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Metabolism of glutamine and glutamate in human lenses
Exp. Eye Res. (1990) 50, 597-601
Metabolism
of Glutamine HOWARD
Departments
of Biochemistry
and Glutamate M. JERNIGAN,
and Ophthalmology,
University
in Human
Lenses
JR” of Tennessee,
Memphis,
TN 38163,
U.S.A.
Glutamate is important to lenses as a central intermediate in amino acid metabolism, as well as for synthesis of proteins and glutathione. In rat and calf lenses, the principal source of lenticular glutamate is glutamine. which enters the lens and is deamidated to form glutamate. In contrast, monkey lenses use external glutamate more readily than glutamine. Amino acid metabolism was studied in human lenses by incubating them with amino-labeled [15N]glutamine or [15N]glutamate. The lenticular free amino acids were then isolated and analysed by gas chromatography-mass spectrometry to determine 15Nlabeled products. The results were compared with those of similar experiments with lenses from other species. Label was measured in aspartate. alanine. serine and proline, as well as lenticular glutamine and glutamate. Glutamine enters lenses more readily than glutamate in all the species examined. Nevertheless, aspartate, alanine and serine were more rapidly labeled by incubating human lenses in [15N]glutamate than in [‘SN]glutamine. This observation is similar to reports of experiments with monkey lenses, which unlike rat lenses, preferentially utilize glutamate rather than glutamine. In contrast, human lens proline was more rapidly labeled by incubating lenses with [‘5N]glutamine than with [‘5N]glutamate. In human lenses, the relatively slow utilization of glutamine for the transamination reactions which form aspartate, alanine and serine appears to result from slow deamidation. The relative preference for glutamine over glutamate as a precursor for proline synthesis in human lenses may be related to the mitochondrial location of the enzymes involved. Keu words: human lens: rat; monkey; amino acids: metabolism: ammonia: glutamate: glutamine : glycine : proline ; serine ; alanine ; aspartate. 1. Introduction The concentration of glutamate is higher in the lens than in the aqueous humor, and the glutamate concentration in most species exceeds that of all other lenticular amino acids with the exception of taurine (Reddy and Kinsey, 1962 ; Reddy, 1967 ; Dickinson, Durham, and Hamilton, 1968; Kinoshita et al., 1969). Although lenses are capable of synthesizing glutamate from other metabolites (Kinoshita and Merola, 1961), the principal source of glutamate for rat and calf lens metabolism is from glutamine, which is transported from the aqueous humor and deamidated (Kern and Ho, 1973; Trayhurn and Van Heyningen, 1973). When rat lenses (Kern and Ho, 1973; Jernigan and Laranang, 1984a) or bovine lenses (Kern and Ho, 1973: Trayhurn and Van Heyningen, 1973) were incubated with labeled glutamine or glutamate, glutamine entered the lenses more readily than glutamate. and was utilized more rapidly for glutamate-requiring biochemical pathways. Thus, in rat bovine lenses, the two-step process of glutamine transport and deamidation (to form glutamate) is more rapid than direct transport of glutamate. In contrast, monkey lenses utilized glutamate more rapidly than glutamine, even though glutamine entered more rapidly (Jernigan and Zigler, 1987). The different results with monkey lenses may have been related to the observation that glutamine was converted to glutamate more slowly in monkey lenses than in rat lenses. The differences observed between rat and primate (rhesus monkey) lenses. together with the relative * For correspondenceat : University of Tennessee,Memphis, 9 56 Court Avenue. Room 11222.Memphis. TN 38163 U.S.A. 0014-4835/90/06(3597+05
$03.00/O
paucity of data on amino acid metabolism in human lenses, led to the present study, which compares human lens amino group metabolism with that of monkeys and rats. Human lenses were incubated with 15N-amino-labeled glutamine or glutamate, and the products were examined to determine the relative rates of utilization of these two amino acids and the distribution of their amino nitrogen among other free amino acids in the lenses. The results from experiments with human lenses were compared with results obtained from experiments with other species. 2. Materials
and Methods
Materials Freshly enucleated human eyes were obtained by the Mid South Eye Bank, and were transported to the laboratory in moist jars in ice. They were stored at 4°C until used. The donors ranged in age from 1 to 99 yr, with median age of 61 yr. Slight haze and small opacities were prevalent in the older lenses: however, no correlation of experimental data with appearance was noted, and the limited number of lenses precluded dividing them into groups by age or appearance. L-Glutamate, 99% 15N, was purchased from KOR Stable Isotopes, Cambridge, MA, and L-glutamine, 99 “/o 15N (amino-labeled) from MSD Isotopes, Rahway, NJ. Other biochemicals were obtained from Sigma Chemical Company, St Louis, MO. incubation of lenses with [‘5N]amino
acids
Human lens incubations were performed by methods previously used for monkey lenses (Jernigan and Zigler, 1987). Within 3-24 hr post-mortem, lenses were dissected posteriorly from the enucleated 0 1990 Academic Press Limited
598
