*Department of Urology, Research Equipment Center, and Department of Biochemistry I, Hamamatsu University School of Medicine, Hamamatsu, Japan; and Department of Urology, University of the Ryukyus, Okinawa, Japan.
Correspondence to Dr. Tatsuya Takayama, Department of Urology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan. Phone: 81-53-435-2306; Fax: 81-53-435-2305;
ABSTRACT. Serine:pyruvate/alanine:glyoxylate aminotransferase(SPT/AGT) is largely located in mitochondria in carnivores,whereas it is entirely found within peroxisomes in herbivoresand humans. In rat liver, SPT/AGT is found in both of theseorganelles, and only the mitochondrial enzyme is markedly inducedby glucagon. Although SPT/AGT is a bifunctional enzyme involvedin the metabolism of both L-serine and glyoxylate, its contributionto L-serine metabolism is independent of mitochondrial or peroxisomallocalization (Xue HH et al., J Biol Chem 274: 16028-16033, 1999).Therefore, the species-specific and food habit-dependent organelledistribution might be required for proper metabolism of glyoxylateat the subcellular site of its formation. Glyoxylate formationfrom glycolate and that from L-hydroxyproline have been shownto occur in peroxisomes and mitochondria, respectively. Thepresent study found that urinary excretion of oxalate was markedlyincreased when a large dose of L-hydroxyproline or glycolatewas administered to rats. Oxalate formation from L-hydroxyprolinebut not that from glycolate was significantly reduced when mitochondrialSPT/AGT had been induced by glucagon. The hydroxyproline contentof collagen is 10 to 13%, and collagen accounts for about 30%of total animal protein; therefore, these results suggest thatan important role of mitochondrial SPT/AGT in carnivores isto convert L-hydroxyproline-derived glyoxylate into glycinein situ, preventing undesirable overflow into the productionof oxalate. E-mail: takayama@hama-med.ac.jp
In herbivores and humans, a major source of glyoxylate, an immediateprecursor of oxalate, is believed to be oxidation of glycolateby glycolate oxidase in liver peroxisomes. Glycolate is an intermediateof photorespiration and is thus much higher in content in plantsthan in animal tissues (1). Glyoxylate is also formed in liverand kidney mitochondria from 4-hydroxy-2-ketoglutarate (HKG),an intermediate of L-hydroxyproline metabolism (2,3). Mitochondrialproduction of glyoxylate from hydroxyproline is assumed to besignificant in carnivores, because the hydroxyproline contentof collagen is about 10 to 13% (4) and collagen accounts forabout 30% of total animal protein. The major metabolic pathwayof hydroxyproline-derived glyoxylate in rat kidney has beensuggested to be transamination to glycine (3). Glycine thusformed may be oxidized to CO2 and ammonia by the mitochondrialglycine cleavage enzyme system, and in conjugation with thisoxidation another molecule of glycine is converted to L-serineby mitochondrial serine hydroxymethyltransferase (5). A candidateenzyme responsible for the transamination of glyoxylate to glycineis a dual-functional enzyme, serine:pyruvate/alanine:glyoxylateaminotransferase (SPT/AGT, alternatively named alanine:glyoxylateaminotransferase-1, AGT-1 or serine:pyruvate aminotransferase,SPT). This enzyme was found as SPT (6), and it was later shownto be a predominant enzyme of L-serine metabolism in human,rabbit, and dog livers (7), but its alanine:glyoxylate aminotransferase(AGT) activity has also been proven to be indispensable forproper metabolism of glyoxylate (8).
The relationship between glyoxylate metabolism and the species-specificand food habit-dependent subcellular distribution of SPT/AGTis of interest. This enzyme is located in liver peroxisomesin herbivores and humans and largely in mitochondria in carnivores(9–13). In the livers of rats and mice, it is locatedin both peroxisomes and mitochondria, but only the mitochondrialenzyme is markedly induced by glucagon (9,11,14–16). It is generally accepted that the peroxisomal presence ofSPT/AGT in herbivores is essential for conversion of glyoxylateformed from glycolate in this organelle into glycine in situ,thus preventing undesirable overflow of it into oxalate.
In the present study, we examined whether mitochondrial SPT/AGTplays a role in removing hydroxyproline-derived glyoxylate fromoxidation to oxalate using rats with or without glucagon inductionof liver mitochondrial SPT/AGT. For this purpose, we investigated(1) the effect of glucagon on enzymes involved in L-hydroxyprolinemetabolism (experiment 1), and (2) the effect of glucagon inductionof mitochondrial SPT/AGT on oxalate formation from L-hydroxyprolineand that from glycolate in vivo (experiment 2).
