ATP-dependent degradation of a mutant serine: pyruvate/alanine:glyoxylate aminotransferase in a primary hyperoxaluria type 1 case.

Primary hyperoxaluria type 1 (PH 1), an inborn error of glyoxylate metabolism characterized by excessive synthesis of oxalate and glycolate, is caused by a defect in serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT). This enzyme is peroxisomal in human liver. Recently, we cloned SPT/AGT-cDNA from a PH 1 case, and demonstrated a point mutation of T to C in the coding region of the SPT/AGT gene encoding a Ser to Pro substitution at residue 205 (Nishiyama, K., T. Funai, R. Katafuchi, F. Hattori, K. Onoyama, and A. Ichiyama. 1991. Biochem. Biophys. Res. Commun. 176:1093-1099). In the liver of this patient, SPT/AGT was very low with respect to not only activity but also protein detectable on Western blot and immunoprecipitation analyses. Immunocytochemically detectable SPT/AGT labeling was also low, although it was detected predominantly in peroxisomes. On the other hand, the level of translatable SPT/AGT-mRNA was higher than normal, indicating that SPT/AGT had been synthesized in the patient's liver at least as effectively as in normal liver. Rapid degradation of the mutant SPT/AGT was then demonstrated in transfected COS cells and transformed Escherichia coli, accounting for the low level of immunodetectable mutant SPT/AGT in the patient's liver. The mutant SPT/AGT was also degraded much faster than normal in an in vitro system with a rabbit reticulocyte extract, and the degradation in vitro was ATP dependent. These results indicate that a single amino acid substitution in SPT/AGT found in the PH1 case leads to a reduced half-life of this protein. It appears that the mutant SPT/AGT is recognized in cells as an abnormal protein to be eliminated by degradation.

The requirement of heat shock cognate 70 protein for mitochondrial import varies among precursor proteins and depends on precursor length.

The cytosolic heat shock cognate 70-kDa protein (hsc70) is required for efficient import of ornithine transcarbamylase precursor (pOTC) into rat liver mitochondria (K. Terada, K. Ohtsuka, N. Imamoto, Y. Yoneda, and M. Mori, Mol. Cell. Biol. 15:3708-3713, 1995). The requirement of hsc70 for mitochondrial import of various precursor proteins and truncated pOTCs was studied by using an in vitro translation import system in which hsc70 was completely depleted. hsc70-dependent import of pOTC was about 60% of the total import, while import of the aspartate aminotransferase precursor, the serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase was about 50, 30, and 0%, respectively. The subunit sizes of these four precursor proteins were 40 to 47 kDa. When pOTC was serially truncated from the COOH terminal, the hsc70 requirement decreased gradually and was not evident for the shortest truncated pOTCs of 90 and 72 residues. These truncated pOTCs were imported and proteolytically processed rapidly in 0.5 to 2 min at 25 degrees C, and the processed mature portions and the presequence portion were rapidly degraded. Sucrose gradient centrifugation analysis followed by import assay showed that pOTC synthesized in rabbit reticulocyte lysate forms an import-competent complex of about 11S in an hsc70-dependent manner. S values of import-competent forms of aspartate aminotransferase precursor, serine:pyruvate aminotransferase precursor, and 3-oxoacyl coenzyme A thiolase were 9S, 9S, and 4S, respectively. Thus, the S value decreased as the hsc70 dependency decreased. Precursor proteins were coimmunoprecipitated from the reticulocyte lysate containing the newly synthesized precursor proteins with an hsc70 antibody. The amount of coimmunoprecipitated proteins was much larger in the absence of ATP than in its presence. Among the four precursor proteins, the amount of coimmunoprecipitated protein decreased as the hsc70 dependency decreased.

The formation of oxalate from glycolate in rat and human liver.

In this study, we attempted to elucidate the metabolic pathway and enzymes actually involved in oxalate formation from glycolate in rat and human liver. In rat liver, the formation of oxalate from glycolate appeared to take place predominantly via glyoxylate. The oxalate formation from glycolate observed with crude enzyme preparations was almost entirely accounted for by the sequential actions of glycolate oxidase and xanthine oxidase (XOD) or lactate dehydrogenase (LDH). Under the conditions used, no significant activity was attributable to glycolate dehydrogenase, an enzyme reported to catalyze the direct oxidation of glycolate to oxalate. Among the three enzymes known to catalyze the oxidation of glyoxylate to oxalate, glycolate oxidase and XOD showed much lower activities (a higher Km and lower Vmax) toward glyoxylate than those with the respective primary substrates. As to LDH, none of the LDH subunit-deficient patients examined showed profoundly lowered urinary oxalate excretion. Based on the results obtained, the presumed efficacies in vivo of individual enzymes, as catalysts of glyoxylate oxidation, and the in vivo conditions assumed to allow their catalysis of oxalate production are discussed.