globes and were incubated, anterior side up, at 3 7°C in 95 % air, 5 % CO, for 24 hr in 8 ml of an amino acidfree, bicarbonate buffered, balanced salt medium with 20 U ml-’ of penicillin G and 20 LLg ml-’ of streptomycin. This medium is based on that of Kinoshita, Merola, and Tung (1968) which was modified and prepared as previously described (Jernigan, 19 8 3). [‘jN]glutamine or [15N]glutamate. 5 ITIM. was added as required. Free amino acids were extracted from lenses with 6 % trichloroacetic acid (TCA), trifluoracetyl-n-butyl (TAB) derivatives were prepared (Roach and Gehrke, 1969), and the relative amounts of amino 15N and 14N in the amino acid derivatives were determined by gas chromatography-mass spectrometry, as previously described (Jernigan andzigler, 1987; Jernigan, 1983). The enrichment of ‘“N in individual amino acids was expressed as percent excess 15N (a measurement analogous to specific radioactivity in radioisotope studies). It is important to note that the derivatization method produces identical derivatives for dicarboxylic acids and their amides. Therefore, the procedure determines 15N enrichment data for the total lenticular GLX (glutamine + glutamate) and ASX (aspartate + asparagine) pools, rather than for these individual amino acids. For selected samples, the glutamate was separated from an aliquot of the TCA lens extract using ion exchange chromatography on Dowex-lformate, and derivatized separately to determine the enrichment of ‘jN in glutamate alone, rather than in the mixture, GLX (Nissim, Yudkoff and Segal, 1985). Measurement of Ammonia Production by Lenses The formation of ammonia by lenses in a glutaminecontaining medium was measured by a method similar to that used for monkey lenses (Jernigan and Zigler, 198 7). Modified TC-199 medium without phenol red. serum, or glutamine (Kinoshita, et al., 1968; Jernigan and Laranang, 1984b) was equilibrated with 5 ‘j& CO,. and lo-ml portions were placed in Erlenmeyer flasks. Each human lens was placed in an individual flask, and other flasks were used as controls without lenses. Glutamine was added to the flasks containing one lens of each pair and to one control flask, to obtain a final concentration of 2.0 mM. Immediately after adding the glutamine, 2-ml aliquots from each flask were frozen at - 70°C for later assay of ammonia, and the flasks were stoppered and incubated for 24 hr at 3 7°C. Aliquots of each medium, taken before and after incubation, were simultaneously assayed for ammonia (which is primarily in the form of ammonium ion at physiological pH) using glutamate dehydrogenase and a modification of the method of Reichelt, Kvamme and Tveit ( 1964) as previously described (Jernigan and Laranang, 1984a). The ammonia content of the unincubated samples and the TCA extracts of the lenses was found to be negligible, and control experiments showed that the results were not affected by frozen storage of the samples.
H. M. JERNIGAN,
JR.
3. Results Incorporation of ‘jN into Free Amino Acids of‘ Human LensesCultured with ‘5N-amino-JabeJed Glutamate or Glutamine Table I shows the incorporation of 13N into the amino groups of the free amino acids of human lenses incubated for 24 hr in balanced salt medium containing 5mM of either [15N]glutamate (column A) or amino-labeled [15N]glutamine (column B). Data from similar experiments with monkey lenses(Jernigan and Zigler, 1987) or rat lenses (Jernigan and Laranang, 1984a) are included for comparison. In general. incorporation of label into lenticular amino acids from either glutamine or glutamate was slower in human lensesthan in either rat or monkey lenses. Because the human lenses were from eye bank donors ranging in age from 1 to 99 yr, and because eye bank lenses are unavoidably variable in many respects which might affect their metabolism, the human lens data were tabulated in two groups for Table I. The mean data are shown for all lensestested, and separate means are shown for those lenses for which pairs of eyes from the same donor were incubated, one with glutamine, and the other with glutamate. Within experimental error, the means calculated from the paired lenses are identical to the means calculated from all the lensestested. Thus, the different results from lensesincubated with [“Nlglutamine or [15N]glutamate do not arise from differences between donors. As indicated by the rate of labeling of the lenticular GLX pool, glutamine entered the human lens more rapidly than glutamate. This observation is consistent for all species studied (Table I). However, human lensesmore rapidly utilize externally added [15N]glutamate than [‘“Nlglutamine for the transamination reactions, which result in formation of [‘5N]ASX, [15N]alanine and [15N]serine. In this respect, human lenses are similar to monkey lenses (Table I and Jernigan and Zigler, 1987) and differ from rat lenses (Table I and Jernigan and Laranang, 1984a). which preferentially utilize glutamine as a source of amino groups. Another difference between primate and rat lenses is the lack of labeling of lenticular glycine in the primate lenses. Human and monkey lenses did not synthesize measurable amounts of [15N]glycine when incubated with either [15N]glutamine or [“N]glutamate. As explained below, this doesnot indicate a lack of glycine synthesis or an absence of serine hydroxymethyltransferase in the primate lenses. Unlike the transamination reactions, which provide amino groups for synthesis of aspartate, alanine and serine, proline synthesis in human lensesappears to use glutamine more readily than glutamate. The proline in human lenses incubated with [“Nlglutamine contained 109% 15N ( kO.8 s.E.M.) which is higher (P < 0.01) than the 7.0% 15N (f 1.0 s.E.M.) found in the proline of lensesincubated with [‘“Nlglut-
AMINO
ACID
METABOLISM
IN HUMAN
599
LENSES TABLE
Percent excess 15N ( f
s.E.M.)
