Ornithine synthesis from glutamate in rat small intestinal mucosa

OF BIOCHEMISTRY 219, No. 1, November,

ARCHIVES

Vol.

Ornithine

AND BIOPHYSICS pp. 186-197, 1982

Synthesis JERRY

Department

from

G. HENSLEE

of Biochemistry Received

Glutamate

School Chapel

March

in Rat Small

AND

of Medicine, Hill, North

MARY University Carolina

15, 1982, and in revised

ELLEN of North 27514

form

July

Intestinal

Mucosa’

JONES” Carolina

at Chapel

Hill,

16, 1982

The de nouo synthesis of ornithine from glutamate by the rat small intestine has been investigated in order to establish conditions necessary for the reaction to proceed optimally. The formation of [14C]ornithine from [‘4C]glutamate was found in a subcellular fraction enriched in mitochondria. Ornithine formation required glutamate, ATP, NADPH, and Mg2+; phosphocreatine in the presence of endogenous creatine phosphokinase was stimulatory. Glutamate and ATP exhibited typical saturation kinetics with apparent K,,, values of about 8 mM and 60 PM, respectively. Ornithine synthesis was increased by NADPH concentrations up to 0.2 mM but higher concentrations were inhibitory. The maximal rate observed was 1 to 4 nmol of ornithine/min . mg protein. Synthesis of ornithine from glutamate was not observed with liver or kidney homogenates. Although the intestinal mitochondrial fraction contained ornithine aminotransferase (OAT), partially purified rat liver OAT stimulated ornithine synthesis. In the absence of exogenous liver OAT, pyridoxal 5’-phosphate or pyridoxamine 5’phosphate stimulated both ornithine synthesis and endogenous OAT activity. These observations support the hypothesis that ATP and NADPH were utilized to reduce glutamate to glutamate-y-semialdehyde which was then aminated by OAT to yield ornithine [Ross, G., Dunn, D., and Jones, M. E. (1978) Biochem. Biophys. Res. Commun. 85, 140-1471. Pyridoxal 5’-phosphate concentrations greater than 0.01 mM inhibited ornithine formation whereas pyridoxamine 5’-phosphate showed no inhibitory effect at this or higher concentrations. Neither coenzyme inhibited endogenous OAT activity. These results suggest that pyridoxal 5’-phosphate inhibits the synthesis of glutamatey-semialdehyde.

Homogenates of small intestinal mucosa from germ-free rats were capable of converting glutamate to ornithine when MgC&, ATP, and NADPH were present as substrates (5). The requirement for these substrates is compatible with the intermediate formation of glutamate-y-semialdehyde, which has been shown to be important in the metabolism of glutamate, proline, and ornithine in microorganisms (6, 7) and mammals (8). OAT4 catalyzes the reaction: ornithine

Rat small intestine has the capacity to synthesize ornithine from glutamate. When rat jejunum was perfused from either the luminal surface or via the arterial bed with glutamate or glutamine, ornithine and citrulline were recovered as products (1-4). 1 This work was supported by NIH grant HD12878. A preliminary account of some of this work was presented at the 72nd Annual Meeting of the American Society of Biological Chemists, June 1981, St. Louis, Missouri. * Current address: Abbott Laboratories, Abbott Park, Bldg. AP7 A, Dept 902, Rt. 43 and Buckley Road, N. Chicago, III. 60064. 3 Author to whom correspondence should be addressed. 0003-9861/82/130186-12$02.00/O Copyright All rights

0 1982 by Academic Press. Inc. of reproduction in any form reserved.

4 Abbreviations used: OAT, ornithine ferase; P5C, pyrroline-5-carboxylate; hydroxyethyl).l-piperazineethanesulfonic OTCase, ornithine transcarbamylase. 186

aminotransHepes, 4-(Zacid;

ORNITHINE

SYNTHESIS

FROM

GLUTAMATE

+ a-ketoglutarate + pyridoxal 5’-phosphate s glutamate-y-semialdehyde + glutamate + pyridoxal 5’-phosphate. The equilibrium in vitro strongly favors the formation of the semialdehyde (9). Therefore, the physiological role of OAT in the production of ornithine has been frequently discussed (10-15); these discussions have primarily been focused on data derived from liver and kidney which have the highest levels of OAT activity (11). There is no evidence that ornithine is produced from glutamate in these tissues. Indeed the conversion of glutamate to ornithine or glutamate-y-semialdehyde has not as yet been observed with liver and kidney homogenates, whereas the reverse reaction, formation of glutamate from glutamatey-semialdehyde, does occur in these two organs (14). Rat small intestine also has a high level of OAT activity (11, 13). A subcellular preparation from this tissue containing OAT activity was able to produce ornithine from P5C (13); this enamine forms spontaneously from glutamate-y-semialdehyde. OAT could therefore be catalyzing the last step in the observed formation of ornithine from glutamate in the small intestine. In liver OAT is a mitochondrial enzyme (16). In the present investigation we have studied the optimal conditions for the conversion of glutamate to ornithine since these conditions were not established by Ross et al. (5). We have utilized for these studies a subcellular fraction of the small intestinal mucosa enriched in mitochondria. MATERIALS