Induction by peroxisome proliferators and triiodothyronine of serine:pyruvate/alanine:glyoxylate aminotransferase of rat liver.

In rat liver, a single serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) gene is transcribed from two transcription initiation sites. Transcription from the upstream site generates the mRNA encoding the precursor for mitochondrial SPT (pSPTm) and is markedly enhanced by the administration of glucagon or cAMP. In this report we show the increase in the downstream transcript, the peroxisomal SPT (SPTp) mRNA, caused by peroxisome proliferators and triiodothyronine (T3). In the case of T3, the pSPTm mRNA was also increased 72 h after a single administration of the hormone in addition to an earlier increase in SPTp mRNA.

Involvement of CCAAT/enhancer-binding protein in regulation of the rat serine:pyruvate/alanine:glyoxylate aminotransferase gene expression.

In the rat liver, transcription of the serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) gene occurs from two sites, +1 and +66, in exon 1, resulting in the formation of two mRNAs, one for a precursor of mitochondrial SPT/AGT and the other for peroxisomal SPT/AGT, respectively. In this study, we attempted to characterize the downstream promoter responsible for generation of peroxisomal SPT/AGT. The minimal downstream promoter was confined to the +21-+90 region. We demonstrated that C/EBPalpha and C/EBPbeta bound around the downstream start site (+66) contribute to the promoter activity. The downstream promoter activity is also regulated positively by a short inverted repeat, located 20-30 bp upstream of the downstream start site, through a protein factor(s) bound to this region. On the other hand, the sequence just downstream of the start site may negatively regulate the promoter activity.

Primary structure of the alpha-subunit of bovine adenylate cyclase-inhibiting G-protein deduced from the cDNA sequence.

The primary structure of the alpha-subunit of the adenylate cyclase-inhibiting G-protein (Gi) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. A much higher degree of amino acid sequence homology is observed between the alpha-subunits of Gi and transducin (68%) than between those of Gi and the adenylate cyclase-stimulating G-protein (Gs) (43%) or between those of transducin and Gs (42%).

Primary structure of porcine cardiac muscarinic acetylcholine receptor deduced from the cDNA sequence.

The complete amino acid sequence of the porcine cardiac muscarinic acetylcholine receptor has been deduced by cloning and sequencing the cDNA. The tissue location of the RNA hybridizing with the cDNA suggests that this muscarinic receptor species represents the M2 subtype.

Increased level of atrial natriuretic peptide messenger RNA in the hypothalamus and brainstem of spontaneously hypertensive rats.

OBJECTIVE: The aim of this study was to investigate atrial natriuretic peptide (ANP) gene expression in the central nervous system (CNS) during hypertension. METHODS: We measured and compared immunoreactive atrial natriuretic peptide (irANP) and ANP messenger RNA (mRNA) in the hypothalamus and brainstem of 17-week-old spontaneously hypertensive rats (SHR) with those of age-matched Wistar-Kyoto (WKY) rats using ribonuclease (RNase) protection assay for ANP mRNA and a specific radioimmunoassay for irANP. RESULTS: RNase protection assay revealed that the concentrations of ANP mRNA in the hypothalamus and brainstem of SHR were higher than those of WKY rats. IrANP concentrations in the hypothalamus and brainstem of SHR were determined by a specific radioimmunoassay and found to be higher than those of WKY rats. Elevated mRNA levels in the hypothalamus and brainstem of SHR indicated that increased level of irANP in the CNS resulted from increased synthesis of ANP. CONCLUSION: We propose that increased synthesis of brain ANP in SHR may reflect a compensatory mechanism induced by hypertension.

Primary hyperoxaluria type 1 in Japan.

