Studies on N5-Methyltetrahydrofolate-Homocysteine Methyltransferase in Normal and Leukemic Leukocytes RENATE PEYTREMANN, JANET THORNDiKE, and WInI.AM S. BECK From the Department of Medicine, Harvard Medical School, and the Hematology Research Laboratory of the Massachusetts General Hospital, Boston, Massachusetts 02114 system A B S T R A C T A cobalamin-dependent N5-methyltetra- reducing CH3-FH4 + L-homocysteine hydrofolate-homocysteine methyltransferase (methyl- cobalamin,rcaing SAMsAm transferase) was demonstrated in unfractionated ex- FH4 + methionine. (1) tracts of human normal and leukemic leukocytes. Ac- Analogous enzymes have been characterized in Esche- tivity was substantially reduced in the absence of an richia coli added cobalamin derivative. Presumably, this residual (2-4) and in various cells of animal origin activity reflects the level (5-15). Some evidence suggests that a major function endogeneous of holoenzyme. of the enzyme is the maintenance of Enzyme activity was notably higher in lymphoid cells methionine levels in tissues. According to other evidence, the pathway has an than in myeloid cells. Thus, mean specific activities essential role in producing FH4, which can enter into (+SD) were: chronic lymphocytic leukemia lympho- essential transfers of "one-carbon" units in various normal cytes, 2.15+1.16; lymphocytes, 0.91+0.59; nor- other pathways, e.g., the folate-dependent synthesis of mal mature granulocytes, chronic myelo- 0.15+0.10; thymidylate (16). Doubtless, both views are correct, cytic leukemia granulocytes, barely detectable activity. the relative importance of the two Properties functions differing of leukocyte enzymes resembled those of perhaps under different conditions. methyltransferases previously studied in bacteria and Despite extensive discussion of an hypothesis that other animal cells. Granulocytes and chronic myelocytic gives this pathway critical importance in the impair- leukemia cells contain a factor or factors that inhibits ment of DNA synthesis associated with the megalo- Escherichia coli enzyme. The data suggest that the blastosis of vitamin Bi2 deficiency (reviewed in refer- prominence of this cobalamin-dependent enzyme in ence 17), little is known of the distribution and char- lymphocytes and other mononuclear cell types may be acteristics of methyltransferase in human blood cells. related to their potential for cell division. This lack has motivated the present investigation. Human leukocytes were chosen for study since they INTRODUCTION include cells that are grossly altered morphologically by vitamin B12 deficiency (granulocytes) and cells that Many studies have shown that the final step in the co- are not evidence of methionine in (lymphocytes); certainly though, is balamin-dependent pathway synthesis available to suggest that vitamin B12 has ef- bacteria and in animal cells is catalyzed by N5-methyl- deficiency tetrahydrofolate-homocysteine fects on the immunologic function (18, 19) and chromo- methyltransferase (meth- somal patterns (20, of lymphocytes. according to equation 1 (reference 21) yltransferase)1 1): In the present study, methyltransferase activity was Dr. Peytremann's present address, Division of Onco- assayed in crude extracts of isolated granulocytes and Hematology, Department of Medicine, H6pital Cantonal, lymphocytes, and kinetic properties of the enzymes in Geneva, Switzerland. these extracts were characterized. The data show that Received for publication 3 September 1974 and in revised lymphocytes contain higher enzyme levels than granulo- form 14 July 1975. 1Abbreviations used in this paper: ALL, acute lympho- cytes, that lymphocytes from subjects with chronic lym- cytic leukemia; CH3-FH4, N5-methyltetrahydrofolate; CLL, chronic lymphocytic leukemia; CML, chronic myelocytic ML, myelomonocytic leukemia; PBS, potassium phosphate leukemia; FH4, tetrahydrofolate; methyltransferase, N5- buffer, 10 mM, pH 7.4, in 0.9% NaCl; PV, polycythemia methyltetrahydrofolate-homocysteine methyltransferase; M- vera; SAM, S-adenosylmethionine. The Journal of Clinical Investigation Volume 56 November 1975-1293-1301 1293 phocytic leukemia (CLL) had even higher enzyme suspensions contained 0-2% granulocytes in CLL blood levels than normal lymphocytes, and that granulocytes and 8-12% in blood of normal donors. Granulocyte prepara- contain one or more factors that inhibit methyltrans- tions contained less than 5% lymphocytes. Lymphocytes and granulocytes were then suspended in ferase activity. 