthesisof the alanine which occurs in pumpkin cotyledons during germination. (Received September 17, 1975; )     

Factors Influencing Alanine Aminotransferase Activity in Leaves of Lolium temulentumL.: II. EFFECTS OF GROWTH REGULATORS AND PROTEIN BIOSYNTHESIS INHIBITORS

Effects of kinetin (K), gibberellin A₃ (GA₃), and 2-(chloroethyl)-trimethylammoniumchloride (CCC) on levels of alanine aminotransferase (GPT) andrates of protein synthesis were studied with both intact plantsand isolated leaf segments of Lolium temulentum L. In intactplants CCC stimulated and CA₃ reduced GPT activity, the effectsbsing much greater in 8.h than in 16-h photoporiods. CCC showedmaximum stimulatory effects at 10^–2 M and K at 5 x 10⁵M. No effect of GA₃ could be demonstrated with concentrationsup to 10^–4M. Both K and CCC retarded GPT decline in leafsections, the latter without associated effects upon pigmentbreakdown. Cycloheximide was highly effective in reducing proteinsynthesis in leaf sections. A close correlation between rateof protein synthesis and GPT activity was found over an inhibitorconcentration range from 10^–6 to 10^–4 M. The resultsare discussed in terms of possible methods of in vivo regulationof GPT activity.     

delta-Aminolevulinic Acid Formation from gamma,delta-Dioxovaleric Acid in Extracts of Euglena gracilis

γ,δ-Dioxovaleric acid (DOVA) has been proposed as a precursor to heme and chlorophyll in plants and algae. DOVA transaminase activity was found in extracts of the unicellular green alga Euglena gracilis Klebs strain Z Pringsheim. Optimum conversion of DOVA to δ-aminolevulinic acid (ALA) occurred at pH 6.8. ALA formation was linear with time for at least 30 minutes at 37° C and was proportional to amount of cell extract in the incubation mixture. Boiled cell extract was inactive. DOVA transaminase from either wild-type or aplastidic derivative strain W₁₄ZNaIL ran as a single band in agarose gel permeation chromatography, with a calculated molecular weight of 98,000 ± 3,000. l-Glutamic acid was the most effective amino donor. d-Glutamic acid was inactive. K_m values for l-glutamic acid and DOVA were 11 and 1.1 millimolar, respectively. Pyridoxal phosphate stimulated activity maximally at 30 micromolar, and (aminooxy)acetate was strongly inhibitory. Glyoxylic acid was a competitive inhibitor with respect to DOVA, with an inhibition constant of 0.62 millimolar. Wild-type and aplastidic cells vielded equal activity, 31 ± 1 nanomoles ALA per 30 minutes per 10⁷ cells, whether grown in light or dark. DOVA transaminase could not be separated from glyoxylate transaminase activity by agarose gel permeation or diethylaminoethyl-cellulose column chromatography. In all fractions, glyoxylate transaminase activity was at least 75 times greater than DOVA transaminase activity. DOVA transamination appears to be catalyzed by glyoxylate transaminase, and not to be of physiological significance with respect to chlorophyll synthesis in Euglena.     

Glyoxylate Inhibition of Ribulosebisphosphate Carboxylase/Oxygenase Activation State in vivo

Photosynthetic CO₂ exchange in photorespiration mutants of Arabidopsisthaliana showed a time-dependent inhibition at 350 µl/literCO₂ in 50% O₂ but not in 2% O₂. In a glycolate-P phos-phatasedeficient mutant, inhibition of photosynthesis was due to adepletion of ribulosebisphosphate. In the remaining mutants,which have defects in photorespiratory enzymes which metabolizeamino acids, reduced photosynthesis was accompanied by a declinein the activation level of ribulosebisphosphate carboxylase/oxygenase(Chastain and Ogren 1985), a decline in ribulosebisphosphateconcentration, and an accumulation of glyoxylate. Addition ofglyoxylate at submillimolar concentrations to intact spinach(Spinacea oleracea L.) chloroplasts inhibited light activationof ribulosebisphosphate carboxylase/oxygenase (rubisco) andCO₂ fixation. Similar concentrations of glyoxylate had no effecton A. thaliana rubisco activity in vitro. These results suggestthat glyoxylate accumulation indirectly inhibited rubisco activationstate in vivo. The inhibition of photosynthesis in mutants whichaccumulate glyoxylate may be attributed to a decline in ribulosebisphosphateconcentration, a reduction in rubisco activation state, or acombination of both phenomena. ³Present address: CSIRO, Division of Plant Industry, GPO Box1600, Canberra, ACT 2601, Australia. (Received May 12, 1989; Accepted July 8, 1989)     

Influence of Nitrogen Availability on Aminotransferases in Lemna minor L.