Glyoxylate is an immediate precursor of oxalate, but in its metabolism the conversion into glycine catalyzed by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) appears to be the main route. When SPT/AGT is missing as in the case of primary hyperoxaluria type 1 (PH1) more glyoxylate is used for the oxalate production, resulting in calcium oxalate urolithiasis and finally systemic oxalosis. SPT/AGT is a unique enzyme of species-specific dual organelle localization; it is located largely in mitochondria in carnivores and entirely in peroxisomes in herbivores and man. For herbivores, the peroxisomal localization of SPT/AGT is indispensable to avoid massive production of oxalate, probably because liver peroxisomes are the main site of glyoxylate production from glycolate, and plants contain glycolate much more than animal tissues. Recently, we took charge of laboratory examination for 8 cases of primary hyperoxaluria in Japan, and felt that symptoms of some of the Japanese PH1 patients are apparently milder than those of Western patients. The reason of this is not clear, but from the above mentioned seemingly indispensable association of grass-eating with the peroxisomal localization of SPT/AGT it may be related, at least in part, to the food habit of Japanese, especially that of old generation, that they prefer boiled greens rather than frying or raw vegetables.

Peroxisomal and mitochondrial targeting of serine:pyruvate/alanine:glyoxylate aminotransferase in rat liver.

Serine:pyruvate/alanine:glyoxylate aminotransferase (SPT or SPT/AGT) of rat liver is a unique enzyme of dual subcellular localization, and exists in both mitochondria and peroxisomes. To characterize a peroxisomal targeting signal of rat liver SPT, a number of C-terminal mutants were constructed and their subcellular localization in transfected COS-1 cells was examined. Deletion of C-terminal NKL, and point mutation of K2 (the second Lys from the C-terminus), K4 and E15 caused accumulation of translated products in the cytoplasm. This suggests that the PTS of SPT is not identical to PTS1 (the C-terminal SKL motif) in that it is not restricted to the C-terminal tripeptide. In vitro synthesized precursor for mitochondrial SPT was highly sensitive to the proteinase K digestion, whereas peroxisomal SPT (SPTp) was fairly resistant to the protease. In in vitro import experiment with purified peroxisomes, however, SPTp recovered in the peroxisomal fraction was very sensitive to the protease. These results suggest that the mitochondrial precursor is synthesized as an unfolded form and is translocated into the mitochondrial matrix, whereas SPTp is synthesized as a folded form and its conformation changes to an unfolded form just before translocation into peroxisomes.

High-performance liquid chromatographic determination of glyoxylate in rat liver.

A high-performance liquid chromatographic method was developed for the determination of glyoxylate in the liver. Alpha-keto acids in charcoal-treated acid-extract of the liver were converted to the corresponding 2,4-dinitrophenylhydrazones and purified as the derivatives by successive extractions with ethyl acetate and sodium bicarbonate solution. The dinitrophenylhydrazones were then quantitatively converted to the corresponding substituted 2-hydroxyquinoxalines by reaction with o-phenylenediamine, followed by analysis by high-performance liquid chromatography with fluorescence detection. As a control to correct the recovery of tissue glyoxylate, an acid-extract of the liver prepared with the addition of standard glyoxylate (25-50 nmol/g wet weight of tissue) was simultaneously subjected to the analytical procedure. The maximum sensitivity of the glyoxylate measurement as 2-hydroxyquinoxaline (the quinoxaline derivative corresponding to glyoxylate) was defined as the peak area reading five times as high as the blank value obtained without sample and was approximately 10 pmol per injection. Glyoxylate in the addition compound with tris(hydroxymethyl)aminomethane was quantitatively recovered as 2-hydroxyquinoxaline. The addition compounds of glyoxylate with bisulfite and cysteine did not react with 2,4-dinitrophenylhydrazine under the conditions employed and were not detectable as glyoxylate by this method, while the adduct-forming substances added to the acid-extract of the liver did not interfere with the glyoxylate determination. No glyoxylate was detected when the liver extract had been incubated at neutral pH with a large excess of cysteine, indicating that little artificial production of glyoxylate occurred during the analytical procedure. Among 64 compounds tested for possible artificial production of glyoxylate or possible interference with the chromatographic determination of 2-hydroxyquinoxaline, p-hydroxyphenylpyruvate was the only compound which was converted to glyoxylate during the procedure. However, p-hydroxyphenylpyruvate was easily removed from the acid-extract of the tissue by charcoal treatment. The amount of glyoxylate in the liver of fasted rat was measured by the present method to be approximately 5 nmol per g of wet weight.