AND

METHODS

Male Sprague-Dawley rats (ZOO-250 g) were obtained from Charles River Breeding Laboratories and maintained on regular laboratory chow. AGl-X8 resin, 200-400 mesh, acetate form and AG50W-~8 resin, 200-400 mesh, hydrogen form obtained from Bio-Rad Laboratories were used for ion exchange chromatography. All columns had a 0.7 X 17-cm bed volume. L-[“C(U)]Glutamate and L-[3-3H(N)]ornithine were purchased from New England Nuclear; both were further purified by ion-exchange chromatography before use. [i4C]Glutamate was loaded on a column of AGl-X8 resin equilibrated in HzO. The column was washed with 10 ml Hz0 followed by elution with 0.2 M acetic acid during which the

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187

[‘4C]glutamate was collected. This fraction was then evaporated to dryness and redissolved in H,O. [3H]Ornithine was loaded on a column of AG50WX8 resin equilibrated in 0.1 N HCl. The column was washed with 30 ml of 1.5 N HCl followed by elution of the (3H]ornithine with 10 ml of 6 N HCl which was evaporated to dryness and redissolved in 0.1 N HCl. All biochemicals were purchased from Sigma Chemical Co.; NADPH was also purchased from Boehringer Mannheim Biochemicals. Rat liver OAT was partially purified (17) from rats fed 3 days on high protein test diet from the United States Biochemical Corporation. Preparation of the mitochondrial fraction from small intestinal mucosa. One to three rats were starved overnight and killed the next morning by cervical dislocation. The small intestine was removed, flushed with saline, and then gently pressed to remove luminal contents. The washed small intestine, placed on a cold glass plate, was divided into lo-cm segments which were trimmed of mesenteric adipose tissue and then slit. The mucosa was removed with a glass slide; 4 g wet weight of mucosal scrapping was obtained per rat. The mucosal scrapping were placed in 5 ml of cold 50 mM Tris-HCl pH 7.4, 150 mM KCl, 5 mM EDTA, 2 mM dithiothreitol, and 0.05% bovine serum albumin (isolation buffer) to be combined (when more than one rat was used) and to be weighed by difference. The mucosal scrappings were homogenized at 0°C as a 5% w/v suspension by making 10 passes with a loosely fitting pestle in a 40-ml capacity Dounce homogenizer. All of the following procedures were performed at 0 to 4’C in this buffer. The homogenate was centrifuged for 10 min at 10,OOOg and the supernatant fraction discarded to remove soluble mucin and protease activity. The pellets were resuspended in the initial volume of isolation buffer using a Dounce homogenizer as described above and the suspension was then centrifuged at 1OOOg for 10 min. The pellet fraction (A) was retained and treated as described below. The supernatant fraction was filtered through double-layered cheesecloth and then centrifuged for 10 min at 10,OOOg. The supernatant fraction was discarded, and the pellet fraction B was retained. Pellet fraction A was resuspended in the initial homogenate volume as described above, and the centrifugation steps at 1OOOg and 10,OOOg were repeated to obtain pellet fraction C. Pellet fractions B and C were combined by adding 0.5 ml of isolation buffer to each tube and dislodging the pellets. The combined pellets were resuspended using a 7-ml capacity Dounce homogenizer. The volume of the mitochondrial suspension was adjusted to one-quarter of the initial homogenate volume with isolation buffer, and the suspension was then centrifuged for 10 min at 1OOOg. The supernatant fraction was carefully decanted, filtered through cheesecloth, and then centrifuged at 10,OOOg for 10 min. The supernatant fraction was discarded. This pellet, the final mitochondrial fraction, was kept on ice and