I
found in free amino acids of lenses after incubating 24 hr with 5 mM [15Njglutamate or ([15Njamino)-gZutamine
Amino
acid in lens
Human lenses (all) GLX ASX Alanine Proline Serine Glycine Human lenses (pairs) GLX ASX Alanine Proline Serine Glycine Monkey lenses* GLX ASX Alanine Proline Serine Glycine Rat lenses (two to three
[ljN]glutamate added (A) --______ n= 18 29.7 ( + 1.7) 19.1 ( f 2.4) 13.9 (f 1.6) 7.O(kl.O) 4.0 (kO.5) < 1.0 n= 12 29.7(+1.5) 18.8 (+ 3.0) 12.4 (+ 1.6) 6,2 ( +O%) 4.0 (kO.7) < 1.0 n=6 37.6(fl.O 47.8 (+ 1.1) 43.4 ( + 2.0) 7.0 (kO.6) 11.1 (kO.7) < 1.0
per group)
n=3 60.0 ( + 2.4) 57.8 (f 1.5) 35.7 ( f 5.5) 29.4 (kO.6) 30.3 (+ 2.9) 13.4 (+1.8)
GLX ASX Alanine Proline Serine Glycine * Data on and Laranang
monkey lenses were taken from Jemigan
and Zigler
(1987)
[15N]glutamine added (B) n= 31 51.9 (&- 1.5) 13.9 11.5 10.9
( * 1.0) (fl.1) ( + 0.8)
2.4 ( + 0.3)
Ratio A/B 0.6 1.4 1.2 0.6 1.7
< 1.0 n=
12
49.9 ( * 1.7) 13.0 (fl.6) 10.6 (+ 1.3) 9.4(fl.l) 2.6 (sO.5)
0.6 1.4 1.2 0.7 1.5
< 1.0
n=6 63.3 (f0.9) 29.2 ( + 1.5) 33.2 (f2.3) 6.4 ( k 0.8) 5.8 ( + 1.0)
0.6 1.6 1.3 1.1 1.9
< 1.0 n:
86.5 70.5 42.0 57.5 34.6 19.4
5 + 1.2) * 1.7) + 2.3) + 0.6) + 2.4) + 1.5)
and data on rat lenses were previously
0.7 0.8 0.8 0.5 0.9 0.7 used in graphs
by Jemigan
(1984a).
amate. Comparing the incorporation of 15N into amino acids for those lenses incubated with either [15N]glutamate (column A, Table I) or [15N]glutamine (column B, Table I), proline synthesis shows the greatest preference for glutamine (lowest ratio, A/B) for all three species studied. Conversion of Glutamine to Glutamate in Human Lenses To study the rate of deamidation of glutamine in human lenses, the rate of ammonia production was measured in lenses incubated in TC-199 medium containing 2 mM unlabeled glutamine. The methods used were identical to those previously used to measure ammonia release by rat (Jernigan and Laranang, 1984a) and monkey (Jernigan and Zigler, 1987) lenses. However, with human lenses the ammonia release was variable and, in most lenses, barely detectable. To further study the rate of equilibration between glutamate and glutamine in human lenses, an ion exchange method (Nissim et al., 1985) was used to separate glutamate from GLX in aliquots of selected human lens extracts prior to derivatization for deter-
mining the 15N content by GCMS. The isolated glutamate was derivatized and analyzed to determine [15N]glutamate. Separate aliquots of the TCA extracts were used to obtain data for Table I (which includes [15N]GLX). In five human lenses from the group incubated in [15N]glutamate, the ratio of the (% ‘jN in glutamate)/@, 15N in GLX) was 1.02 (kO.03 s.E.M.). indicating that the labeled amino groups in glutamine and glutamate had approximately equilibrated. In nine human lenses from the group incubated in [15N]glutamine the ratio of the (% 15N in glutamate)/(% 15N in GLX) was only 0.84 (+0*03 s.E.M.). indicating that the labeled glutamine and glutamate in these lenses had not equilibrated within the 24-hr period of the experiment. 4. Discussion Glutamate occupies a central position in amino acid metabolism as a result of its involvement in numerous transamination, deamination, and biosynthetic reactions. In the lens, glutamate is necessary for the synthesis of products including glutamine, glutathione, and proline, as well as proteins such as the
H. M. JERNIGAN,
glutamate-rich crystallins (Trayhurn and van Heyningen, 1973; Kern and Ho, 1973). Studies with lenses of rats (Jernigan, 198 3 ; Jernigan and Laranang. 1984a) and rhesus monkeys (Jernigan and Zigler, 1987) have shown that 15N-labeled amino nitrogen from glutamate or glutamine can be transferred to other amino acid pools by transamination and other reactions, resulting in formation of labeled aspartate, alanine, serine, glycine and proline. In all studies using rat lenses, the principal source of lenticular glutamate was found to be glutamine, which was rapidly transported from the surrounding fluids and deamidated by glutaminase (forming glutamate), whereas the direct transport of glutamate into the lens was relatively slow. In contrast, as a result of less rapid deamidation of glutamine, the various metabolic pathways in monkey lenses appeared to use glutamate obtained directly from the external glutamate pool more rapidly than glutamate derived indirectly from glutamine (Jernigan and Zigler, 1987). The present study compared the utilization of glutamate and glutamine in human lenses, and the results are somewhat different from those obtained with rats or monkeys. The transamination reactions in human lenses appeared to preferentially involve amino groups derived from glutamate. As with monkey lenses, the slower use of glutamine by human lenses may result from its slow rate of deamidation compared with rat lenses. On the basis of wet weight, the ability of monkey lenses to release ammonia from