Fidelity of translation initiation of mRNA for the precursor of rat mitochondrial serine:pyruvate/alanine:glyoxylate aminotransferase.

Serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) of rat liver is localized in both mitochondria and peroxisomes. The rat SPT/AGT gene is single, but there are two species of mRNA which differ at their 5' termini due to transcription from two alternative initiation sites. The longer mRNA is translated from the first AUG codon and thereby directs synthesis of the 45 kDa precursor of mitochondrial SPT/AGT, which includes a mitochondria-targeting N-terminal signal sequence. Peroxisomal SPT/AGT is synthesized as a product of mature size (43 kDa) from the shorter mRNA, which starts 3' to the first AUG codon and thus is translated from a downstream AUG codon. In our previous immunocytochemical study, SPT/AGT was found to be localized only in peroxisomes, when a cDNA encoding 43 kDa SPT/AGT was expressed in COS cells. When a cDNA encoding the 45 kDa precursor was expressed, on the other hand, SPT/AGT was localized mostly in mitochondria, but a small number of peroxisomes were also positively stained [Yokota, S., Funai, T., and Ichiyama, A. (1991) Biomed. Res. 12, 53-59]. We show in this paper that 43 kDa SPT/AGT is also synthesized from the longer mRNA in an in vitro translation system through a leaky scanning mechanism. Although the first AUG initiator codon is in a suboptimal context, the amount of 43 kDa SPT/AGT synthesized from the longer mRNA was small, probably because a downstream stem-loop structure facilitates recognition of the first AUG initiator codon.

Induction of mitochondrial serine:pyruvate aminotransferase of rat liver by glucagon and insulin through different mechanisms.

Studies were performed in the rat liver to examine whether or not insulin as well as glucagon causes the induction of mitochondrial serine:pyruvate aminotransferase (SPTm) [EC 2.6.1.51] and if so, whether the mechanisms of induction are similar or different for the two hormones. Not only glucagon but also insulin induced SPTm. Cell-free translation assaying and RNA blot analysis showed that both hormones cause an increase in the hepatic level of mRNA for the precursor of SPTm. Their effects were virtually additive, and the time course of the increase in the mRNA level differed between the hormones. The maximal increase induced by glucagon was observed 3.5 h after the hormone injection while that by insulin was found after 6 h. The increase in the mRNA due to insulin was completely inhibited by the co-administration of cycloheximide, while that due to glucagon was not. The finding suggests that a newly synthesized, insulin-dependent protein(s) is involved in the regulation of the mRNA level by insulin. On the other hand, hydrocortisone treatment selectively suppressed the increase in the mRNA due to glucagon. These data indicate that the synthesis of the mRNA for SPTm is regulated by glucagon and insulin through different mechanisms. The size of the hormone-induced mRNA for SPTm gradually decreased with time, but the cell-free translation products did not exhibit size alteration. RNase H digestion to remove the poly(A) tail of the mRNA indicated that shortening of the poly(A) sequence might be responsible for the time-dependent size alteration of the mRNA.

Pyridoxal 5'-phosphate binding of a recombinant rat serine: pyruvate/alanine:glyoxylate aminotransferase.

Serine: pyruvate/alanine: glyoxylate aminotransferase in the liver is a class IV amino-transferase. The present study was undertaken to characterize the pyridoxal 5'-phosphate (PLP) binding to a recombinant rat serine: pyruvate/alanine: glyoxylate aminotransferase (SPT10), which is a homodimer of 44.4 kDa subunits. Purified SPT10 exhibited absorption maxima at approximately 330 nm in addition to a 278 nm protein peak and a approximately 420 nm peak of PLP bound via Schiff base, and contained 0.56-0.69 mol of PLP per mol of subunit. Apo-SPT10 without measurable bound PLP did not exhibit the absorbance at approximately 420 nm, but still showed the approximately 330 nm peak. Upon reconstitution, 0.73-0.79 mol of PLP per mol of subunit was bound to apo-SPT10 with an apparent Kd of approximately 0.1 microM, resulting in a holo-SPT10 preparation whose specific activity and A approximately 420/A approximately 330 absorbance ratio were higher than those of the original SPT10. On SDS/PAGE of BrCN-cleavage peptides of NaBH4-reduced SPT10, 22-23 kDa fragments migrated as a pair of bands. On amino acid sequencing, the approximately 22 and approximately 23 kDa pair gave the same sequence, except that Lys was released only from the approximately 22 kDa band material in the cycle corresponding to Lys209. NaB3H4-treated SPT10 also migrated as a pair of 44-45 kDa bands and 3H was incorporated only into the approximately 45 kDa band. It appears that SPT10 has the capacity to bind 1 mol of PLP to Lys209 of every subunit, but usually binds less PLP in a Schiff base structure, probably due to the presence of a 330 nm-absorbing chromophore.