Protein contents and glutamate: glyoxylate, serine: glyoxylate,alanine: glyoxylate and glutamate: pyruvate aminotransferaseactivities per gram fresh weight declined sharply when Lemnaminor L., previously grown on nitrate medium, was starved ofnitrogen. Nitrogen replenishment after 5 d caused complete recoveryof these parameters with higher values in ammonium-fed thannitrate-fed plants 7 d after transfer of plants from nitrogen-freemedium. Glutamate: glyoxylate and alanine: glyoxylate aminotransferasespecific activities (based on total extracted protein) showedlittle change with nitrogen availability. Serine: glyoxylateaminotransferase increased slowly during nitrogen starvationand decreased following nitrogen replenishment whether withammonium or nitrate. After 1 d of nitrogen starvation the specificactivity of glutamate: pyruvate aminotransferase declined; itincreased following nitrogen replenishment and ammonium gaverise to agreater activity than nitrate. The results are discussed in relation to the differences instability of the various enzymes relative to the overall proteinturnover rate. Key words: Aminotransferases, Nitrogen source, Photorespiration     

Inhibition of glutamate:glyoxylate aminotransferase activity in tobacco leaves and callus by glycidate, an inhibitor of photorespiration

The effect of glycidate (2,3-epoxypropionate), an inhibitor of glycolate synthesis and photorespiration in leaf tissue, was studied on glutamate:glyoxylate and serine:glyoxylate aminotransferases and glycine decarboxylase activities in particulate preparations obtained from tobacco (Nicotiana tabacum L.) callus and leaves. Glycidate specifically and effectively inhibited glutamate:glyoxylate aminotransferase. The inhibition was dependent on glycidate concentration and, to a lesser extent, on substrate concentration. The enzyme was not protected by either substrate. Even with saturating substrate concentrations the glycidate inhibition was only partially reversed. Under the in vitro assay conditions, glycidate inhibition of the aminotransferase was reversible. Glutamate:glyoxylate aminotransferase is the only enzyme of the glycolate pathway thus far examined which is severely inhibited by glycidate. However, in leaf discs, pretreatment with glycidate decreased both glutamate:glyoxylate and serine:glyoxylate aminotransferase activities suggesting binding by glycidate in vivo.

Glycidate increased the pool sizes of both glutamate and glyoxylate in leaf discs. It has been shown that increases in concentration of either of these metabolites decrease photorespiration and glycolate synthesis and increase net photosynthesis. It is proposed that glycidate inhibits photorespiration indirectly by increasing the internal concentrations of glutamate and glyoxylate, as a consequence of the inhibition of glutamate:glyoxylate aminotransferase activity.

     

Glycolate and glyoxylate metabolism in HepG2 cells

Oxalate synthesis in human hepatocytes is not well defined despite the clinical significance of its overproduction in diseases such as the primary hyperoxalurias. To further define these steps, the metabolism to oxalate of the oxalate precursors glycolate and glyoxylate and the possible pathways involved were examined in HepG2 cells. These cells were found to contain oxalate, glyoxylate, and glycolate as intracellular metabolites and to excrete oxalate and glycolate into the medium. Glycolate was taken up more effectively by cells than glyoxylate, but glyoxylate was more efficiently converted to oxalate. Oxalate was formed from exogenous glycolate only when cells were exposed to high concentrations. Peroxisomes in HepG2 cells, in contrast to those in human hepatocytes, were not involved in glycolate metabolism. Incubations with purified lactate dehydrogenase suggested that this enzyme was responsible for the metabolism of glycolate to oxalate in HepG2 cells. The formation of ¹⁴C-labeled glycine from ¹⁴C-labeled glycolate was observed only when cell membranes were permeabilized with Triton X-100. These results imply that peroxisome permeability to glycolate is restricted in these cells. Mitochondria, which produce glyoxylate from hydroxyproline metabolism, contained both alanine:glyoxylate aminotransferase (AGT)2 and glyoxylate reductase activities, which can convert glyoxylate to glycine and glycolate, respectively. Expression of AGT2 mRNA in HepG2 cells was confirmed by RT-PCR. These results indicate that HepG2 cells will be useful in clarifying the nonperoxisomal metabolism associated with oxalate synthesis in human hepatocytes. liver; peroxisomes; hepatocytes; hyperoxaluria; alanine:glyoxylate aminotransferase; glyoxylate reductase     