glutamine is only about 10% that of rat lenses (Jernigan and Zigler, 1987). Measurements of ammonia release by human lenses indicate an even slower rate than in monkey lenses, and the rate appears to be more variable. The slow rate of deamidation of glutamine in human lenses is further supported by the observation that the lenticular glutamine and glutamate pools had not equilibrated, with respect to ‘jN enrichment, after 24 hr of incubation. It is possible that differences in diffusion kinetics due to lens size may have contributed to the observed species differences in amino acid metabolism. Also, it is difficult to compare ages in such different species as rats and monkeys. The lenses of both species were obtained from relatively young or ‘juvenile ’ animals, i.e. 2-3-yr-old monkeys and 80-100 g rats. The human lenses were from donors ranging in age from 1 to 99 yr, and there was no apparent correlation of 15N incorporation with age, although there were too few young human lenses for an adequate analysis of early age changes. The lack of labeling of glycine in the primate lenses, as reported in Table I, results from the low incorporation of 15N into serine, which is the immediate precursor for glycine synthesis. Experiments in which human, monkey, or rat lenses were incubated directly with [15N]serine or [*5N]glycine clearly demonstrated that these lenses are capable of the reversible inter-
JR
conversion of serine and glycine (Geller. %igler and Jernigan, 1990). This reaction is catalyzed by the enzyme serine hydroxymethyltransferase. Although the lens may be able to obtain adequate supplies of glycine from other sources, the serine hydroxymethyltransferase reaction may be important as a source of 5. lo-methylenetetrahydrofolate. which is necessary for several biosynthetic reactions and which is formed from the hydroxymethyl group of serine during the formation of glycine. The observation that glutamate used for human lens proline biosynthesis, unlike that used for the transamination reactions in human lenses, is derived primarily from glutamine (Table I, see ratio of columns A/B) may arise from the subcellular location of the enzymes involved. Glutaminase, which converts glutamine to glutamate. is located in mitochondria in all tissues examined (Errera and Greenstein, 1949), and rat lens glutaminase also appears to be mitochondrial (Vallari, Macleod and Jernigan. 1987). Likewise, pyrroline-5-carboxylate synthase, which carries out the first step in the conversion of glutamate to proline, is also located in mitochondria (Wakabayashi and Jones. 1983). Therefore, the glutamate at the site where proline synthesis begins (i.e. in mitochondria) may arise primarily from deamidation of glutamine by glutaminase. In the experiments with ‘jN-labeled glutamine or glutamate, the enrichment of ‘;‘N in mitochondrial glutamate would more closely reflect the enrichment in lenticular glutamine than the enrichment in glutamate in the lens as a whole. Acknowledgements The excellent technical assistance of MS A. S. Vallari, MS A. Rustom and MS P. S. Blum is gratefully acknowledged. The author is also indebted to the Mid South Eye Bank,
Memphis,TN, for the human eyes,and to DrsC. Dasand D. M. Desiderio of the Stout Neurosciences Laboratory, University of Tennessee, Memphis, for their assistance in use of the mass spectrometer, which was obtained with the help of NIH Shared Equipment Grant RR01651. The project was supported in part by National Institutes of Health Research Grants EY02665 and EY07938.
References Dickinson, J. C.. Durham. D. G. and Hamilton, P. B. ( 1968). Ion exchangechromatography of free amino acidsin aqueous fluid and lens of the human eye. Invest. Ophthalmol. 7, 551-63. Errera. M. and Greenstein, J. P. (1949). Phosphate activated glutaminase in kidney and other tissues. 1. Biol. Chem. 178, 49 S-502. Geller, A. M., Zigler, Jr., J. S. and Jernigan. Jr., H. M. ( 1990). Serine hydroxymethyltransferase : Evidence for its presence in human monkey and rat lenses. Exp. Eye Res. 50. 149-55. Jernigan. Jr., H. M. (1983). Metabolism of [15N]-labeled amino acids in rat lens. Exp. Eye Res. 37, 77-84. Jernigan, Jr., H. M. and Laranang. A. S. (1984a). Metabolism of the alpha-amino nitrogen of glutamine in rat lens. Exp. Eye Res. 39, 113-22. Jernigan, Jr., J. M. and Laranang, A. S. (1984b). Effects of
AMINO
ACID
METABOLISM
IN
HUMAN
LENSES