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resuspended immediately before use in a final volume of 1 to 2 ml using a Dounce homogenizer. Protein concentration of the mitochondrial fraction was determined by a modification of the Lowry procedure (18) and was normally 10 to 20 mg/ml. The several steps using filtration through cheesecloth and the final lOOOg-10 min centrifugation step were necessary to minimize aggregation of the mitochondria. Assay conditions for measuring ornithine synthesis from glutamate. Ornithine synthesis from glutamate was determined by measuring the conversion of [14C]glutamate to [14C]ornithine. This conversion was assayed in a final volume of 1.0 ml at 37°C in the presence of 0.1 M Hepes, 20.0 mM [U-‘4C]glutamate (0.05-0.10 &i/ymol), 0.4 mM NADPH, 1.0 mM ATP, 6.0 mM MgCls, 10.0 mM phosphocreatine, 10 units of creatine phosphokinase, and 0.05% (w/v) Nonidet P40. Potassium Hepes (0.4 ml of a 0.25 M solution, pH 7.56 at 22°C) was added to the rest of the reaction mixture at 37°C to yield a pH of 7.4. The reaction was started by adding 0.075 ml of mitochondrial fraction, about 1 mg protein, to the reaction mixture which had been preincubated for 3 min at 37°C. A blank incubation was similarly prepared omitting the addition of the mitochondrial fraction. The incubation was for 20 min unless indicated. The assay mixture was prepared for ion-exchange chromatography according to a modified procedure of Ross et al. (5). The reaction was stopped by adding concentrated HCIOl to a final concentration of 3.5%. [3H]Ornithine, 18,000 cpm, to serve as internal standard and 7 pmol of carrier ornithine were added. The reaction mixture was centrifuged to remove the denatured protein and the supernatant fraction carefully transferred to a clean tube and kept in ice. The pH of the deproteinized extract was adjusted in the range of 5.0 to 5.3 using an amount of 10.0 N KOH equivalent to 90% of the perchloric acid followed by sufficient 0.2 N KOH to reach the pH indicated. The KClO.,precipitate was removed by centrifugation. The neutralized extract was then evaporated to dryness after which it was redissolved in 0.5 ml of 0.116 M Na citrate, pH 5.3. The initial anion exchange column used by Ross et al. (5) was not used in this study; however, the two cation exchange columns were utilized in sequence as described (5). The neutralized, acid-soluble extract was fractionated on a column of AG50W-X8 resin equilibrated in 0.116 M Na citrate, pH 5.3. Glutamate is not retained on this column while ornithine is. The ornithine fraction from this column was purified further on a HCl equilibrated AG50W-X8 resin column to obtain the final ornithine fraction in 10 ml of 6 N HCl. This fraction was evaporated to dryness and redissolved in 1.0 ml of deionized Hz0 which was transferred to a scintillation vial containing 9.0 ml of scintillation fluid (5). [14C]Ornithine and [sH]ornithine were measured in a Beckman LS-100

AND

JONES

C scintillation spectrometer. Recovery of the [3H]ornithine internal standard was about 70%. Between 100 and 200 14C cpm were usually recovered from the blank incubation compared to several thousand 14C cpm from incubations containing 1 mg mitochondrial protein. Any deviations in a specific experiment from the normal assay procedure will be explained in the figure legends. Conversion of ornithine to citrulline. To confirm that the product was ornithine, the final ornithine fraction was enzymatically converted to citrulline by OTCase using a modified procedure (5). Carrier ornithine was not added to the reaction mixture, and [3H]ornithine internal standard was added only after the ornithine fraction had been dissolved in 1.0 ml H,O. Two 0.4-ml aliquots of the putative ornithine fraction were removed, evaporated to dryness, and redissolved in 0.4 ml of 0.2 M Tris-HCl, pH 8.5. To the test sample was added 6 rmol ornithine, 5 units OTCase, 46 pmol carbamyl phosphate, and 0.2 M Tris-HCl, pH 8.5, such that the final buffer concentration was 0.17 M Tris-HCl in a final volume of 1.2 ml. The control sample was the same except carbamyl phosphate was omitted. Both samples were incubated for 2 h at 37°C and then stopped with 1.0 ml of 4.5 N HCl. After centrifugation the supernatant fluids were evaporated to dryness and redissolved in 0.5 ml of 0.116 M Na citrate, pH 5.3. The pH of each sample was adjusted to 5.3 and each loaded on an AG50WX8 resin column equilibrated in the same Na citrate buffer. The columns were eluted with the Na citrate buffer collecting 3-ml fractions. Measurement of ornithine aminotransferase activity in the mitochondrial fraction. OAT activity was measured in the direction of P5C formation. Except for the omission of [14C]glutamate, the components used to assay ornithine synthesis from glutamate were included in the OAT assay mixture. The assay mixture also contained 35 mM ornithine, 5 mM a-ketoglutarate, and 5.5 pM pyridoxal5’-phosphate in a final volume of 1.0 ml. About 0.3 mg protein of the mitochondrial fraction was added to initiate the reaction which was allowed to proceed for 15 min at 37°C. The reaction was terminated by the addition of 30 /.d of 100% trichloroacetic acid followed by 100 /.d of 0.1 M o-aminobenxaldehyde in 40% ethanol. The assay mixture was centrifuged to remove precipitated protein and the supernatant fluid quantitatively removed. It was neutralized with 30 ~1 of 4.5 mM K&03 and kept at room temperature for 30 min. Neutralization was necessary because together NADPH and o-aminobenzaldehyde produced an interfering orange color (19) at low pH. The sample was centrifuged again before measuring the absorbance of the supernatant fluid at 440 nm. A molar extinction coefficient of 2580 (20) was used to determine micromoles of P5C produced. OAT units are expressed as micromoles of P5C formed/minute.