Plant leaf alanine: 2-oxoglutarate aminotransferase. Peroxisomal localization and identity with glutamate:glyoxylate aminotransferase.

The distribution of alanine:2-oxoglutarate aminotransferase (EC 2.6.1.2) in spinach (Spinacia oleracea) leaf homogenates was examined by centrifugation in a sucrose density gradient. About 55% of the total homogenate activity was localized in the peroxisomes and the remainder in the soluble fraction. The peroxisomes contained a single form of alanine:2-oxoglutarate aminotransferase, and the soluble fraction contained two forms of the enzyme. Both the peroxisomal enzyme and the soluble predominant form (about 90% of the total soluble activity) were co-purified with glutamate:glyoxylate aminotransferase to homogeneity; it had been reported to be present exclusively in the peroxisomes of plant leaves and to participate in the glycollate pathway in leaf photorespiration [Tolbert (1971) Annu. Rev. Plant Physiol. 22, 45-74]. The evidence indicates that alanine:2-oxoglutarate aminotransferase and glutamate:glyoxylate aminotransferase activities are associated with the same protein. The peroxisomal and soluble enzyme preparations had nearly identical properties, suggesting that the soluble predominant alanine aminotransferase activity is from broken peroxisomes and about 96% of the total homogenate activity is located in peroxisomes.     

Poly(A) polymerase in Dormant Embryos of Triticum durum

Triticum durum‘Cappelli’ has a ‘relative’dormancy which can be broken by dry after-ripening at room temperature.The breakage of dormancy in the embryos of T. durum , is accompaniedby a decline in content and a different degree of synthesisof poly(A)⁺RNA. This work studies the activity of poly(A) polymerase(E.C. 2.7.7.19), the enzyme which permits polyadenylation. Anincrease in the activity of this enzyme in parallel with theenhanced rate of germination is revealed. Since poly(A) polymeraseactivity is the same in dormant and non-dormant dry embryos,it seems that the activity of the enzyme is not involved inthe breakage of dormancy. The use of cycloheximide and cordycepinshows the presence of enzymes with different origins: a storedenzyme and one bound to a long lived mRNA, present in dormantand non-dormant embryos, plus an enzyme bound to newly synthesizedmRNA which is mainly active in non-dormant embryos. Since dormancycould be the result of an interaction between hormones, thiswork analyses the effects of GA₃and ABA on poly(A) polymerase.GA₃enhanced poly(A) polymerase activity only in dormant embryoswhile ABA inhibited this activity only in non-dormant embryos.Cycloheximide applied to excised wheat embryos represses thestimulatory and inhibitory effects of GA₃and ABA, respectively.The hormone action on poly(A) polymerase activity is thus dependenton de novo protein synthesis. Results using cordycepin suggestthe presence of a stored mRNA for poly(A) polymerase, togetherwith hormonal regulation of enzyme activity at a translationallevel. Copyright 1999 Annals of Botany Company Triticum durum , wheat, dormancy breakage, poly(A) polymerase, GA₃, ABA, germination.     