Experiment 1
All animal experiments (experiments 1 and 2) were done at 26°Cunder a light and dark cycle of 12:12. Glucagon was injectedintraperitoneally into 24 h-fasted (deprived of food with freeaccess to water) male Wistar rats of about 170 g at a dose of300 µg/100 g body wt. Where indicated, the same dose ofglucagon was given twice after 24 h and 48 h of fasting. Therats were killed after another 24 h of fasting by exsanguinationfrom the carotid arteries after being rendered unconscious bya blow to the neck, in accordance with the Guidlines for AnimalExperimentation of Hamamatsu University School of Medicine.Livers were immediately removed and homogenized using a Potter-Elvehjemhomogenizer in 4 vol of 0.25 M sucrose (pH 7.2) containing 3mM imidazole/HCl, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride,and 10 µg/ml E-64c for assay of the SPT activity of SPT/AGT,and in 4 vol of chilled water for assay of hydroxyproline oxidaseand HKG aldolase. The homogenates were then passed through alayer of cheesecloth and subjected to sonication 5 times for15 s each with a Blonson Sonifier B-12. When 1-pyrroline-5-carboxylate(P5C) dehydrogenase was to be assayed, acetone dried powderwas prepared from a liver specimen of each rat, and the powderwas extracted with 10 vol of chilled water as described by Strecker(17). The reaction mixture (1.0 ml) for the assay of P5C dehydrogenasecomprised 50 mM Tris-HCl, 50 µM K-phosphate, 1.2 mM pyrroline5-carboxylate and acetone powder extract, final pH being closeto 8.2. The extract was added last, and the activity was measuredat room temperature by the increase in absorbance at 340 nm.The SPT activity of SPT/AGT was determined as described previously(18,19). SPT/AGT catalyzes transamination of glyoxylate toglycine with L-alanine or L-serine as an amino acid (rat SPT/AGTshows wider substrate specificity), but the tissue level ofthis enzyme was represented by the SPT activity in this study,because a single catalytic site is responsible for the threeenzymatic activities and determination of SPT activity was freeof interference by AGT-2, an isoenzyme of SPT/AGT or AGT-1.Hydroxyproline oxidase and HKG aldolase activities were assayedas described by Carnie et al. (20).
Experiment 2
Male Wistar rats weighing about 170 g were divided into sixgroups (groups A, B, C, D, E, and F) and fasted for 72 h withfree access to water. After 24 h of fasting, rats of groupsA, C, and E were given 300 µg/100 g body wt of glucagonintraperitoneally to induce liver mitochondrial SPT/AGT (14,18). After 48 h of fasting (24 h after the injection of glucagon),630 mg (4.8 mmol) of L-hydroxyproline in 2 ml water was administeredto rats from groups C and D via a stomach tube. Rats from groupsE and F were similarly given 100 mg (1.02 mmol) of Na-glycolatein 1 ml water. Rats from groups A and B did not receive L-hydroxyproline,glycolate, or water, but preliminary experiments showed thatoral administration of water caused no significant change inurinary excretion of oxalate and glycolate, especially whentheir excretion was expressed as the ratios to creatinine. Eachanimal was then individually housed in a glass metabolic cage,and 24 h-urine was collected (usually from 8:00 to 8:00 of thenext day) into glass bottles containing 500 µl of 6 NHCl while the fasting was continued. Then rats were killed andthe SPT activity in liver homogenates was determined as describedabove.
Measurement of Urinary Substances
Oxalate was determined by high performance capillary electrophoresis(HPCE) as described previously (21,22), except that urine samplestreated with stearate-deactivated charcoal (23) were subjectedto HPCE. In another experiment in this study, in which oxalatewas determined by both HPCE and an enzymatic method with oxalateoxidase (23), the values measured by the two methods reasonablyagreed with each other. Creatinine and glycolate were determinedby the Jaffe reaction and HPCE (21,22), respectively. Ureaand ammonia were determined enzymatically with glutamate dehydrogenaseand a combination of urease-S and glutamate dehydrogenase, respectively(24,25). Hydroxyproline, proline, glycine, and serine wereanalyzed by PICO-TAG chromatography.
Materials
Sodium DL-4-hydroxy-2-ketoglutarate (substrate for HKG aldolase)was prepared as described by Carnie et al. (20), except thatits barium salt was converted to the sodium salt by exchangingBa2+ for H+ with a small amount of Dowex 50W-H+ slurry, followedby neutralization of the supernatant to about pH 6 with NaOH.The 2,4-dinitrophenylhydrazone derivative of P5C was prepared,and P5C was generated from the hydrazone as described by Mezland Knox (26), the concentration of generated P5C being determinedwith o-aminobenzaldehyde. The 2-aminobenzaldehyde agent andglucagon were obtained from Wako Pure Chemical Industries (Osaka,Japan) and NOVO Nordisk Pharma (Denmark), respectively. Sourcesof reagents and enzymes used for the assay of the SPT activityof SPT/AGT (18,19) have been described in the publicationscited.
Statistical Analyses
Values are expressed as mean ± SEM. Intergroup comparisonswere made by two-tailed t test for unpaired data, and intragroupcomparisons were made by two-way ANOVA, followed by the Newman-Keulsas post hoc test using the SPSS statistical package (SPSS, Inc.,Chicago, IL). P < 0.05 was considered statistically significant.