ORNITHINE

SYNTHESIS

FIG. 1. Electron micrograph indicate intact mitochondria material”; vesicles containing sosomes (L).

FROM

of mitochondrial (M); mitochondria “fuzzy material”

GLUTAMATE

IN

SMALL

INTESTINE

fraction from rat small intestinal in the process of swelling (mf) (f). Also present are structures

RESULTS

Characterization of the mitochondrial fraction from small intestinal mucosa. The enzyme(s) catalyzing the synthesis of ornithine from glutamate was always distributed as the mitochondrial marker enzymes, i.e., the activity was distributed between the cellular debris fraction and the putative mitochondrial fraction. No activity was observed in the 10,OOOg supernatant even in the presence of exogenous rat liver OAT. The mitochondrial marker enzyme activities were recovered in nearly equal amounts in the cellular debris fraction (the final IOOOg-IO min pellet) and in a putative mitochondrial fraction (lO,OOOg-10 min pellet). This high recovery of mitochondrial marker enzymes in the cellular

189

mucosa. The letters that contain “fuzzy which resemble Iy-

debris fraction was probably caused by entrapment or aggregation of mitochondria since over 90% of the cytosolic enzyme marker activity was always recovered in the lO,OOOg-10 min supernatant, an indication that cell breakage was efficient. A significant portion of microsomal and lysosomal marker enzyme activities was also recovered in the cellular debris fraction. Further attempts to free mitochondria from the cellular debris resulted in mitochondrial breakage as measured by the release of glutamate dehydrogenase activity into the soluble fraction, In our review of the literature we have seen no electron micrographs of subcellular fractions obtained from the small intestinal mucosa. The nature of the fraction we obtained is demonstrated in Fig. 1. Mitochondria with condensed matrices are

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evident. A few round, electron-dense structures are present that are probably intact lysosomes. Also present are irregularly shaped vesicles containing a fuzzy material which may be damaged mitochondria. Similar structures were observed by Matlib and Srere (21) of swollen rat liver mitochondria. Several small intestinal mitochondria appear to be in the process of swelling since they contain both cristae and fuzzy material. The electron micrograph indicates that an enrichment of mitochondria was achieved. Identification of putative ornithine. The mitochondrial fraction from intestinal mucosa produced ornithine as shown by its conversion to citrulline in the presence of OTCase and carbamyl phosphate (Fig. 2). t3H]Ornithine internal standard (88.2% recovery) and putative [ 14C]ornithine (86.5% recovery) coeluted as a basic amino acid when carbamyl phosphate was omitted in the control incubation (Fig. 2A). When carbamyl phosphate was included so that citrulline could be formed, both [3H]ornithine internal standard (91.5% recovery) and the putative [14C]ornithine (92.6% recovery) coeluted as a neutral amino acid, i.e., citrulline, in the first 12 ml elution volume (Fig. 2B). Effects of assay component concentrations. The concentration of each assay component was varied to study conditions necessary for optimal ornithine synthesis from glutamate by the mitochondrial fraction. The effects of glutamate and ATP concentrations appeared to follow typical saturation kinetics (Figs. 3 and 4). The apparent K,,, values for glutamate obtained from two experiments were 7.1 and 9.1 mM and the respective apparent V values, 3.5 and 4.0 nmol ornithine/min * mg protein. These K, values suggest that at least 90 mM glutamate would be required to saturate the reactions synthesizing ornithine. To avoid the inhibitory effect (unpublished data) of a high ionic strength, the concentration of glutamate used for the results reported below was 20 mM. The V expected with 20 mM glutamate is 2.7 nmol ornithine/min . mg protein; values observed ranged from 1 to 4 nmol ornithine/ min. mg protein.

AND JONES

FIG. 2. Conversion of putative ornithine to citrulline in the presence of OTCase and carbamyl phosphate. Mitochondrial protein, 1.5 mg, was incubated under conditions for ornithine synthesis from glutamate in the presence of 10 aCi [‘*C]glutamate. [aH]Ornithine (20,500 cpm in a lOO+d aliquot) was added as internal standard to the final putative ornithine fraction which was incubated with StreptoCOCCLLS OTCase as described under Materials and Methods. 3H cpm (O), “C cpm (0). Minus carbamyl phosphate (A); plus carbamyl phosphate (B).