Enxymological characterization of a putative canine analogue of primary hyperoxaluria type 1

This paper concerns an enzymological investigation into a putative canine canalogue of the human autosomal recesive disease primary hyperoxaluria type 1 (alanine:glyoxylate / serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and seribe:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephripathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including d-glycerate dehydrogenese / glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate / serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal humanb liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is anlogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

Dimethylarginine:pyruvate aminotransferase in rats. Purification, properties, and identity with alanine:glyoxylate aminotransferase 2

Dimethylarginine:pyruvate aminotransferase, which plays a role in the metabolism of dimethylarginines, has been purified to homogeneity from rat kidney. The enzyme has a molecular weight of approximately 200,000 and an isoelectric point at about pH 6.3. The enzyme consists of four similar subunits having a molecular weight of about 50,000. The enzyme catalyzes the effective transaminations of guanidino-N methylated L-arginines (e.g. NG,NG-dimethyl-L-arginine, NG,N'G-dimethyl-L-arginine and NG-monomethyl-L-arginine) and the alpha-amino group of L-ornithine to pyruvate or glyoxylate. The enzyme was always accompanied by the known alanine:glyoxylate amino-transferase activity with the ratios of their specific activities remaining constant during the purification steps. The physicochemical and immunological properties of the purified enzyme were shown to be identical with those of the isozyme of alanine:glyoxylate aminotransferase (EC 2.6.1.44), designated as alanine:glyoxylate aminotransferase 2 (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The distribution profiles in tissues and the negative response to glucagon treatment further supported the identity of the two enzymes. The present data show that alanine:glyoxilate aminotransferase 2 functions in dimethylarginine metabolism in vivo in rats.     

Developmental profiles and properties of hepatic peroxisomal apo- and mitochondrial holoalanine:glyoxylate aminotransferase during chick embryogenesis

Alanine:glyoxylate aminotransferase was present as the apoenzyme in the peroxisomes and as the holoenzyme in the mitochondria in chick embryos. The peroxisomal enzyme predominated in the early stage and gradually decreased during embryonic development and disappeared after hatching. In contrast, the mitochondrial enzyme gradually increased and predominated in the later stage of chick embryos. Peroxisomal alanine:glyoxylate aminotransferase in chick embryos was a single peptide with a molecular weight of about 40,000. The enzyme differed from the mitochondrial enzyme in the embryos, and mammalian alanine:glyoxylate aminotransferases 1 (with a molecular weight of about 80,000 with two identical subunits) and 2 (with a molecular weight of about 200,000 with four identical subunits) in molecular weights and immunological properties. Mitochondrial alanine:glyoxylate aminotransferase in chick embryos had an identical molecular weight and immunologically cross-reacted with mammalian mitochondrial alanine:glyoxylate aminotransferase 2. Pyridoxal 5'-phosphate dissociated easily from the peroxisomal enzyme saturated with pyridoxal 5'-phosphate. Hepatic aspartate:2-oxoglutarate aminotransferase and alanine:2-oxoglutarate aminotransferase in chick embryos, and hepatic alanine:glyoxylate aminotransferases in different animal species were all present as the holoenzyme.     

Enzymological characterization of a putative canine analogue of primary hyperoxaluria type 1.

This paper concerns an enzymological investigation into a putative canine analogue of the human autosomal recessive disease primary hyperoxaluria type 1 (alanine:glyoxylate/serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and serine:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephropathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including D-glycerate dehydrogenase/glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate/serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal human liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is analogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

Nitrogen-Dependent Regulation of the Gene for Alanine Aminotransferase which is Involved in the C4 Pathway of Panicum miliaceum

Levels of protein and mRNA for alanine aminotransferase, whichis involved in the C₄ pathway of Panicum miliaceum, were measuredduring recovery from nitrogen stress. The results indicatethatthe enzyme accumulates selectively in response to nitrogen availabilityprimarily as a consequence of changes in the level of its mRNA. (Received January 6, 1992; Accepted April 1, 1992)     

Purification and Characterization of 4,5-Dioxovalerate Reductase (NADPH) from Chlorella regularis

Two types of 4,5-dioxovalerate reductases (NADPH) were partiallypurified and characterized from green alga, Chlorella regularis.The enzyme was separated by DEAE-Sephacel chromatography intotwo peaks: type I (first peak) and type II (second peak). Theactivity ratio of the type II to type I enzyme varied between5 to 7 with a starting cell material. Both enzymes had the samepH optimum at 6.0 and pI value of 4.9. The molecular weightestimated by gel filtration was 33,000 for type I and 99,000for type II enzyme. Both enzymes used only NADPH, but were notspecific for 4,5-dioxovaleric acid (DOVA). Type I enzyme reducedglyoxylate 68-fold faster than DOVA, whereas type II enzymeacted more specifically on a variety of aldehydes than DOVA.It is suggested that these enzymes may not function primarilyas NADPH-DOVA reductases in the metabolic pathway of DOVA. (Received June 15, 1985; Accepted October 14, 1985)     