Effect of Glucagon Injection on Enzymes Involved in L-Hydroxyproline Metabolism (Experiment 1)
Induction by glucagon of SPT/AGT in liver mitochondria in therat (9,11,14–16) may be useful as a model for studyingthe role of this enzyme in the metabolism of glyoxylate formedfrom L-hydroxyproline in liver mitochondria, provided that otherenzymes of hydroxyproline metabolism are not affected by glucagon.In rat liver, SPT/AGT is located in both mitochondria and peroxisomesin a ratio of about 4:6; of these, only mitochondrial SPT/AGTis markedly induced by glucagon (11,14). Therefore, the fivefoldincrease by glucagon of total SPT/AGT activity represented byits SPT activity suggests that mitochondrial SPT/AGT had beeninduced more than tenfold. We investigated whether hydroxyprolineoxidase and HKG aldolase were affected by glucagon under conditionsin which mitochondrial SPT/AGT was induced; of the four enzymesinvolved in the metabolism of L-hydroxyproline (Figure 1), thesetwo enzymes were thought to be specific to hydroxyproline metabolism(27,28). P5C dehydrogenase contributes to the metabolism ofproline and ornithine in addition to that of hydroxyproline,but its activity was also measured.
As shown in Table 1, hydroxyproline oxidase, P5C dehydrogenase,and HKG aldolase did not respond to the injection of glucagonunder conditions in which SPT/AGT was substantially induced.Rat liver mitochondrial aspartate aminotransferase was alreadydocumented not to respond to 300 µg/100 g body wt of glucagongiven intraperitoneally during fasting (29). These results suggestthat liver mitochondrial SPT/AGT is induced by glucagon withoutaccompanying changes in the metabolism of L-hydroxyproline toglyoxylate.
Effect of Glucagon Induction of SPT/AGT on Formation of Oxalate from L-Hydroxyproline In Vivo (Experiment 2)
We examined whether glyoxylate formed from L-hydroxyprolinein mitochondria was oxidized to oxalate in vivo by orally administeringa high dose of L-hydroxyproline to 48 h-fasted control rats.We also investigated whether the formation of L-hydroxyproline-derivedoxalate was curtailed when liver mitochondrial SPT/AGT was increased,by comparing urinary oxalate excretion in glucagon-treated ratswith that in control rats. In this series of experiments, wetook advantage of the slow rate of turnover of SPT/AGT in ratliver mitochondria (18). We repeatedly observed that glucagoninduced SPT/AGT in mitochondria almost selectively; once induced,the mitochondrial SPT/AGT maintained the high level of activityfor as long as several days (18). In this study, glucagon wasinjected under fasting conditions, and L-hydroxyproline wasgiven orally 24 h after the glucagon injection, when the inducedlevel of SPT/AGT had reached a plateau as judged from preliminaryobservation of the time course of the glucagon induction. Urinewas then collected for 24 h while fasting was continued andoxalate, glycolate, and other compounds were determined.
Two-way analysis of variation showed that SPT activity was significantlyaffected by glucagon injection (P = 0.0001) but not by administrationof L-hydroxyproline (P = 0.7252). Urinary excretion of oxalateand glycolate was significantly affected by administration ofL-hydroxyproline (P = 0.0001 and 0.0012, respectively) and glucagoninjection (P = 0.0001 and 0.0019, respectively), and there wasa significant interaction between the effect of L-hydroxyprolineand glucagon on excretion of oxalate and glycolate (P = 0.0001and 0.0001, respectively). Urinary excretion of glycine, serine,hydroxyproline, proline, ammonia, and urea was also significantlyaffected by administration of L-hydroxyproline (P = 0.0025,0.0004, 0.0001, 0.0001, 0.0001, and 0.0009, respectively) butnot by glucagon injection, especially when data were expressedas the ratios to creatinine (P = 0.9949, 0.6885, 0.0882, 0.1752,0.4995, and 0.3674, respectively).
Administration of L-hydroxyproline caused a large increase inurinary hydroxyproline excretion, as expected (Table 2). Recoveryof the administered dose (4.8 mmol) of L-hydroxyproline in urinewas 13 to 17%. Because L-hydroxyproline is not utilized as aprecursor amino acid for protein synthesis, its only metabolicfate is expected to be catabolism (Figure 1). We therefore presumedthat a fairly large amount of L-hydroxyproline had been catabolizedin this experiment. Interestingly, a small increase in urinaryexcretion of proline was also observed (Table 2), suggestingthat L-hydroxyproline administration somehow interfered withoxidation of proline.
Table 2. Urinary excretion of ammonia, urea, and amino acids after administration of L-hydroxyproline or glycolatea
Excretion of glycine, serine, ammonia, and urea was also increased1.6 to 3.4-fold by administration of L-hydroxyproline (Table 2).Although the amount of glycine and serine excreted intourine was very small, increase in excretion of ammonia plusurea by L-hydroxyproline administration accounted for more than40% of administered dose of L-hydroxyproline, irrespective ofprior induction by glucagon of mitochondrial SPT/AGT. About15% of administered dose of L-hydroxyproline was excreted intourine unchanged; therefore, the increase in urinary ammoniaplus urea represented as much as 50% of the lost (remaining85%) L-hydroxyproline. These results indicate that a large fractionof administered L-hydroxyproline was metabolized through HKGand glycine in both glucagon-treated and control rats (Figure 2A).It is possible that glyoxylate was metabolized throughglycine fairly efficiently, even in the absence of glucagoninduction of liver mitochondrial SPT/AGT (Tables 1 to 3).
