794

Biochimica et Biophysica Aeta, 444 (1976) 794--80.1 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 28029 SUBCELLULAR LOCALIZATION OF 7~LUTAMYL CARBOXYPEFYIDASE AND OF FOLATES

YOON SOOK SHIN *, CATHERINE CHAN **, AMANDA J. VIDAL ***, TOM BRODY and E.L.R. STOKSTADT

Department of Nutritional Sciences, University of California, Berkeley, Calif. 94 720 (U.S.A.) (Received June 1st, 1976)

Summary

The subcellular distributions of glutamyl carboxypeptidase, folate specific activities,and radioactive metabolites of injected [:~H]folicacid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal f r a c t i o n was near o r g r e a t e r t h a n f o u r t i m e s t h a t i n t h e o t h e r subcellular f r a c tions.

The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 9 5 % folate polyglutamates, and microsomal and soluble folates were 85--90% folate polyglutamates. Injected [3H]folic acid was initiallyconcentrated in the microsomal fraction, as measured by 3H c p m per ng folate. Initially,injected [3H]folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less,as measured by the percentage of total 3H c p m determined to be [3H]folate penta- and hexaglutamates. At 24 h, the conversion of [3H]folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates. Injected [3H]folic acid after 2 h was found to consist of 9 4 % reduced folates

* P r e s e n t a d d r e s s : K i n d e r K l l n i k d e r U n i v e r s l t ~ t M / i n c h e n , 8 M f l n c h e n 2, L i n d w u r m str. 4, W. Germany. ** P r e s e n t a d d r e s s : D e p a r t m e n t o f M e d i c i n e , B o s t o n UniverSity M e d i c a l S c h o o l , 7 5 0 H a r r i s o n A v e . ( R m . E - 4 8 8 ) , B o s t o n Mass. 0 2 1 1 8 , U . S . A : *** P r e s e n t a d d r e s s : F o o d a n d D r u g L a b o r a t o r y , C a l l f o m i a S t a t e D e p a r t m e n t o f H e a l t h , 2 1 5 1 B e r k e l e y W a y , B e r k e l e y , Calif. 9 4 7 0 4 , U . S . A . t To whom reprint requests should be addressed. Abbreviation: PteGlu, pteroylg]utarr/ate.

795 in the soluble fraction, 56% in the mitochondria!, 55% in the nuclear, 20% in the lySouomal, and 15% in the m ~ o m a I ~ b t ~ ,

Introduction

Folates are cofactors in the enzymatic transfer of one-carbon units. It is well established that folates in nature are metabolized to reduced forms [1], to pteroyl-~/-L-oligoglutamates (folate polyglutamates) [2--5], and that the oligoglutamate moiety is hydrolyzed by glutamyl carboxypeptidase, EC 3.4.12.10 [1,6--8]. Even though glutamyl carboxypeptidases from several sources have been characterized, their physiological significance in folate metabolism is only partially understood. We report the subcellular localization of rat liver glutamyl carboxypeptidase a n d of folates. Radioactivity metabolites of a single injection of [3H]PteGlu in various subcellular fractions were examined for total uptake, polyglutamate chain length, and state of reduction. Materials and M e t h o d s Animals. Male Sprague-Dawley rats (Simonson Lab., Grimy, Calif.)were housed singly in stainless steel screen-bottom cages in a constant temperature room at 20°C with a 14 h light cycle. Adult rats weighing 250--300 g were fed water and Purina Rat Chow a d libitum f o r 2 weeks. Rats were then injected intraperitoneally with 20/~Ci [3H]PteGlu which had been purified with QAESephadex A-25 [9]. The rats were fasted until killing by decapitation at 1, 2, 5, 10, or 24 h after injection. Fractionation o f rat liver. The procedure for rat liver fractionation was based on that of de Duve et al. [10] and Appelmans et al. [11]. Rats were killed by decapitation and each liver was perfused through t h e portal vein and inferior vena cava with a b o u t 30 ml ice-cold saline. Livers were removed and immersed in three volumes of ice-cold extraction medium (0.25 M sucrose, 1 mM disodium ethylenediaminetetraacetate, pH 7.4). The tissues were cut and homogenized three times for 15 s each, with interruptions to avoid heating. A known volume of each fraction was boiled for 7 min to destroy liver glutamyl carboxypeptidase and to release folate from proteins, and was used for microbiological assay and Sephadex column chromatography. The remainder of each fraction was assayed for protein, marker enzymes, and glutamyl carboxypeptidase. In experiments on the extent of folate reduction, the extraction and centrifugation medium used was 0.25 M sucrose, 0.15% ascorbate, adjusted to pH 7.0 with NaOH. The extent of reduction of the metabolized [3H]PteGlu was determined by injecting four rats with [3H]PteGlu, killing after 2 h, and fractionating a pool of the homogenates. Each fraction was brought to pH 4.7 with 10% ascorbic acid, autoclaved 10 rain, treated with hog kidneY glutamyl carboxypeptidase, and frozen overnight. Each fraction was thawed, adjusted to pH 6.0 with NaOH and chromatographed on DEAE-cellulose columns [12] along with 10-CHOPteGlu, H4PteGlu, and PteGlu standards.