3-8 vol of 20 mM PBS, pH 7.4 with 1 mM fi-mercapto- ethanol and disrupted for 60 s in a cooled 18-20 kcycle METHODS MSE ultrasonic disintegrator (Measuring & Scientific Reagents. Chemicals were obtained from the following Equipment Ltd., London). Phase microscopy showed essen- sources: L-homocysteine thiolactone hydrochloride, S-aden- tially complete disruption. Sonic extracts were centrifuged osylmethionine chloride, folic acid, soybean and beef pan- at 27,000 g for 30 min, and supernatant fractions were as- creas trypsin inhibitors, and bovine pancreas trypsin from sayed for methyltransferase. Experiments showed that all Sigma Chemical Co., St. Louis, Mo., casein (Hammarsten activity was recoverable with these fractions. quality) from Nutritional Biochemicals Corp., Cleveland, Leukocytes from the blood of CLL patients with > 80% Ohio; cyanocobalaminandhydroxycobalamin from Schwarz- lymphocytes or other leukemias were routinely isolated by Mann Div., Becton, Dickinson & Co., Orangeburg, N. Y.; dextran sedimentation (28) with omission of the Hypaque- deoxyadenosylcobalamin from Calbiochem, San Diego, Calif. Ficoll gradient. Separate experiments clearly indicated that Samples of methylcobalamin were kindly provided by Dr. similar enzyme levels were obtained when the gradient was Bernard M. Babior, New England Medical Center, Boston, not omitted. Platelet-rich supernatant fractions were again and Dr. Robert H. Abeles, Brandeis University, Waltham. discarded, erythrocytes were lysed osmotically, and cells Propylcobalamin was synthesized by reduction of cyanoco- were washed twice with PBS. Cell extracts were prepared balamin with sodium borohydride, followed by alkylation as described above. Supernatant fractions were assayed for with propyl bromide. The product was isolated by thin- methyltransferase after storage for 18 h or less at 4°C layer chromatography on silica gel, using propanol: water: or for longer periods at -50'C. Enzyme activity decreased ammonium hydroxide (100: 99: 1) as solvent system (22). less than 10% after storage up to 40 days at - 500C. When Nonradioactive d,l-CH3FH,, synthesized according to Blair indicated, cell extracts were dialysed for 18 h against 100- and Saunders (23), was purified by DEAE-cellulose chro- 200 vol of 20 mM PBS, pH 7.4, with 1 mM a-mercapto- matography according to Gupta and Huennekens (24). N5- ethanol. [1"C CH3-FH4 was purchased from Amersham/Searle Corp., Preparation of E. coli extracts. E. coli B was grown in Arlington Heights, Ill. Both products were chromatographi- minimal essential medium (31) except in experiments in cally homogeneous and stable when stored under N2 at which it was grown in minimal medium containing added -20'C for 1 yr. 0.01 AM cyanocobalamin. Cells were harvested in the loga- L-Homocysteine thiolactone hydrochloride was converted rithmic phase of growth. Cells were suspended in 3 vol of to L-homocysteine by exposure to 0.3 M NaOH (7-10 min), Tris-chloride buffer, 50 mM, pH 7.4, containing 1 mM j3- followed by neutralization with 0.3 M KH2PO4 (25). mercaptoethanol and disrupted for 3 min in a cooled 18-20 Preparation of lymphocytes and granulocytes. Venous kcycle MSE ultrasonic disintegrator. Cell debris was re- blood (20-100 ml) was drawn from ostensibly normal moved by centrifugation for 30 min at 27,000 g at 2°C. donors (hospital personnel and patients without blood Aliquots of supernatant fractions (termed E. coli extract) dyscrasias other than mild leukocytosis) and patients with were stored at - 50'C for methyltransferase assay. At this CLL and other hematologic disorders that were untreated temperature activity remained stable for several months. except as noted. Cell morphology in CLL patients studied With each batch of enzyme assays in leukocyte materials, was primarily that of typical small lymphocytes, without methyltransferase was assayed simultaneously in a control nucleoli, nuclear clefting, or pleomorphism. Normal sub- E. coli extract. jects and CLL patients were not matched for age, the mean Methyltransferase assay and other assays. Methyltrans- ages of the two groups being 26±4 and 63±+13 yr. Serum ferase was measured as the amount of ["C]methionine prepared from an aliquot of blood was stored at -100C formed from N6- ["C]CHa-FH, according to Weissbach et for assay of serum folate and vitamin B12 levels by methods al. (3), with minor modifications (7). The standard assay employing Lactobacillus casei (26) and Lactobacillus leich- system contained in a total volume of 0.5 ml: N- [14C]- inannii (27). Normal ranges in this laboratory for serum CH3-FH4, 250 ,M (sp act, 400 cpm/nmol); SAM, 100 folate are 6-16 ng/ml and for serum vitamin B12 are ,uM; L-homocysteine freshly prepared from the tliiolactone 150-450 pg/ml. Heparin (Liquaemin sodium, The Upjohn hydrochloride, 1.25 mM; cyanocobalamin, 100 jiM; ,B-mer- Company, Kalamazoo, Mich.) was used as anticoagulant captoethanol, 200 mM; PBS, 50 mM, pH 7.4; and 0.5-1.0 for blood samples (10-20 U/ml). mg of leukocyte protein or 0.2-0.3 mg of E. coli protein.