Figure 2. Transfer of amino group in the metabolism of L-hydroxyproline (A) and glycolate (B) in rat liver. In the metabolism of L-hydroxyproline (A), amino group derived from this imino acid is removed as L-aspartate in the mitochondrial aspartate aminotransferase-catalyzed reaction. On the other hand, stoichiometric amount of L-alanine is required for conversion of glyoxylate into glycine. The major source of L-alanine in mammalian cells is alanine aminotransferase-catalyzed transfer of amino group from L-glutamate to pyuvate. L-glutamate is in equilibrium with L-aspartate in cells through rapid interconversion catalyzed by aspartate aminotransferase, which is also responsible for transfer of amino group from 4-hydoxy-L-glutamate to oxaloacetate generating HKG and L-aspartate. When a large amount of L-aspartate is supplied from 4-hydroxy-L-glutamate, therefore, a considerable amount of L-glutamate should be generated in association with the formation of HKG. The amino group of L-glutamate is then transferred to glyoxylate via L-alanine to form glycine. Glycine is oxidized to NH3 and CO2 by mitochondrial glycine cleavage enzyme system; in conjugation with this oxidation, another molecule of glycine is converted to L-serine by mitochondrial serine hydroxymethyltrasferase. In rat liver, L-serine is dehydrated by serine dehydratase producing NH3 and pyruvate (37). In the metabolism of glycolate (B), on the other hand, neither L-alanine nor L-glutamate is supplied from glycolate. Therefore, about the same amount of L-glutamate as that used for supply of L-alanine for the metabolism of glyoxylate may be saved from glutamate dehydrogenase-catalyzed oxidation to -ketoglutarate and NH3. AlaT, alanine aminotransferase; AspT, aspartate aminotransferase; GCS, glycine cleavage enzyme system; GlDH, glutamate dehydrogenase; -KG, -ketoglutarate; Me-THF, 5,10-methylenetetrahydrofolate; mAspT, mitochondrial aspartate aminotranferase; OAA, oxaloacetate; OH-L-Glu, 4-hydroxy-L-glutamate; P5C-DH, P5C dehydrogenase; SDH, serine dehydratase; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate.
Table 3. Effect of glucagon induction of mitochondrial SPT/AGT on urinary excretion of oxalate and glycolate after administration of L-hydroxyproline or glycolatea
In control rats, urinary excretion of oxalate and glycolatewas significantly increased (oxalate, 5.6-fold; glycolate, 2.5-fold)by administration of L-hydroxyproline, although fractional conversionof the "lost" L-hydroxyproline to oxalate plus glycolate wasonly 0.36%. The increase in oxalate excretion due to L-hydroxyprolineadministration was distinctly less in glucagon-treated ratsthan in control rats, and the small amount of L-hydroxyproline-derivedglycolate formation was prevented by prior induction of mitochondrialSPT/AGT (Table 3).
Effect of Glucagon Induction of SPT/AGT on Formation of Oxalate from Glycolate (Experiment 2)
Glyoxylate is also produced from glycolate in liver peroxisomes,and according to Harris and Richardson (1) glycolate is muchmore abundant in plants than in animal tissues; therefore, theperoxisomal presence of SPT/AGT in herbivores may be requiredto prevent excessive oxalate production by effectively metabolizingglycolate-derived glyoxylate to glycine in situ. If this isthe case, induction of mitochondrial SPT/AGT by glucagon wouldnot significantly affect oxalate production from glycolate.To test this hypothesis, 100 mg (1.02 mmol) of Na-glycolatewas administered orally to control rats and glucagon-treatedrats, and the urinary excretion of oxalate and glycolate wasmeasured.
Two-way analysis of variation showed that SPT activity was significantlyaffected by glucagon injection (P = 0.0001) but not by administrationof glycolate (P = 0.8924). Urinary excretion of oxalate, glycolate,and glycine was significantly affected by administration ofglycolate (P = 0.0001, 0.0001, and 0.0046, respectively) butnot by glucagon injection (P = 0.2736, 0.5184, and 0.8339, respectively).No significant intragroups difference was detected in urinaryexcretion of serine, hydroxyproline, proline, ammonia, and urea.
As shown in Table 3, glycolate administration caused a markedincrease (about eightfold) in oxalate excretion into urine;as expected, glucagon induction of mitochondrial SPT/AGT didnot affect the glycolate-derived oxalate. A large amount ofglycolate was also excreted into urine, and the mean recoveryof administered glycolate in urine was about 43 to 46%, suggestingthat a fairly large amount of administered glycolate had beenmetabolized through glyoxylate and glycine under the conditionsused. In this case, no significant increase was observed inurinary excretion of ammonia and urea, consistent with the absenceof amino group in administered glycolate (Table 2; Figure 2B).