NADP+-Dependent Sorbitol Dehydrogenase Found in Apple Leaves

An NADP⁺-dependent sorbitol dehydrogenase that catalyzes sorbitoland glucose was found in apple leaves. The partially purifiedenzyme had optimum activity at pH 9.6 and a K_m value of 128mM for sorbitol. Among the polyols studied, this enzyme showedthe most activity for sorbitol. ¹This paper is contribution A-173 of the Fruit Tree ResearchStation. (Received June 4, 1984; Accepted July 31, 1984)     

Photorespiratory N donors,aminotransferase specificity and photosynthesis in a mutant of barley deficient in serine: glyoxylate aminotransferase activity

A mutant of Hordeum vulgare L. (LaPr 85/84) deficient in serine: glyoxylate aminotransferase (EC 2.6.1.45) activity has been isolated. The plant also lacks serine: pyruvate aminotransferase and asparagine: glyoxylate aminotransferase activities. Genetic analysis of the mutation strongly indicates that these three activities are all carried on the same enzyme protein. The mutant is incapable of normal rates of photosynthesis in air but can be maintained at 0.7% CO₂. The rate of photosynthesis cannot be restored by supplying hydroxypyruvate, glycerate, glutamate or ammonium sulphate through the xylem stream. This photorespiratory mutant demonstrates convincingly that photorespiration still occurs under conditions in which photosynthesis becomes insensitive to oxygen levels. Two major peaks and one minor peak of serine: glyoxylate aminotransferase activity can be separated in extracts of leaves of wild-type barley by diethylaminoethyl-sephacel chromatography. All three peaks are missing from the mutant, LaPr 85/84. The mutant showed the expected rate (50%) of ammonia release during photorespiration but produced CO₂ at twice the wild-type rate when it was fed [¹⁴C]glyoxylate. The large accumulation of serine detected in the mutant under photorespiratory conditions shows the importance of the enzyme activity in vivo. The effect of the mutation on transient changes in chlorophyll a fluorescence initiated by changing the atmospheric CO₂ concentration are presented and the role of the enzyme activity under nonphotorespiratory conditions is discussed.Abbreviations DEAE diethylaminoethyl - PFR photon fluence rate - SGAT serine:glyoxylate aminotransferase     

Purification and Properties of Sweet Potato NADPH-Cytochrome c (P-450) Reductase

NADPH-cytochrome c reductase, strictly NADPH-cytochrome P-450reductase, was purified by chromatography through DEAE-cellulose,2',5'-ADP-Sepharose, and Sephadex G-100 columns after solubilizationfrom microsomes from Ceratocystis fimbriata-infected sweet potatoroot tissue with Emulgen 913. The enzyme existed in three formsafter solubilization which migrated to positions correspondingto molecular weights of 81,000, 75,000 and 72,000 on an SDS-polyacrylamidegel. Trypsin treatment of the enzyme species with the largestpolypeptide yielded the species with the smallest one. Aftersucrose density gradient centrifugation of the pellet fractionobtained by centrifugation at 100,000?g of the crude extract,the enzyme species with the largest polypeptide was presentin the particulate fractions, whereas that with the smallestone was only found at the top of the gradient. We conclude thatthe enzyme species with the largest polypeptide is in an intact,amphipathic form, whereas that with the smallest one, and probablyalso the other species, is its hydrophilic domain produced byan endogenous protease(s). The K_m values of the enzyme in theintact form for NADPH and cytochrome c were 7.7 and 2.3 µM,respectively. ¹ Present address: Laboratory of Food Hygienics, Faculty ofAgriculture, Kagawa University, Miki-cho, Kida-gun, Kagawa 761-07,Japan. (Received September 6, 1984; Accepted December 27, 1984)