A spectrophotometric method for the determination of glycolate in urine and plasma with glycolate oxidase.

An enzymatic assay was developed for the spectrophotometric determination of glycolate in urine and plasma. Glycolate was first converted to glyoxylate with glycolate oxidase, and the glyoxylate formed was condensed with phenylhydrazine. The glyoxylate phenylhydrazone formed was then oxidized with K(3)Fe(CN)(6) in the presence of excess phenylhydrazine, and A(515) of the resulting 1, 5-diphenylformazan was measured. Since glycolate oxidase also acts on glyoxylate and L-lactate, the incubation of samples with glycolate oxidase was carried out in 120-170 mM Tris-HCl (pH 8.3) to obtain glyoxylate as its adduct with Tris. The pyruvate formed from lactate was removed by subsequent brief incubation with alanine aminotransferase in the presence of L-glutamate, and alpha-ketoglutarate formed was converted back to L-glutamate by glutamate dehydrogenase and an NADPH generating system. Thus the specificity of the assay relies principally on the substrate specificity of glycolate oxidase, and high sensitivity is provided by the high absorbance of 1,5-diphenylformazan at 515-520 nm. Plasma was deproteinized with perchloric acid, and then neutralized with KOH. Plasma and urine samples were then incubated with approximately 5 mM phenylhydrazine, and then treated with stearate-deactivated activated charcoal to remove endogenous keto and aldehyde acids as their phenylhydrazones. The normal plasma glycolate and urinary glycolate/creatinine ratio for adults determined by this method are approximately 8 microM and approximately 0.036, respectively.

Tryptophan hydroxylase from mouse mastocytoma P-815. Reversible activation by ethylenediaminetetraacetate.

Tryptophan hydroxylase from mouse mastocytoma P-815 is activated by ethylenediaminetetraacetate (EDTA). The activation proceeds in a pH-, temperature-, and time-dependent manner and leads to a 30-fold higher activity at maximum. The optimal EDTA concentration is 10 microns. The activation requires solely EDTA with desalted crude enzyme solution in the absence of any cellular metabolites. There are no indications that the activation is due to proteolysis, modification of protein-bound sulfhydryl groups or other covalent modifications such as phosphorylation and methylation. In the presence of appropriate stabilizing agents, the activated state is maintained after the removal of EDTA by gel-filtration. The activation is reversible under conditions in which bound metal(s) is dissociated from the complex with EDTA. These results imply that the role of EDTA is metal chelation. A model is proposed in which the enzyme has at least two interconvertible states, the activated state and ground state, corresponding to the free and metal-bound forms, respectively. The metal is probably derived from the cell. The assay method for tryptophan hydroxylase utilized a rapid and sensitive (5 pmol/injection) determination of 5-hydroxytryptophan by high performance liquid chromatography.

Assay of tryptophan hydroxylase and aromatic L-amino acid decarboxylase based on rapid separation of the reaction product by high performance liquid chromatography.

A rapid separation of 5-hydroxytryptophan by high performance liquid chromatography (HPLC) was achieved for the assay of tryptophan hydroxylase. "Bulk separation" of the product from all other components in the reaction mixture by HPLC was achieved by 1) the choice of a suitable column-solvent system so as to elute the reaction product ahead of other components in the sample mixture, 2) the use of a monitor selective for the reaction product, 3) minimization of the column length so as to achieve rapid separation of the product from the substrate. The method finally employed a reversed phase column of 5 cm length, relatively rapid elution at 2 ml/min and fluorescence detection at 350 nm with an excitation at 302 nm. The assay is convenient and as sensitive as the radioisotope method. The advantages of the method are 1) almost no pretreatment of samples, 2) repeatability every 2 min, 3) wide latitude of product determination from picomole to nanomole amounts per assay. The method was extended to the assay of 5-hydroxytryptophan decarboxylase by essentially the same procedures.