ribotlavin-sensitized photooxidation on choline metabolism in cultured rat lenses. Cur-r. Eye Res. 3, 121-6. Jernigan, Jr., J. M. and Zigler. Jr., J, S. (1987). Metabolism of glutamine and glutamate in monkey lens. Exp. Eye Res. 44, 871-6. Kern, H. L. and Ho, C. K. (1973). Transport of L-glutamic acid and L-glutamine and their incorporation in lenticular glutathione. Exp. Eye Res. 17, 455-62. Kinoshita, J. H., Barber, G. W., Merola, L. 0. and Tung, B. (1969). Changes in the levels of free amino acids and myo-inositol in the galactose-exposed lens. Invest. Ophthalmol. 8, 625-32. Kinoshita. J. H. and Merola, L. 0. (1961). The utilization of pyruvate and its conversion to glutamate in calf lens. Exp. Eye Res.1, 53-9. Kinoshita,J. H., Merola, L. 0. andTung, B. (1968). Changes in cation permeability in the galactose-exposed rabbit lens.Exp. Eye Res.7, 80-90. Nissim,I., Yudkoff, M. and Segal,S. (1985). Metabolismof glutamine and glutamate by rat renal tubules. Study with 15Nand gaschromatography-massspectrometry. 1. Biol. Chem. 260, 13955-67. Reddy,D. V. N. (1967). Distribution of free amino acidsand
601
relatedcompoundsin ocular fluids, lens,and plasmaof various mammalian species.Invest. Ophthnlmol.6, 478-83. Reddy, D. V. N. and Kinsey, V. E. (1962). Studieson the crystalline lens,IX. Quantitative analysisof free amino acids and related compounds.Invest. Ophthalmol.1, 63541. Reichelt, K. L., Kvamme, E. and Tveit. B. (1964). The enzymatic determination of ammonia in blood and tissues.Scnnd.J. Clin. Lab. Invest. 16, 433-9. Roach,D. and Gehrke,C. W. (1969). Direct esterificationof the protein amino acids.Gas-liquidchromatographyof N-TPA n-butyl esters.1. Chromatogr.44, 269-78. Trayhurn. P. and van Heyningen, R. (1973). The metabolism of glutamine in the bovine lens: glutamine as a sourceof glutamate.Exp. Eye Res. 17, 149-54. Vallari, A. S..Macleod,R. M. andJernigan.H. M.. Jr. ( 1987). Rat lensglutaminase: separationand characterization of solubleand particulate fractions. Elcp.Eye Res.45, 491-500. Wakabayashi, Y. and Jones. M. E. (1983). Pyrroline-Scarboxylate synthesisfrom glutamateby rat intestinal mucosa.I. Biol. Chem. 258, 3865-72.
Metabolism
of Glutamine HOWARD
Departments
of Biochemistry
and Glutamate M. JERNIGAN,
and Ophthalmology,
University
in Human
Lenses
JR” of Tennessee,
Memphis,
TN 38163,
U.S.A.
Glutamate is important to lenses as a central intermediate in amino acid metabolism, as well as for synthesis of proteins and glutathione. In rat and calf lenses, the principal source of lenticular glutamate is glutamine. which enters the lens and is deamidated to form glutamate. In contrast, monkey lenses use external glutamate more readily than glutamine. Amino acid metabolism was studied in human lenses by incubating them with amino-labeled [15N]glutamine or [15N]glutamate. The lenticular free amino acids were then isolated and analysed by gas chromatography-mass spectrometry to determine 15Nlabeled products. The results were compared with those of similar experiments with lenses from other species. Label was measured in aspartate. alanine. serine and proline, as well as lenticular glutamine and glutamate. Glutamine enters lenses more readily than glutamate in all the species examined. Nevertheless, aspartate, alanine and serine were more rapidly labeled by incubating human lenses in [15N]glutamate than in [‘SN]glutamine. This observation is similar to reports of experiments with monkey lenses, which unlike rat lenses, preferentially utilize glutamate rather than glutamine. In contrast, human lens proline was more rapidly labeled by incubating lenses with [‘5N]glutamine than with [‘5N]glutamate. In human lenses, the relatively slow utilization of glutamine for the transamination reactions which form aspartate, alanine and serine appears to result from slow deamidation. The relative preference for glutamine over glutamate as a precursor for proline synthesis in human lenses may be related to the mitochondrial location of the enzymes involved. Keu words: human lens: rat; monkey; amino acids: metabolism: ammonia: glutamate: glutamine : glycine : proline ; serine ; alanine ; aspartate. 1. Introduction The concentration of glutamate is higher in the lens than in the aqueous humor, and the glutamate concentration in most species exceeds that of all other lenticular amino acids with the exception of taurine (Reddy and Kinsey, 1962 ; Reddy, 1967 ; Dickinson, Durham, and Hamilton, 1968; Kinoshita et al., 1969). Although lenses are capable of synthesizing glutamate from other metabolites (Kinoshita and Merola, 1961), the principal source of glutamate for rat and calf lens metabolism is from glutamine, which is transported from the aqueous humor and deamidated (Kern and Ho, 1973; Trayhurn and Van Heyningen, 1973). When rat lenses (Kern and Ho, 1973; Jernigan and Laranang, 1984a) or bovine lenses (Kern and Ho, 1973: Trayhurn and Van Heyningen, 1973) were incubated with labeled glutamine or glutamate, glutamine entered the lenses more readily than glutamate. and was utilized more rapidly for glutamate-requiring biochemical pathways. Thus, in rat bovine lenses, the two-step process of glutamine transport and deamidation (to form glutamate) is more rapid than direct transport of glutamate. In contrast, monkey lenses utilized glutamate more rapidly than glutamine, even though glutamine entered more rapidly (Jernigan and Zigler, 1987). The different results with monkey lenses may have been related to the observation that glutamine was converted to glutamate more slowly in monkey lenses than in rat lenses. The differences observed between rat and primate (rhesus monkey) lenses. together with the relative * For correspondenceat : University of Tennessee,Memphis, 9 56 Court Avenue. Room 11222.Memphis. TN 38163 U.S.A. 0014-4835/90/06(3597+05