In this study, we showed that urinary excretion of oxalate andglycolate was markedly increased when a large dose of L-hydroxyprolinewas administered to rats (Table 3), suggesting that glyoxylateformed from L-hydroxyproline in mitochondria can be oxidizedto oxalate. A portion of glyoxylate oversupplied in mitochondriamay leak out of mitochondria and serve as a substrate for lactatedehydrogenase (30) and glyoxylate reductase, resulting in theformation of oxalate and glycolate, respectively, and theirexcretion into the urine. We also observed that oxalate formationfrom L-hydroxyproline was significantly reduced when mitochondrialSPT/AGT had been induced by prior injection of glucagon. Glucagondid not affect other enzymes of hydroxyproline metabolism; wetherefore think it likely that in glucagon-treated rats glyoxylatesupplied from L-hydroxyproline in mitochondria is very efficientlymetabolized to glycine in situ, reducing the harmful formationof oxalate.
Urinary excretion of glycine, serine, ammonia, and urea wasalso increased by L-hydroxyproline administration (Table 2).Especially, the increase in the excretion of ammonia plus ureaaccounted for as much as 43 to 45% of the administered doseof L-hydroxyproline not only in glucagon-treated rats but alsoin control rats, suggesting that hydroxyproline-derived glyoxylatewas metabolized fairly rapidly via glycine, even in the absenceof glucagon-induction of liver mitochondrial SPT/AGT. Nevertheless,the hydroxyproline-derived oxalate formation was distinctlyless in glucagon-treated rats than in control rats. In thiscontext, it is important that Km of SPT/AGT for glyoxylate isas low as 0.01 mM (31). It appears that a fairly high activityof such a low Km aminotransferase is necessary to maintain tissueconcentration of glyoxylate sufficiently low, even after ingestionof a large amount of L-hydroxyproline. The increase in the excretionof glycine and serine was small, probably because they are physiologicallyimportant amino acids that are used in a variety of ways incells, and their intracellular concentrations are maintainedwithin a given range by serine hydroxymethyltransferase-catalyzedinterconversion and their rapid metabolism by the mitochondrialglycine cleavage enzyme system, serine dehydratase, and SPT/AGT(7). In this study, we did not attempt to measure glyoxylateexcretion, because in our previous study (at University of theRyukyus) excretion of glyoxylate after administration of glycolatewas as much as two orders of magnitude lower than that of oxalate(21), and measurement of total amount of glyoxylate excretedinto urine was not simple enough due to the properties of glyoxylateto form adducts with various compounds (32).
The dose of L-hydroxyproline administered (630 mg) was about5 times the amount that would be ingested if a rat had to supplydaily heat production from total animal protein alone. For thiscalculation, heat production by a 170-g rat was assumed to be17 kcal/d (33), and collagen content of total animal proteinand hydroxyproline content of collagens were assumed to be 30%(wt/wt) and 10 to 13% (wt/wt) (4), respectively. Therefore,the experimental conditions applied to rats in this study werefar from physiologic, but it is evident that oxalate can beformed from L-hydroxyproline in vivo, via its metabolism toglyoxylate in mitochondria.
We also observed that induction of mitochondrial SPT/AGT byglucagon had little effect on oxalate production from glycolate(Table 3). This finding is in accordance with our predictionand the report by Danpure et al. (34) that SPT/AGT misroutedto mitochondria in a group of patients with primary hyperoxaluriatype 1 cannot fulfill its metabolic role of detoxicating glyoxylateproperly.
Concerning the species-specific and food habit-dependent organelledistribution of SPT/AGT, it is important to recall that oxalateis an apparently useless and even toxic end-product of metabolism,because its insoluble calcium salt forms deposits in tissues,disrupting their functions. In patients with primary hyperoxaluriatype 1, a hereditary oxalate stone disease caused by a functionaldeficiency of SPT/AGT (8), progressive renal deposition of calciumoxalate in the form of urolithiasis, and/or nephrocalcinosisleads to renal failure and systemic oxalosis, finally resultingin death, usually before the third decade of age (35,36). Itis also important that glyoxylate is formed either from glycolatein peroxisomes or from L-hydroxyproline in mitochondria. Inaddition to its dual organelle distribution, SPT/AGT plays adual role in glyoxylate metabolism and serine metabolism, althoughour recent studies showed that this enzyme contributed substantiallyto serine metabolism irrespective of mitochondrial or peroxisomallocalization (7,37). Therefore, peroxisomal localization ofSPT/AGT in herbivores and humans may be important for removalof glycolate-derived glyoxylate, because plants contain muchmore glycolate than animal tissues, although the content ofglycolate in vegetables and fruits is not very high, at 1 to7.5 mg/100 g wet weight (1), and glycolate is converted to glyoxylatein liver peroxisomes. Likewise, the present study suggests thatmitochondrial presence of SPT/AGT in carnivores is importantfor the metabolism of glyoxylate formed from L-hydroxyprolinein mitochondria, because animal proteins are rich in hydroxyprolineas a constituent amino acid of collagens and elastins. Thisspecies-specific dual organelle localization of SPT/AGT is causedeither by transcription of the SPT/AGT gene from two differentstart sites in exon 1 (16,38,39) or loss of the upstream translationinitiation ATG codon as a result of mutations (40–42). It is attractive to hypothesize that those animal speciesthat succeeded in providing SPT/AGT for the proper organelleas a result of gene mutations have been advantageous in survivingevolutionary selection. To investigate further the hypothesis,it would be highly desirable if rat peroxisomal SPT/AGT couldbe markedly and selectively induced; because no such means areavailable at present, we are going to undertake metabolic studiesin rabbits and dogs.