Uptake and processing of serine: pyruvate aminotransferase precursor by rat liver mitochondria in vitro and in vivo.

Processing and uptake of the precursor of serine: pyruvate aminotransferase [EC 2.6.1.51] by mitochondria were studied in vitro and in vivo. Serine: pyruvate aminotransferase was synthesized mainly on free ribosomes as judged by immunoprecipitation of puromycin-labeled nascent peptides prepared from free and bound ribosomes. The precursor of rat liver serine:pyruvate aminotransferase (pSPT) synthesized in vitro was post-translationally processed to an apparently mature form by isolated rat liver mitochondria. Available evidence indicated that the processed product was localized in the matrix of mitochondria. Mature serine:pyruvate aminotransferase did not inhibit the in vitro processing, suggesting that the extra peptide was necessary for the mitochondrial uptake of the precursor. In the livers of rats fed a vitamin B6-deficient high-protein diet, the induction by glucagon of serine:pyruvate aminotransferase occurred and most of the induced enzyme existed in mitochondria as the apo-form, suggesting that pSPT was taken up by mitochondria and processed in the apo-form under the conditions employed. In the in vitro system, on the other hand, the processing of pSPT proceeded both in the absence and presence of pyridoxal 5'-phosphate. Should the precursor also bind the prosthetic molecule, therefore, it would be transported into mitochondria in both the apo- and holo-forms. When isolated rat hepatocytes were labeled with [35S]methionine, labeled pSPT appeared in the cytosolic fraction and was transported rapidly into mitochondria in association with the processing. This uptake and processing were inhibited by a fluorescent laser dye, rhodamine 123, and the precursor accumulated in the cytosol in the presence of the dye.

Spectrophotometric determination of oxalate in urine and plasma with oxalate oxidase.

In order to establish a standard procedure for the spectrophotometric determination of urinary and plasma oxalate with oxalate oxidase (Laker, M.F., et al. (1980) Clin. Chem. 26, 827-830; Sugiura, M., et al. (1980) Clin. Chim. Acta 105, 393-399) and to define the limitations of the method, the procedures and reactions involved in the assay have been examined. Among the chromogenic hydrogen donors for peroxidase tested, a combination of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and sodium N-sulfopropylaniline (HALPS) was found to be best for the oxalate determination under the conditions used. Urine contained substance(s) which were inhibitory to the measurement of hydrogen peroxide by the peroxidase-catalyzed oxidative condensation of MBTH and HALPS, but they were largely removed by charcoal treatment at pH 5.6 without significant loss of oxalate. Deproteinization of plasma was carried out by ultrafiltration through a membrane cone (Centriflo CF-25) at neutral pH. The plasma oxalate ultrafiltrability under the conditions employed was calculated to be approximately 95%. A standard assay system for oxalate in these urine and plasma samples was then set up based on a series of studies on the reactions involved in the assay. In the case of normal plasma, however, the absorbance change was very small due to the low concentration of oxalate, and in addition, pretreatment of plasma with excess oxalate decarboxylase followed by the ultrafiltration and oxalate determination did not abolish completely the oxalate oxidase-dependent absorbance increase. It was concluded that the enzymic method was useful for the assay of urinary oxalate and in detecting elevated levels of plasma oxalate such as those in hemodialysis patients but was not sensitive enough to determine accurately the normal or decreased level of oxalate in plasma. The apparent concentration of oxalate in normal human plasma was measured in this work as 3.5 +/- 0.8 microM (mean +/- S.D., n = 8), and this result was interpreted to mean that the concentration of plasma oxalate was less than approximately 3.5 microM, as estimated by the present method.

Studies on biologically active pteridines. III. The absolute configuration at the C-6 chiral center of tetrahydrobiopterin cofactor and related compounds.

Tetrahydrobiopterin, the natural pteridine cofactor for aromatic amino acid hydroxylases, was produced stereopecifically with reference to the C-6 chiral center from 7,8-dihydrobiopterin by the action of dihydrofolate reductase. Similarly, 7,8-dihydro-6-methylpterin was reduced to tetrahydro-6-methylpterin having the same 6-configuration by the same enzyme. The absolute configuration of these tetrahydropterins at the C-6 chiral center was determined to be L, as in (2S)-1,2,3,4-tetrahydro-2-methylquinoxaline, which was servied from L-alanine, by comparison of the CD spectra.