The apparent K,,, value for ATP was about 60 PM. The ATP concentration which was saturating (1.0 mM) was selected for the assay mixture (Fig. 4). MgC& concentration was varied at an initial ATP concentration of 1.0 mM (data not shown). Ornithine synthesis increased rapidly from 0 to 3 mM MgClp presumably as Mg2+-ATP was formed. Between 3 and 8 mM MgCl,, ornithine synthesis was optimal, while at concentrations greater than 8 mM the rate of ornithine synthesis decreased somewhat. A MgCl, concentration of 6.0 mM was used for assay. As the NADPH concentration was increased from 0 to 0.2 mM, ornithine synthesis increased. However, ornithine production decreased as the NADPH concentration was increased further (Fig. 5). This decrease in activity does not appear to be caused by the production of NADP for the time courses observed for ornithine synthesis in the presence of 0.2 and 2.0 mM NADPH (Fig. 5, inset) were linear for 25 min, after an initial lag period. If NADP was inhibiting, a decrease in ornithine synthesis with respect to time should be observed as NADP accumulated and the

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SYNTHESIS

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[Glu] mM FIG. 3. Glutamate concentration curve for ornithine synthesis from glutamate. Ornithine synthesis from [“C]glutamate was measured as described under Materials and Methods. An Eadie-Hofstee plot of the data is the inset. Mitochondrial protein, 1.0 mg, was added per assay vessel. After reactions were terminated with perchloric acid, the glutamate concentrations were adjusted with nonradioactive glutamate so that all assay mixtures were about 21 mM glutamate to equalize carrier glutamate concentrations prior to ion-exchange chromatography. The concentration of all other substrates is given under Materials and Methods.

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[ATPI mM FIG. 4. ATP concentration curve for ornithine synthesis from glutamate. The reaction was assayed as in Fig. 3. An Eadie-Hofstee plot of the data is the inset. Mitochondrial protein, 1.2 mg, was added per assay. The concentration of the substrates other than ATP is given under Materials and Methods. The units for the V/[s] axis of the Eadie-Hofstee plot are nanomoles ornithine per milligram. minute per micromolar ATP.

191

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6 0

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[NADPH] FIG. 5. NADPH concentration curve for assayed as in Fig. 3. Mitochondrial protein, shown in the inset were performed in the (0) with 1.4 mg mitochondrial protein. The under Materials and Methods.

JONES

I 20

mM

ornithine synthesis from glutamate. 1.2 mg, was added per assay vessel. The presence of 0.2 mM NADPH (0) or concentration of substrates other than

NADP/NADPH ratio increased, and ornithine synthesis at early time periods should be greater in the presence of 2.0 mM NADPH. Both of these predictions are not observed. The same inhibitory effect was observed with NADPH obtained from either Sigma or Boehringer Mannheim. Since the presence of 0 to 2.0 mM NADPH had no effect on the OAT activity endogenous to the mitochondrial fraction, the enzyme(s) affected must be those concerned with the synthesis of glutamate-y-semialdehyde. The initial NADPH concentration used for assay was set. at 0.4 mM. Ross et al. (5) included regenerating systems for ATP and NADPH when measuring ornithine synthesis from glutamate by mucosa homogenates. They used isocitrate dehydrogenase and isocitrate to regenerate NADPH; we find that this system reduces ornithine synthesis by half. The production of a-ketoglutarate by isocitrate dehydrogenase should shift the equilibrium of the OAT reaction from ornithine synthesis toward P5C synthesis. A NADPHregenerating system was, therefore, not

30

The reaction was two experiments 2.0 mM NADPH NADPH is given

used. Phosphocreatine enhanced ornithine synthesis about twofold when the phosphocreatine concentration was between 5 and 15 mM (data not shown). Mitochondria from the rat small intestinal mucosa have creatine phosphokinase activity (22) which can complete an ATP-regenerating system in the presence of phosphocreatine; addition of 10 units of exogenous creatine phosphokinase, in an assay containing 1.4 mg mitochondrial protein, had no effect. on ornithine synthesis suggesting that the mitochondrial creatine phosphokinase activity was sufficient.. Nevertheless, exogenous creatine kinase was included in all assays to ensure optimal ATP regeneration; the phosphocreatine concentration was set at 10 mM. The rate of ornithine synthesis remained constant as the potassium Hepes concentration was varied between 25 and 200 mM suggesting that this salt is not inhibitory and that a variation in ionic strength over this range had no effect. However, in the presence of 100 mM Hepes, ornithine synthesis was inhibited by KC1

ORNITHINE

SYNTHESIS

FROM

GLUTAMATE

TimeCmin)