Noguchi et al. (43) showed that mitochondrial extracts fromthe livers of dogs, cats, rats, and mice contained two formsof alanine:glyoxylate aminotransferase (AGT). The form-designatedisozyme 1 is SPT/AGT or AGT-1, the subject enzyme of this study,and the other form-designated isozyme 2 (AGT-2) is a separateaminotransferase specific for L-alanine and glyoxylate. Accordingto their results, the activity of AGT-2 in mitochondria of normalrat liver was much higher than the AGT activity of SPT/AGT,but administration of glucagon caused SPT/AGT activity to markedlyexceed AGT-2 activity. In mitochondria from dog and cat livers,AGT activity of SPT/AGT exceeded that of AGT-2 activity, asin the case of glucagon-treated rats (43). In addition, thereported apparent Km of AGT-2 for glyoxylate (1.0 mM) (43) wasmuch higher than that of SPT/AGT (0.01 mM) (31). We do not yetknow how SPT/AGT and AGT-2 share the role of metabolizing glyoxylatein mitochondria, but SPT/AGT may be a more efficient catalystdue to its extremely low Km for glyoxylate.
In kidney mitochondria, glyoxylate is efficiently convertedto L-serine via transamination to glycine, followed by furthermetabolism by mitochondrial glycine cleavage enzyme system andserine hydroxymethyltransferase (3). In the dog and cat, kidneymitochondria contain both SPT/AGT and AGT-2, SPT/AGT being predominant,whereas pig, rat, and monkey kidney mitochondria contain onlyAGT-2 (44). Thus, mitochondrial presence of SPT/AGT in carnivoresmay also be highly desirable for efficient metabolism in thekidney of hydroxyproline-derived glyoxylate to glycine, preventingoxidation to oxalate.
Acknowledgments
This study was supported in part by a Grant-in-Aid for Encouragementof Young Scientists from the Ministry of Education, Culture,Sports, Science and Technology of Japan (to TT, #13770876).We thank Ms. Keiko Sato, Department of Biochemistry I, HamamatsuUniversity School of Medicine (HUSM), and Ms. Tomoko Maeda,Department of Urology, University of the Ryukyus, for technicalassistance in the enzymatic determination of oxalate and forcapillary electropheretic determination of oxalate and glycolate,respectively. We thank Dr. Masao Kanamori, Department of PublicHealth, HUSM, for advice on statistical analysis of data. Weare also grateful to Dr. Masatoshi Kitagawa, Department of BiochemistryI, HUSM, Dr. Satoshi Yamaguchi, Department of Urology, AsahikawaMedical College, and Dr. Yusaku Okada, Department of Urology,Shiga University of Medical Science for their useful discussion.
Harris KS, Richardson KE: Glycolate in the diet and its conversion to urinary oxalate in the rat. Invest Urol 18: 106–109, 1980[Medline]
Maitra U, Dekker EE: Purification and properties of rat liver 2-keto-4-hydroxyglutarate aldolase. J Biol Chem 239: 1485–1491, 1964[Free Full Text]
Lowry M, Hall DE, Brosnan JT: Hydroxyproline metabolism by the rat kidney: Distribution of renal enzymes of hydroxyproline catabolism and renal conversion of hydroxyproline to glycine and serine. Metabolism 34: 955–961, 1985[CrossRef][Medline]
Neuman RE, Logan MA: The determination of hydroxyproline. J Biol Chem 184: 299–306, 1950[Free Full Text]
Snell K: Enzymes of serine metabolism in normal, developing and neoplastic rat tissues. Adv Enzyme Regul 22: 325–400, 1984[CrossRef][Medline]
Sallack HJ: Formation of serine from hydroxypyruvate and L-alanine. J Biol Chem 223: 1101–1108, 1956[Free Full Text]
Xue HH, Sakaguchi T, Fujie M, Ogawa H, Ichiyama A: Flux of the L-serine metabolism in rabbit, human, and dog livers. Substantial contributions of both mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase. J Biol Chem 274: 16028–16033, 1999[Abstract/Free Full Text]
Danpure CJ, Jennings PR: Peroxisomal alanine:glyoxylate aminotransferase deficiency in primary hyperoxaluria type I. FEBS Lett 201: 20–24, 1986[CrossRef][Medline]
Noguchi T: Amino acid metabolism in animal peroxisomes. In: Peroxisomes in Biology and Medicine, edited by Fahimi HD, Sies H, Berlin, Springer-Verlag, 1987, pp 234–243
Noguchi T, Takada Y: Peroxysomal localization of serine: pyruvate aminotransferase in human liver. J Biol Chem 253: 7598–7600, 1978[Abstract/Free Full Text]