$03.00/O
paucity of data on amino acid metabolism in human lenses, led to the present study, which compares human lens amino group metabolism with that of monkeys and rats. Human lenses were incubated with 15N-amino-labeled glutamine or glutamate, and the products were examined to determine the relative rates of utilization of these two amino acids and the distribution of their amino nitrogen among other free amino acids in the lenses. The results from experiments with human lenses were compared with results obtained from experiments with other species. 2. Materials
and Methods
Materials Freshly enucleated human eyes were obtained by the Mid South Eye Bank, and were transported to the laboratory in moist jars in ice. They were stored at 4°C until used. The donors ranged in age from 1 to 99 yr, with median age of 61 yr. Slight haze and small opacities were prevalent in the older lenses: however, no correlation of experimental data with appearance was noted, and the limited number of lenses precluded dividing them into groups by age or appearance. L-Glutamate, 99% 15N, was purchased from KOR Stable Isotopes, Cambridge, MA, and L-glutamine, 99 “/o 15N (amino-labeled) from MSD Isotopes, Rahway, NJ. Other biochemicals were obtained from Sigma Chemical Company, St Louis, MO. incubation of lenses with [‘5N]amino
acids
Human lens incubations were performed by methods previously used for monkey lenses (Jernigan and Zigler, 1987). Within 3-24 hr post-mortem, lenses were dissected posteriorly from the enucleated 0 1990 Academic Press Limited
598
globes and were incubated, anterior side up, at 3 7°C in 95 % air, 5 % CO, for 24 hr in 8 ml of an amino acidfree, bicarbonate buffered, balanced salt medium with 20 U ml-’ of penicillin G and 20 LLg ml-’ of streptomycin. This medium is based on that of Kinoshita, Merola, and Tung (1968) which was modified and prepared as previously described (Jernigan, 19 8 3). [‘jN]glutamine or [15N]glutamate. 5 ITIM. was added as required. Free amino acids were extracted from lenses with 6 % trichloroacetic acid (TCA), trifluoracetyl-n-butyl (TAB) derivatives were prepared (Roach and Gehrke, 1969), and the relative amounts of amino 15N and 14N in the amino acid derivatives were determined by gas chromatography-mass spectrometry, as previously described (Jernigan andzigler, 1987; Jernigan, 1983). The enrichment of ‘“N in individual amino acids was expressed as percent excess 15N (a measurement analogous to specific radioactivity in radioisotope studies). It is important to note that the derivatization method produces identical derivatives for dicarboxylic acids and their amides. Therefore, the procedure determines 15N enrichment data for the total lenticular GLX (glutamine + glutamate) and ASX (aspartate + asparagine) pools, rather than for these individual amino acids. For selected samples, the glutamate was separated from an aliquot of the TCA lens extract using ion exchange chromatography on Dowex-lformate, and derivatized separately to determine the enrichment of ‘jN in glutamate alone, rather than in the mixture, GLX (Nissim, Yudkoff and Segal, 1985). Measurement of Ammonia Production by Lenses The formation of ammonia by lenses in a glutaminecontaining medium was measured by a method similar to that used for monkey lenses (Jernigan and Zigler, 198 7). Modified TC-199 medium without phenol red. serum, or glutamine (Kinoshita, et al., 1968; Jernigan and Laranang, 1984b) was equilibrated with 5 ‘j& CO,. and lo-ml portions were placed in Erlenmeyer flasks. Each human lens was placed in an individual flask, and other flasks were used as controls without lenses. Glutamine was added to the flasks containing one lens of each pair and to one control flask, to obtain a final concentration of 2.0 mM. Immediately after adding the glutamine, 2-ml aliquots from each flask were frozen at - 70°C for later assay of ammonia, and the flasks were stoppered and incubated for 24 hr at 3 7°C. Aliquots of each medium, taken before and after incubation, were simultaneously assayed for ammonia (which is primarily in the form of ammonium ion at physiological pH) using glutamate dehydrogenase and a modification of the method of Reichelt, Kvamme and Tveit ( 1964) as previously described (Jernigan and Laranang, 1984a). The ammonia content of the unincubated samples and the TCA extracts of the lenses was found to be negligible, and control experiments showed that the results were not affected by frozen storage of the samples.
H. M. JERNIGAN,
JR.