FIG. 6. Time course of ornithine synthesis from glutamate. Mitochondrial protein, 0.64 mg, was assayed in the presence (0) and absence (0) of 1.2 units exogenous OAT. The assay conditions are given under Materials and Methods.

or potassium phosphate at concentrations as low as 2.5 mM.5 At constant potassium concentrations, the inhibition by potassium phosphate was greater than by KCl. These results suggest that the inhibitory effects of KC1 or potassium phosphate cannot be attributed to ionic strength alone. Attempts to maintain intact intestinal mucosa mitochondria during incubation at 37°C were unsuccessful. To avoid the problem of having a mixed population of intact and permeable mitochondria, Nonidet P-40 was used to make all mitochondria permeable. Nonidet P-40 was included in the assay incubations at a final concentration of 0.05% (w/v). At 0.05% Nonidet P-40,90% of the mitochondrial matrix enzyme glutamate dehydrogenase is soluble when the protein concentration is 1 mg/ml. Ornithine synthesis from proline by rat liver mitochondria was dependent upon the presence of rotenone which inhibits the conversion of P5C to glutamate (15). Rotenone, 5 pM had no effect on ornithine synthesis from glutamate by rat small intestinal mucosa mitochondria when assayed as described under Materials and Methods. Effect of exogenous OAT on ornithine 5 J. G. Henslee periments.

and M. E. Jones,

unpublished

ex-

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193

synthesis. The mitochondrial fraction isolated from the small intestinal mucosa had OAT activity. The average OAT activity, measured in the direction of P5C formation, from seven mitochondrial preparations was 0.10 (f0.05 SD) pmol P5C formed/min * mg of mitochondrial protein. This endogenous OAT should be necessary to convert glutamate-y-semialdehyde formed from glutamate to ornithine, however, it is difficult to prove that this is so without purification of the enzymes required to convert glutamate to ornithine. A large, approximately lo-fold, excess of rat liver OAT stimulates ornithine synthesis by the small intestinal mitochondrial fraction (Figs. 6 and 7). This stimulation by exogenous OAT suggests that glutamate-y-semialdehyde is an intermediate in the synthesis of ornithine from glutamate and that endogenous OAT activity is somewhat limiting. The stimulatory effect of exogenous OAT on ornithine synthesis was studied with respect to both time and protein concentration. In the absence of exogenous OAT, ornithine synthesis was linear from 10 to 60 min after an initial lag period. No lag period occurred in the presence of a lo-

FIG. 7. Ornithine synthesis from glutamate with respect to protein concentration. (0), 1.2 units exogenous OAT added, (@) no exogenous OAT added. The assay conditions are given under Materials and Methods.

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fold excess of exogenous OAT (Fig. 6). The lag period might represent the interval required for the glutamate-y-semialdehyde/ P5C concentration to reach a sufficiently high steady-state concentration so that the small amount of endogenous OAT can produce ornithine at a linear rate. The amount of ornithine formed in 20 min (Fig. 7) was low in the absence of exogenousOAT when was the mitochondrial tein concentration less than 0.4 promg/ ml. The rate of ornithine svnthesis increased linearly, however, as mitochondrial protein increased above 0.4 mg/ml. If a lo-fold excess of rat liver OAT was added, the amount of ornithine formed at each protein concentration was increased, but the rate of ornithine formation again did not increase linearly until the mitochondrial protein concentration exceeded 0.4 mg/ml. In the region of the linear response the amount of ornithine formed in the vessels containing exogenous OAT was 4.27 nmol/min . mg of mitochondrial protein while in the absence of exogenous OAT the rate is 2.22 nmol/min. mg of mitochondrial protein. Effect of pyridoxal 5’-phosphate on ornithine synthesis. Since OAT requires pyridoxal5’-phosphate as a coenzyme, we studied the effects of this coenzyme and pyridoxamine 5’-phosphate on ornithine synthesis from glutamate in the absence of exogenous OAT. Both coenzymes stimulated ornithine synthesis twofold (Fig. 8). The maximal activity attained with each coenzyme was similar, but the maximal activity was reached with only 0.005 mM exogenous pyridoxal 5’-phosphate while 0.05 mM exogenous pyridoxamine 5’-phosphate was required to give the same rate. Ornithine synthesis decreased as the concentration of exogenous pyridoxal5’-phosphate increased above 0.01 mM; at 1.0 mM the rate of ornithine synthesis was only 10% of the maximal activity. No inhibition of ornithine synthesis, however, was observed with increasing concentrations of exogenous pyridoxamine 5’-phosphate. Inhibition by pyridoxal phosphate of ornithine synthesis from glutamate is not due to an inhibition of OAT (Fig. 8) and must,