796

Assay of: marker enzymes. Cytochrome a3 oxidase, a m i ~ c h o n d r i a l marker [10], was assayed by the optical test of Cooperstein and Lazarow [13]. Horse heart cytochrome c was obtained from Sigma. Glucose-6-phosphatase, the microsomal marker [10], was assayed according to Harper [14], using the method of Fiske and SubbaRow [15] for inorganic phosphate. Acid phosphatase, a lysosomal marker, was assayed by the procedure of Appelmans et al. [1~] and Hiibscher and West [16]. Column chromatography of folic acid derivatives. The boiled portion of each fraction was ~centrifuged ,~ a n d the supernatant applied t o the column, the isalt concentration of the sample was adjusted approximately to that of the eluant buffer (0~1 M potassium phosphate buffer, pH 7.0, with 0.2 M 2-mercapto. ethanol). The separation of folate derivatives by Sephadex G-25 c o l u m n chromatography was described~ in a previous publication [17]. DEAE-ceUulose column chromatography was according to Thenen a n d Stokstad [ 12 ]. Microbiological assay. The microbiological assay procedure with three organisms, Lactobacillus casei (ATCC 7469), Streptococcus faecalis (8043) and Pediococcus cerevisiae (8081) was based on the method by Waters and Mollin [18], as modified by Tamura et al. [19]. Glutamyl carboxypeptidase was pre, pared from hog kidney [20], purified over Sephadex G-25, and handled according to Bird et al. [21]. Assay o f glutamyl carboxypeptidase. E n z y m e activity in each subcellular fraction was determined by its ability to hydrolyze synthetic PteGlu-[~4C]Glu Glu .to PteGlu [22]. The assay System contained 0.2 ml pteroyltri[14C]gluta mate (approx. 1000 cpm equivalent to approx. 50 ng), 0.4 ml 0 . 1 M~sodium acatate buffer, pH 4.7, 0.4 rnl 1% sodium ascorbate, pH 4.7, and 0:2 ml of t h e re' spective fraction of rat liver. After 1 h incubation at 37°C, 0.1 ml of 50% trichloroacetic acid and 0.1 ml 5% charcoal solution (in 0.1 M sodium acetate buffer~ pH 4.7) were added to remove unreacted PteGlu-[ ~4C]Glu-Glu from t h e medium. The medium wasfiltered through a Millipore filter and the filtrate was counted irt a Beckman: CPM-100 liquid scintillation counter using Aquasol counting solution (from New England Nuclear). Preparation of folate standard compounds. Pteroyltri[~4C]glutamate was synthesized by the s o l i d phase synthesis technique of Krumdieck a n d coworkers [22,23]. NS-Methyl-[3H]tetrahydropteroylpolyglutamate (5-CH3H4PteGlun) was obtained by the incubation of L. casei with [3H]PteGlu L23]. Tetrahydrofolic ~acid (H4PteGlu) was prepared by the m e t h o d o f Davis [~4]. Synthesis o f 10-CHO.PteGlu was according to Beavon and Blair [25]: [3H]PteGlu labeled in the 9, 3' a n d 5' positions (specific activity: 20 Ci/mmol) was from Amersham-Searle Corp, Determination of protein. Protein was measured by the method o f Lowry et al. [26] with crystalline bt~vine serum albumin (from Sigma) as standard. Results T h e remits of the fractionz¢ion a n d t h e distribution o f the marker e n z y m e s in each'fraction are shown in~rrable L T h e marker enzymes~tppeared predominantly in the corresponding fraction, indicating, that tl~e fractions obtained.