3. Results Incorporation of ‘jN into Free Amino Acids of‘ Human LensesCultured with ‘5N-amino-JabeJed Glutamate or Glutamine Table I shows the incorporation of 13N into the amino groups of the free amino acids of human lenses incubated for 24 hr in balanced salt medium containing 5mM of either [15N]glutamate (column A) or amino-labeled [15N]glutamine (column B). Data from similar experiments with monkey lenses(Jernigan and Zigler, 1987) or rat lenses (Jernigan and Laranang, 1984a) are included for comparison. In general. incorporation of label into lenticular amino acids from either glutamine or glutamate was slower in human lensesthan in either rat or monkey lenses. Because the human lenses were from eye bank donors ranging in age from 1 to 99 yr, and because eye bank lenses are unavoidably variable in many respects which might affect their metabolism, the human lens data were tabulated in two groups for Table I. The mean data are shown for all lensestested, and separate means are shown for those lenses for which pairs of eyes from the same donor were incubated, one with glutamine, and the other with glutamate. Within experimental error, the means calculated from the paired lenses are identical to the means calculated from all the lensestested. Thus, the different results from lensesincubated with [“Nlglutamine or [15N]glutamate do not arise from differences between donors. As indicated by the rate of labeling of the lenticular GLX pool, glutamine entered the human lens more rapidly than glutamate. This observation is consistent for all species studied (Table I). However, human lensesmore rapidly utilize externally added [15N]glutamate than [‘“Nlglutamine for the transamination reactions, which result in formation of [‘5N]ASX, [15N]alanine and [15N]serine. In this respect, human lenses are similar to monkey lenses (Table I and Jernigan and Zigler, 1987) and differ from rat lenses (Table I and Jernigan and Laranang, 1984a). which preferentially utilize glutamine as a source of amino groups. Another difference between primate and rat lenses is the lack of labeling of lenticular glycine in the primate lenses. Human and monkey lenses did not synthesize measurable amounts of [15N]glycine when incubated with either [15N]glutamine or [“N]glutamate. As explained below, this doesnot indicate a lack of glycine synthesis or an absence of serine hydroxymethyltransferase in the primate lenses. Unlike the transamination reactions, which provide amino groups for synthesis of aspartate, alanine and serine, proline synthesis in human lensesappears to use glutamine more readily than glutamate. The proline in human lenses incubated with [“Nlglutamine contained 109% 15N ( kO.8 s.E.M.) which is higher (P < 0.01) than the 7.0% 15N (f 1.0 s.E.M.) found in the proline of lensesincubated with [‘“Nlglut-
AMINO
ACID
METABOLISM
IN HUMAN
599
LENSES TABLE
Percent excess 15N ( f
s.E.M.)
I
found in free amino acids of lenses after incubating 24 hr with 5 mM [15Njglutamate or ([15Njamino)-gZutamine
Amino
acid in lens
Human lenses (all) GLX ASX Alanine Proline Serine Glycine Human lenses (pairs) GLX ASX Alanine Proline Serine Glycine Monkey lenses* GLX ASX Alanine Proline Serine Glycine Rat lenses (two to three
[ljN]glutamate added (A) --______ n= 18 29.7 ( + 1.7) 19.1 ( f 2.4) 13.9 (f 1.6) 7.O(kl.O) 4.0 (kO.5) < 1.0 n= 12 29.7(+1.5) 18.8 (+ 3.0) 12.4 (+ 1.6) 6,2 ( +O%) 4.0 (kO.7) < 1.0 n=6 37.6(fl.O 47.8 (+ 1.1) 43.4 ( + 2.0) 7.0 (kO.6) 11.1 (kO.7) < 1.0
per group)
n=3 60.0 ( + 2.4) 57.8 (f 1.5) 35.7 ( f 5.5) 29.4 (kO.6) 30.3 (+ 2.9) 13.4 (+1.8)
GLX ASX Alanine Proline Serine Glycine * Data on and Laranang
monkey lenses were taken from Jemigan
and Zigler
(1987)
[15N]glutamine added (B) n= 31 51.9 (&- 1.5) 13.9 11.5 10.9
( * 1.0) (fl.1) ( + 0.8)
2.4 ( + 0.3)
Ratio A/B 0.6 1.4 1.2 0.6 1.7
< 1.0 n=
12
49.9 ( * 1.7) 13.0 (fl.6) 10.6 (+ 1.3) 9.4(fl.l) 2.6 (sO.5)
0.6 1.4 1.2 0.7 1.5
< 1.0
n=6 63.3 (f0.9) 29.2 ( + 1.5) 33.2 (f2.3) 6.4 ( k 0.8) 5.8 ( + 1.0)
0.6 1.6 1.3 1.1 1.9
< 1.0 n:
86.5 70.5 42.0 57.5 34.6 19.4
5 + 1.2) * 1.7) + 2.3) + 0.6) + 2.4) + 1.5)
and data on rat lenses were previously
0.7 0.8 0.8 0.5 0.9 0.7 used in graphs
by Jemigan
(1984a).