AND

JONES

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FIG. 8. Effect of coenzymes, pyridoxal5’-phosphate and pyridoxamine 5’-phosphate, on ornithine synthesis. The reaction was followed as in Fig. 3. For ornithine synthesis 1.1 mg mitochondrial protein was added per assay vessel and assayed in the presence of pyridoxal 5’-phosphate (0) or pyridoxamine 5’. phosphate (0). For OAT activity 0.3 mg mitochondrial protein was added per assay vessel and assayed in the presence of pyridoxal 5’-phosphate (A) or pyridoxamine 5’-phosphate (A); NADPH was omitted so that neutralization was not required after termination of the reaction with trichloroacetic acid (see Materials and Methods). At this acidic pH, the absorbance at 440 nm of exogenous pyridoxal 5’.phosphate is insignificant, whereas at neutral pH, this coenzyme absorbs strongly at 440 nm producing a high blank reading.

therefore, be due to an inhibition of glutamate-y-semialdehyde synthesis.6 Effect of preincubation on ornithine synthesis. Preincubating the mitochondrial protein at 37°C in the absence of substrates significantly decreased ornithine formation (Fig. 9). After 2.5 min of preincubation time, ornithine synthesis was decreased by about 50%, and synthesis was ‘To test endogenous OAT activity a much lower amount of the small intestinal mitochondrial protein had to be used to measure OAT activity in the direction of P5C formation. Under these conditions, OAT activity was dependent on the addition of exogenous coenzyme; in contrast ornithine synthesis is not entirely dependent on the addition of pyridoxal phosphate. The quantitative difference in the amount of coenzyme necessary to have enzyme activity in the slow synthesis of ornithine from glutamate and the rapid P5C synthesis from ornithine can be rationalized if the crude undialyzed intestinal mitochondrial homogenate contains only 1 pmol pyridoxal phosphate/mg protein.

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FIG. 9. Effect of preincubation of mitochondrial protein on ornithine synthesis and OAT activity. For ornithine synthesis (0) mitochondrial protein, 0.37 mg, was preincubated at 37°C in 0.82 ml under assay conditions except for the absence of glutamate, ATP, NADPH, phosphocreatine, and [i4C]glutamate. Exogenous OAT, 0.2 unit, was added at the end of the preincubation period followed immediately by the addition (at concentrations listed under Materials and Methods) of the omitted components listed above in one aliquot to initiate ornithine synthesis and to bring the final volume to 1.0 ml. Incubation time was 30 min. For mitochondrial OAT activity (0), 0.18 mg of mitochondrial protein was preincubated in a volume of 0.7 ml as described above. No exogenous OAT was added at the end of preincubation. After the preincubation period OAT activity was measured as described under Materials and Methods by initiating the reaction with ornithine, oc-ketoglutarate, pyridoxal 5’-phosphate, glutamate, ATP, NADPH, and phosphocreatine bringing the final volume to 1.0 ml. Values obtained with no preincubation period were set at 100%.

almost undetectable after a 20-min preincubation period. This rapid decrease suggests the inactivation of one or more enzymes involved in ornithine formation. Endogenous OAT activity decreased during preincubation but at a much slower rate than for ornithine formation. The addition of exogenous OAT immediately before initiating ornithine synthesis should have minimized any effects caused by the decrease in endogenous OAT activity. These results suggest that one or more enzymatic steps catalyzing the formation of glutamate-y-semialdehyde from glutamate is unstable at 37°C in the absence of substrates. Tissue distribution of the ability to syn-

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195

thesize ornithine from glutamate. We prepared 10% homogenates (as well as the mitochondrial fraction of these homogenates) from rat liver and kidney (21) and tested the capacity of both the homogenate and the mitochondria to synthesize ornithine under the conditions described here. When sufficient protein from the homogenate or the mitochondria of either liver or kidney was added to contain at least 1 mg protein per assay vessel no ornithine synthesis was observed. DISCUSSION

Young rats maintained on an argininedeficient diet continue to grow, although at a depressed rate (23-B), suggesting that de nouo synthesis of arginine is occurring. Only two mammalian tissues, liver and the small intestine, have the two enzymes, carbamyl phosphate synthetase I and OTCase, that synthesize citrulline de nouo if ornithine is available (26, 27). Liver might provide citrulline, via the urea cycle, to the plasma for arginine synthesis by nonhepatic tissues (28, 29) but, recently, work has shown that the small intestine from rat can not only synthesize citrulline de nouo but can also synthesize its precursor, ornithine, from glutamate or glutamine (l-5). Glutamine has been identified as a major respiratory fuel for the small intestine (4), and a mitochnondrial phosphatedependent glutaminase in this tissue may initiate glutamine metabolism by converting it to glutamate (30). Glutamate, therefore, may be readily available in small intestinal mitochondria for conversion to both ornithine and citrulline; the latter could be released into the plasma to serve as a substrate for arginine synthesis in tissues that cannot synthesize citrulline de nouo (4). Ornithine was also formed from glutamine by chick intestinal brush border cells (31). The enzymes involved in the synthesis of ornithine and citrulline from glutamate in the small intestine have not been established. However, OAT (11,13) and OTCase (5, 26, 27) are both in the small intestine and could respectively produce or-