Takada Y, Noguchi T: Subcellular distribution, and physical and immunological properties of hepatic alanine: glyoxylate aminotransferase isozymes in different mammalian species. Comp Biochem Physiol 72B: 597–604, 1982
Danpure CJ, Guttridge KM, Fryer P, Jennings PR, Allsop T, Purdue PE: Subcellular distribution of hepatic alanine: glyoxylate aminotransferase in various mammalian species. Cell Sci 97: 669–678, 1990[Abstract/Free Full Text]
Danpure CJ, Fryer P, Jennings PR, Allsop J, Griffiths S, Cunningham A: Evolution of alanine:glyoxylate aminotransferase 1 peroxisomal and mitochondrial targeting. A survey of its subcellular distribution in the livers of various representatives of the classes Mammalia. Aves and Amphibia. Eur J Cell Biol 64: 295–313, 1994[Medline]
Oda T, Yanagisawa M, Ichiyama A: Induction of serine: Pyruvate aminotranferase in rat liver organelles by glucagon and a high-protein diet. J Biochem 91: 219–232, 1982[Abstract/Free Full Text]
Uchida C, Funai T, Oda T, Ohhashi K, Ichiyama A: Regulation by glucagon of serine:pyruvate/alanine:glyoxylate aminotransferase gene expression in cultured rat hepatocytes. J Biol Chem 269: 8849–8856, 1994[Abstract/Free Full Text]
Uchida C, Oda T, Sugiyama T, Otani S, Kitagawa M, Ichiyama A: The role of Sp1 and AP-2 in basal an protein kinase A-induced expression of mitochondrial serine:pyruvate aminotransferase in hepatocytes. J Biol Chem 277: 39082–39092, 2002[Abstract/Free Full Text]
Strecker HJ: The interconvention of glutamic acid and proline. III. 1-pyrroline 5-carboxylate dehydrogenase. J Biol Chem 235: 3218–3223, 1960[Free Full Text]
Fukushima M, Aihara Y, Ichiyama A: Immunochemical studies on induction of rat liver mitochondrial serine:pyruvate aminotransferase by glucagon. J Biol Chem 253: 1187–1194, 1978[Abstract/Free Full Text]
Ishikawa K, Kaneko E, Ichiyama A: Pyridoxal 5'-phosphate binding of a recombinant rat serine:pyruvate/alanine:glyoxylate aminotranferase. J Biochem 119: 970–978, 1996[Abstract/Free Full Text]
Carnie JA, Rowsell EV, Dabbaghian MK, Hobbs DR, Rowsell KV: Comparative and development studies on 4-hydroxy-2-oxoglutarate aldolase and hydroxyproline oxidase. Comp Biochem Physiol 71B: 681–688, 1982
Ogawa Y, Miyazato T, Hatano T: Importance of oxalate precursors for oxalate metabolism in rats. J Am Soc Nephrol 10: S341–S344, 1999
Ogawa Y, Hatano T: Urinary oxalate and citrate determination. Kidney and Dialysis Suppl 43: 151–157, 1997
Ichiyama A, Nakai E, Funai T, Oda T, Katafuchi R: Spectrophotometric determination of oxalate in urine and plasma with oxalate oxidase. J Biochem 98: 1375–1385, 1985[Abstract/Free Full Text]
Gutmann I, Bergmeyer HU: Urea. In: Methods of Enzymatic Analysis. 2nd ed., edited by Bergmeyer HU, New York and London-Weinheim, Verlag Chemie-Academic Press, 1974, pp 1791–1798
Kun E, Kearney EB: Ammonia. In: Methods of Enzymatic Analysis. 2nd ed., edited by Bergmeyer HU, New York and London-Weinheim, Verlag Chemie-Academic Press, 1974, pp 1802–1806
Mezl VA, Knox WE: Properties and analisis of a stable derivative of pyrroline-5-carboxylic acid for use in metabolic studies. Anal Biochem 74: 430–440, 1976[CrossRef][Medline]
Kuttan R, Radhakrishnan AN: Biochemistry of the hydroxyprolines. In: Advances in Enzymology 37, edited by Meister A, New York, Interscience, 1973, pp 273–347
Phang JM, Yeh GC, Scriver CR: Disorders of proline and hydroxyproline metabolism. In: The Metabolic and Molecular Bases of Inherited Disease, 7th Ed, edited by Scriver CR, Beaudet AL, Sly WS, Valle D, New York, McGraw-Hill, 1995, pp 1125–1146
Horio Y, Tanaka T, Taketoshi M, Uno T, Wada H: Rat cytosolic aspartate aminotransferase: Regulation of its mRNA and contribution to gluconeogenesis. J Biochem 103: 805–808, 1988[Abstract/Free Full Text]
Poore RE, Hurst CH, Assimos DG, Holmes RP: Pathways of hepatic oxalate synthesis and their regulation. Am J Physiol 272: C289–C294, 1997
Yanagisawa M, Higashi S, Oda T, Ichiyama A: Properties and possible physiological role of rat liver serine:pyruvate aminotransferase. In: Biochemistry of Metabolic Processes, edited by Lennon DLF, Stratman FW, Zahlten RN, New York-Amsterdam-Oxford, Elsevier Biomedical, 1983, pp 413–426