amate. Comparing the incorporation of 15N into amino acids for those lenses incubated with either [15N]glutamate (column A, Table I) or [15N]glutamine (column B, Table I), proline synthesis shows the greatest preference for glutamine (lowest ratio, A/B) for all three species studied. Conversion of Glutamine to Glutamate in Human Lenses To study the rate of deamidation of glutamine in human lenses, the rate of ammonia production was measured in lenses incubated in TC-199 medium containing 2 mM unlabeled glutamine. The methods used were identical to those previously used to measure ammonia release by rat (Jernigan and Laranang, 1984a) and monkey (Jernigan and Zigler, 1987) lenses. However, with human lenses the ammonia release was variable and, in most lenses, barely detectable. To further study the rate of equilibration between glutamate and glutamine in human lenses, an ion exchange method (Nissim et al., 1985) was used to separate glutamate from GLX in aliquots of selected human lens extracts prior to derivatization for deter-
mining the 15N content by GCMS. The isolated glutamate was derivatized and analyzed to determine [15N]glutamate. Separate aliquots of the TCA extracts were used to obtain data for Table I (which includes [15N]GLX). In five human lenses from the group incubated in [15N]glutamate, the ratio of the (% ‘jN in glutamate)/@, 15N in GLX) was 1.02 (kO.03 s.E.M.). indicating that the labeled amino groups in glutamine and glutamate had approximately equilibrated. In nine human lenses from the group incubated in [15N]glutamine the ratio of the (% 15N in glutamate)/(% 15N in GLX) was only 0.84 (+0*03 s.E.M.). indicating that the labeled glutamine and glutamate in these lenses had not equilibrated within the 24-hr period of the experiment. 4. Discussion Glutamate occupies a central position in amino acid metabolism as a result of its involvement in numerous transamination, deamination, and biosynthetic reactions. In the lens, glutamate is necessary for the synthesis of products including glutamine, glutathione, and proline, as well as proteins such as the
H. M. JERNIGAN,
glutamate-rich crystallins (Trayhurn and van Heyningen, 1973; Kern and Ho, 1973). Studies with lenses of rats (Jernigan, 198 3 ; Jernigan and Laranang. 1984a) and rhesus monkeys (Jernigan and Zigler, 1987) have shown that 15N-labeled amino nitrogen from glutamate or glutamine can be transferred to other amino acid pools by transamination and other reactions, resulting in formation of labeled aspartate, alanine, serine, glycine and proline. In all studies using rat lenses, the principal source of lenticular glutamate was found to be glutamine, which was rapidly transported from the surrounding fluids and deamidated by glutaminase (forming glutamate), whereas the direct transport of glutamate into the lens was relatively slow. In contrast, as a result of less rapid deamidation of glutamine, the various metabolic pathways in monkey lenses appeared to use glutamate obtained directly from the external glutamate pool more rapidly than glutamate derived indirectly from glutamine (Jernigan and Zigler, 1987). The present study compared the utilization of glutamate and glutamine in human lenses, and the results are somewhat different from those obtained with rats or monkeys. The transamination reactions in human lenses appeared to preferentially involve amino groups derived from glutamate. As with monkey lenses, the slower use of glutamine by human lenses may result from its slow rate of deamidation compared with rat lenses. On the basis of wet weight, the ability of monkey lenses to release ammonia from glutamine is only about 10% that of rat lenses (Jernigan and Zigler, 1987). Measurements of ammonia release by human lenses indicate an even slower rate than in monkey lenses, and the rate appears to be more variable. The slow rate of deamidation of glutamine in human lenses is further supported by the observation that the lenticular glutamine and glutamate pools had not equilibrated, with respect to ‘jN enrichment, after 24 hr of incubation. It is possible that differences in diffusion kinetics due to lens size may have contributed to the observed species differences in amino acid metabolism. Also, it is difficult to compare ages in such different species as rats and monkeys. The lenses of both species were obtained from relatively young or ‘juvenile ’ animals, i.e. 2-3-yr-old monkeys and 80-100 g rats. The human lenses were from donors ranging in age from 1 to 99 yr, and there was no apparent correlation of 15N incorporation with age, although there were too few young human lenses for an adequate analysis of early age changes. The lack of labeling of glycine in the primate lenses, as reported in Table I, results from the low incorporation of 15N into serine, which is the immediate precursor for glycine synthesis. Experiments in which human, monkey, or rat lenses were incubated directly with [15N]serine or [*5N]glycine clearly demonstrated that these lenses are capable of the reversible inter-
JR
conversion of serine and glycine (Geller. %igler and Jernigan, 1990). This reaction is catalyzed by the enzyme serine hydroxymethyltransferase. Although the lens may be able to obtain adequate supplies of glycine from other sources, the serine hydroxymethyltransferase reaction may be important as a source of 5. lo-methylenetetrahydrofolate. which is necessary for several biosynthetic reactions and which is formed from the hydroxymethyl group of serine during the formation of glycine. The observation that glutamate used for human lens proline biosynthesis, unlike that used for the transamination reactions in human lenses, is derived primarily from glutamine (Table I, see ratio of columns A/B) may arise from the subcellular location of the enzymes involved. Glutaminase, which converts glutamine to glutamate. is located in mitochondria in all tissues examined (Errera and Greenstein, 1949), and rat lens glutaminase also appears to be mitochondrial (Vallari, Macleod and Jernigan. 1987). Likewise, pyrroline-5-carboxylate synthase, which carries out the first step in the conversion of glutamate to proline, is also located in mitochondria (Wakabayashi and Jones. 1983). Therefore, the glutamate at the site where proline synthesis begins (i.e. in mitochondria) may arise primarily from deamidation of glutamine by glutaminase. In the experiments with ‘jN-labeled glutamine or glutamate, the enrichment of ‘;‘N in mitochondrial glutamate would more closely reflect the enrichment in lenticular glutamine than the enrichment in glutamate in the lens as a whole. Acknowledgements The excellent technical assistance of MS A. S. Vallari, MS A. Rustom and MS P. S. Blum is gratefully acknowledged. The author is also indebted to the Mid South Eye Bank,
Memphis,TN, for the human eyes,and to DrsC. Dasand D. M. Desiderio of the Stout Neurosciences Laboratory, University of Tennessee, Memphis, for their assistance in use of the mass spectrometer, which was obtained with the help of NIH Shared Equipment Grant RR01651. The project was supported in part by National Institutes of Health Research Grants EY02665 and EY07938.
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