196

HENSLEE

nithine from glutamate-y-semialdehyde followed by the formation of citrulline from ornithine. In liver, OAT is a mitochondrial enzyme (16). We have shown here that a fraction enriched in mitochondria from the small intestinal mucosa also has OAT activity and can synthesize ornithine from glutamate at rates ranging from 1 to 4 nmol ornithine per min. mg protein which is about 1000 X greater than the rate reported by Ross et al. (5). Ornithine production by this mitochondrial fraction was stimulated by exogenous OAT, which had been partially purified from rat liver. The stimulatory effect of low concentrations of pyridoxal 5’-phosphate on both small intestinal OAT activity and ornithine formation from glutamate further supports a role for OAT in ornithine synthesis. Higher pyridoxal 5’-phosphate concentrations inhibited ornithine synthesis but had no effect on OAT activity. The inhibition may occur during glutamate-ysemialdehyde synthesis from glutamate. This synthesis may involve y-glutamyl phosphate as an enzyme-bound intermediate (5). Several enzymes which do not utilize pyridoxal5’-phosphate are inhibited by this coenzyme (32-34); the common feature of these enzymes is a specific phosphate binding site. Pyridoxal5’-phosphate is believed to bind at the phosphate binding site and to subsequently form a Schiff base with a neighboring reactive lysine residue. The enzyme(s) catalyzing glutamatey-semialdehyde formation should also possess specific phosphate binding sites since ATP and NADPH are substrates. Clearly the site at which pyridoxal 5’-phosphate binds to inhibit ornithine synthesis has an affinity for this coenzyme that is significantly lower than the active center of OAT. McGivan et al. (15) studied OAT activity in isolated rat liver mitochondria and observed ornithine production from glutamate-y-semialdehyde generated from proline only in the presence of rotenone. They concluded that in liver mitochondria, glutamate-y-semialdehyde was oxidized to glutamate by P5C dehydrogenase and was not available as substrate for OAT. This

AND

JONES

competition for the semialdehyde would not be as marked in the small intestine where the P5C dehydrogenase activity is l/lOOth that of the mature rat liver and there is little or no detectable P5C dehydrogenase activity in fetal small intestine (14). The amount of OAT activity, on the other hand, is similar in liver and small intestine (11). The apparent K,,, of 8 mM for glutamate observed for ornithine synthesis cannot presently be assigned to either the synthesis of glutamate-y-semialdehyde from glutamate or to ornithine synthesis from the semialdehyde. The K,,, values reported for rat liver OAT are 25 and 1.4 mM for glutamate and P5C, respectively (42). This report confirms earlier evidence (5) that glutamate, ATP, NADPH, and Mg2+ are utilized as substrates by small intestinal mitochondria in the production of glutamate-y-semialdehyde which can be converted to ornithine by endogenous or rat liver OAT. In a recent preliminary report, Wakabayashi (43) showed directly that P5C, the chemically formed enamine of glutamate-y-semialdehyde, is a product of the small intestinal enzyme(s) that utilize glutamate, ATP, NADPH, and Mg2+; in that report P5C formed from [14C]glutamate was reduced to proline with either sodium borohydride or yeast P5C reductase and NADH. Glutamate-y-semialdehyde is also the postulated intermediate in the synthesis of proline from glutamate observed in some mammalian cell lines (44). Recently Smith et al. (45) reported formation of P5C from glutamate in homogenates of a Chinese hamster ovary cell line. These authors characterized the P5C by its ability to form a complex with oaminobenzaldehyde; they were not able to observe saturation of this reaction by glutamate. There are a number of important studies to be done. One of these is to ascertain which mammalian tissues have the enzyme(s) that form glutamate-y-semialdehyde from glutamate. Studies reported here and previously (46) suggest that liver and kidney do not have this enzyme and cannot convert glutamate to glutamate-

ORNITHINE

SYNTHESIS

FROM

GLUTAMATE

y-semialdehyde. This result might indicate that certain tissue primarily convert OFnithine to proline or glutamate while others may primarily form ornithine and proline from glutamate. ACKNOWLEDGMENTS The

technical

gratefully

assistance

acknowledged.

Griffith and micrographs chondrial

of We

Susan Hester of the small

Eugenia also

I. Hirsch thank

for preparing intestinal

Dr.

Jack

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