Funai T, Ichiyama A: High-performance liquid chromatographic determination of glyoxylate in rat liver. J Biochem 99: 579–589, 1986[Abstract/Free Full Text]
McMahan T: Size and shape in biology: Elastic criteria impose limits on biological proportions, and consequently on metabolic rates. Science 179: 1201–1204, 1973[Abstract/Free Full Text]
Danpure CJ, Cooper PJ, Wise PJ, Jennings PR: An enzyme trafficking defect in two patients with primary hyperoxaluria type 1: Peroxisomal alanine/glyoxylate aminotransferase rerouted to mitochondria. J Cell Biol 108: 1345–1352, 1989[Abstract/Free Full Text]
Latta K, Brodehl J: Primary hyperoxaluria type I. Eur J Pediatr 149: 518–522, 1990[CrossRef][Medline]
Danpure CJ, Purdue PE: Primary hyperoxaluria. In: The Metabolic and Molecular Bases of Inherited Disease. 7th Ed, edited by Scriver CR, Beaudet AL, Sly WS, Valle D, New York, McGraw-Hill, 1995, pp 2385–2424
Xue HH, Fujie M, Sakaguchi T, Oda T, Ogawa H, Kneer NM, Lardy HA, Ichiyama A: Flux of the L-serine metabolism in rat liver. The predominant contribution of serine dehydratase. J Biol Chem 274: 16020–16027, 1999[Abstract/Free Full Text]
Oda T, Funai T, Ichiyama A: Generation from a single gene of two mRNAs that encode the mitochondrial and peroxisomal serine:pyruvate aminotransferase of rat liver. J Biol Chem 265: 7513–7519, 1990[Abstract/Free Full Text]
Sugiyama T, Uchida C, Oda T, Kitagawa M, Ichiyama A: Involvement of CCAAT/enhancer binding protein in regulation of the rat serine:pyruvate/alanine:glyoxylate aminotransferase gene expression. FEBS lett 508: 16–22, 2001[CrossRef][Medline]
Takada Y, Kaneko N, Esumi H, Purdue PE, Danpure CJ: Human peroxisomal L-alanine: glyoxylate aminotransferase. Evolutionary loss of a mitochondrial targeting signal by point mutation of the initiation codon. Biochem J 268: 517–520, 1990[Medline]
Purdue PE, Lumb MJ, Danpure CJ: Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular targeting. Analysis of the marmoset and rabbit genes. Eur J Biochem 207: 757–766, 1992[Medline]
Lumb MJ, Purdue PE, Danpure CJ: Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular targeting. Analysis of the feline gene. Eur J Biochem 221: 53–62, 1994[Medline]
Noguchi T, Okuno E, Takada Y, Minatogawa Y, Okai K, Kido R: Characteristics of hepatic alanine-glyoxylate aminotransferase in different mammalian species. Biochem J 169: 113–122, 1978[Medline]
Takada Y, Noguchi T: Kidney alanine:glyoxylate aminotranferase isoenzymes; Species distribution, subcellular distribution and properties. Comp Biochem Physiol 65B: 133–138, 1980[CrossRef]
Received for publication January 2, 2002.
Accepted for publication January 10, 2003.
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Abstract
Introduction
1-pyrroline-5-carboxylate (P5C) dehydrogenase was to be assayed, acetone dried powder was prepared from a liver specimen of each rat, and the powder was extracted with 10 vol of chilled water as described by Strecker (17). The reaction mixture (1.0 ml) for the assay of P5C dehydrogenase comprised 50 mM Tris-HCl, 50 µM K-phosphate, 1.2 mM pyrroline 5-carboxylate and acetone powder extract, final pH being close to 8.2. The extract was added last, and the activity was measured at room temperature by the increase in absorbance at 340 nm. The SPT activity of SPT/AGT was determined as described previously (18,19 ). SPT/AGT catalyzes transamination of glyoxylate to glycine with L-alanine or L-serine as an amino acid (rat SPT/AGT shows wider substrate specificity), but the tissue level of this enzyme was represented by the SPT activity in this study, because a single catalytic site is responsible for the three enzymatic activities and determination of SPT activity was free of interference by AGT-2, an isoenzyme of SPT/AGT or AGT-1. Hydroxyproline oxidase and HKG aldolase activities were assayed as described by Carnie et al. (20). 
-ketoglutarate and NH3. AlaT, alanine aminotransferase; AspT, aspartate aminotransferase; GCS, glycine cleavage enzyme system; GlDH, glutamate dehydrogenase;