797 TABLE I RELATIVE SPECIFIC ACTIVITIES OF SUacELLULAR

FRACTION' MARKER ENZYMES

Fraction

Cytochrome oxidase

Acid phesphatace

Glueose-6-phosphatase

Nucleus M/toehondria Lysosome Microsome Soluble fraction

0,23 3.05 1.20 0.20 0.18

0.20 0.77 3.94 1.04 0.63

0.41 0.25 1.01 2.85 0.62

-

from this procedure were sufficiently pure for this study. Theprotein content in each fraction is shown in Table II. The recovery of fractiorration was calculated by the comparison of the value before and after the fractionation. The protein recovery was approx. 85% and a similar value was observed f o r glutamyl, carboxypeptidase and the marker enzymes. The ,specific activity of glutamyl carboxypeptidase in each fraction is shown in Table II. The specific activity in the various fractions roughly follows t h a t Qf acid phosphatase (Tables I and II). ~The unusually high and low levels of prot e i n in the lysosomal and mitochondrial fractions, respectively, indicate poor separation of these fractions . . . . . . The results o f the microbiological assay of folate derivatives in boiled, portions o f each fraction are shown in Table III. Folates are concentrated mainly in the soluble fraction, and the specific folate activiW with respect to protein is also higher, here than in other fractions. -: The uptake of [SH]PteGlu by the various fractions 1, 5, 10 a n d 2 4 . h after the injection i s shown in Table IV. Over 55% of the radioactivity in the liver .1 h after injection was found i n the,microsomal fraction. Nuclear, mito-; chondrial, and lysosomal fractions contained very little radioactivity at this time period; however, the proportion gradually 'increased witth time as t h e radioactivity in the microsomal fraction decreased. The specific radioactivity of, folic acid~ (cpm/ng folate) therefore increased with time in all f~actions except the micr0somal fraction (Table IV). The specific activity i n n u c l e i , mitochondrial, and lysosomal fractions reached approximately the same value 24 h after t h e injection, while the values for: the soluble and microsomal fractions are about half as much as for the other fractions.

T A B L E II GLUTAMYL CARBOXYPEPTIDASE

A C T I V I T Y IN R A T L I V E R F R A C T I O N S ~

R e s u l t s a r e e x p r e s s e d a s t h e m e a n Of five a n i m a l s (+ S . D . ) •

Fraction

P r o t e i n ( m g / g l/vet)

A c t i v i t y (%) ¢.o~m~""-"

Nucleus ; MitoehOndria • Lysoson~e Microsome Soluble fraction

60.4 10.2 27.6 56.7

± ± + +

6.9 2.0 5.9 4.4 89:2 ± 8.9

0.4 1.2, 4.6 0.43 0.6

(%)

i

Speelfle aetlvity ( c p m X 10 2 ~ m s protein)

Total activity (~)

2.25 6.16 22.66 2.63 3.45

9,95 5.03 52.62 9.84 22.56

± + ~ + +

0.20 0.65 1.24 0.27 0.31

'

798 TABLE nl FOLATE CONTENTS IN RAT LIVER FRACTIONS DETERMINED BY MICROBIOLOGICAL ASSAY W I T H L. CASEI A F T E R T R E A T M E N T W I T H H O G K I D N E Y G L U T A M Y L C A R B O X Y P E P T I D A S E ........ / Fraction

Percent o f t o t a l folate activity per g liver *

ng folic acid per m g p r o t e i n * *

Nucleus Mitochondria Lysosome Micxosome S o l u b l e fraction

14.3 10.0 10.0 3.3 62.4

30 78 54 22 102

* Average o f t e n animals. ** Average o f five an/mals.

To investigate the folate oligc~lutamate length distributionsin the fractions, liver fractions 2, 5, and 24 h after the injection of [3H]PteGlu were prepared and chromato~phed on Sephadex G,25 columns. Fig. 1 shows an example of the radioactive and microbiological assay elution patterns of folatesusing the soluble fraction. The radioactivityin all fractions at the end of the 2 h period contained predominantly monoglutamate; however, monoglutamate derivatives represented only about 20% of total radioactivity at the end of the 5 h period in nuclear and mitochondrial fractions.On the other hand, endogenous folates, which are assumed to show a similar pattern regardless of time interval after the injection, are predominantly polyglutamates. The concentration of endogenous monoglutamate in microsomal and soluble fractions was higher than in the other fractions. Endogenous polyglu~ate eluted somewhat earlierthan the radioactive polyglutamates at all time periods for all subcellular fractions, indicating that the metabolized [3H]PteGlu did not closely reflect the pattern of endogenous forms even at 24 h after the injection. An aliquot of a pool from the Sephadex G-25 2 h monoglutamate peak from each subceUular fraction was rechromatographed on a DEAE-ceUulose column. The resulting pattern for the microsomal fraction demonstrated that most of the monoglutamate derivatives (about 80%) consisted of unchanged [3H]PteGlu, in contrast to the results with other subcellular fractions. Only in the soluble fraction were the radioactive and the endogenous elution patterns (of

TABLE IV THE PERCENTAGE OF TOTAL RADIOACTIVITY A T E S IN R A T L I V E R F R A C T I O N S Fraction

Nucleus Mitoehondria Lyaosome Mlerosome Soluble fraction

AND THE SPECIFIC RADIOACTIVITY

OF FOL-

Specific activity ( c p m / n 8 folate *)

Percent o f t o t a l radioactivity lh

5h

10h

24h

lh

5h

10h

24h

9.9 0.8 5.4 55.4 28.4

11.3 3.6 7,8 29.1 47.8

17.1 4.0 8.5 8.5 61.8

19.0 7.9 17.6 3.0 52.5

3.3 1.3 4.3 75.3 3.0

9.0 3.3 3.5 21.8 7.0

11.0 5.8 5.5 10.3 15.5

42.5 48.2 50.8 22.5 23.5

* A u a y e d w i t h L. casei a f t e r t l ~ a t m e n t w i t h h o g kidney g l u t a m y l c a r b o x y p e p t i d a s e .

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