15// 454 BIOCHEMICAL SOCIETY TRANSACTIONS

Ammonia stimulation of malate dehydrogenase from Bacilllls slearolhermophillls: generation of an apparent aspartate dehydrogenase activity

C. N. G. SCHMIDT* and L. JER VISt Attempts to purify aspartate dehydrogenase by salt fraction• • Departmel/t 0/ Botal/Y. Rothamsted Experimel/tal Statiol/. ation. chromatography on Cibacron Blue 3GA-Sepharose or \ Harpel/del/. Herts. A L5 2JQ. U.K .. al/d t Departmel/t 0/ Procion Red HE-3B-Scpharose. affinity chromatography on Biology. Paisley College o/Tee/mology. High Street. Paisley 5'-AMP-Sepharose and gel filtration through Sephacryl S-300 PA I 2BE. Re/l(rewshire. Seollal/d. U.K. always resulted in co-purification of malate dehydrogenase. ' Purification of malate dehydrogenase to near-Ifomogeneity by During investigations into the enzymology of assimi• the procedure of Wright & Sundaram (1979) yielded a lation by Baeil/lls stearolhermophillls strain PH24 (Buswell & that was more than 90% pure but still appeared to contain Twomey. 1975). we detected high amounts of an apparent aspartate dehydrogenase in that the addition of ammonia to the aspartate dehydrogenase activity. The appeared to be malate dehydrogenase assay system caused a 2-fold increase in capable of catalysing the reductive ami nation of oxaloacetic acid the rate of NADH oxidation. Attempts to stain polyacrylamide with NA DH as . but could not catalyse the oxidative gels for aspartate dehydrogenase activity by using I.-aspartate. deamination of I.-. At low concentrations of NI~4C1 NAD+., Nitro Blue Tetrazolium and phenazonium metho• as the sole nitrogen source in the culture medium. the most sulphate were unsuccessful. active amino acid dehydrogenase activity appeared to be Paper chromatography of the aspartate dehydrogenase aspartate dehydrogenase (specific activity 0.194 unit/mg of reaction products revealed no trace of I.-aspartate. but malate protein). Both N AD+ -dependent was present. It appears. therefore. that although ammonia (0.03 unit/mg) and alanine dehydrogenase (0.04 unit/mg) were stimulates the rate of NADH oxidation. the ammonia is not also present. as were glutamine synthetase (2.60 units/mg) and incorporated into an amino acid product but is merely glutamate synthase (0.02 unit/mg). Whereas the activity of stimulating the malate dehydrogenase activity. This conclusion alanine dehydrogenase rises considerably with I.-alanine as sole was confirmed by the finding that Mg2+ can also bring about a nitrogen source (2.80 units/mg). indicating a catabolic role for 2-fold increase in the rate of the malate dehydrogenase catalysed this enzyme. the activity of aspartate dehydrogenase rose only reaction. Other ions.' notably MnH and CaH inhibited the slightly when I.-aspartate was the sole nitrogen source malate dehydrogenase activity. (0.27unit/mg). and cell growth was poor. These results Other arc known to catalyse an aspartate dehydro• suggested that aspartate dehydrogenase could be involved in genase reaction. particularly GDH (Fahien & Smith. 1974: ammonia assimilation in B. slearolhermophillls PH24. Conse• Fah'ien et al .. 1977). but only in the direction of oxidative quently. we further investigated this enzyme activity in order to deamination. Malate dehydrogenase from several animal determine its physical and kinetic characteristics. sources has been reported to catalyse the oxidative deamination Aspartate dehydrogenase has been reported previously. of aspartate (Shaw & Koen. 1964). but only at 3% of the malate notably from A I/abael/a eylil/driea (Batt & Brown. 1974). The dchydrogenase activity. The reverse reaction was not alTected by activity is usually assayed by malate dehydrogenase-assay ammonia. systems supplemented with ammonia. the reaction being lri view of the rcsults presented here. it is clear. that. in some monitored by measuring NADH oxidation. Malate dehydro• species. malate dehydrogenase can be considerably stimulated genase activity is subtracted from the total activity to yield the by ammonia in the direction of oxaloacetate reduction. to aspartate dehydrogenase activity. This procedure is similar to generate an apparcnt aspartate dehydrogenase activity. Assays that used for alanine dehydrogenase (Epstein & Grossowicz. for the latter enzyme require more rigorous controls than simply 1976). By using the procedure we found that. on addition of subtracting malate dehydrogenase activity from the total rate of NH.CI (100 mM) to an assay system containing oxaloacetate NADH oxidation. (IOO,uM) and NADH (60 pM). the rate of NADH oxidation was stimulated 2-4-fold. When NADH was replaced by NADPH. C. N. G. S. thanks the Science Research Council ror a Research which can also be used by B. stearothermophillls malate Studentship. dehydrogenase. addition of ammonia caused no increase in the rate of NADPH oxidation. Attel!'pts to assay the reverse reaction. i.e. the oxidative deami,iation of I.-aspartate. were unsuccessful. Thus cell extracts appeared to contain an Batt. T. & Brown. D. II. (1974) Plall/a 116.27-37 Buswell. J. A. & Twomey. D. G. (1975).1. Gell. Microhiol. 87. 377-379 irreversible NA DH-dependent aspartate dehydrogenase. Epstein. I. & Grossowicz. N. (1976) Rioehi",. Biophys. Ac/a 445. When cells were grown under dilTerent conditions. activities of 549-557 . aspartate dehydrogenase and malate dehydrogenase were found Fahien. L. A. & Smith. S. E. (1974).1. Bioi. Che",. 249. 2696-2703 to alter in parallel. As the former enzyme utiliz.es oxaloacetate Fahien. L. A .• Hsu. S. L. & Kmiotek. E. (1977) .I. Riol. Che",. 252. whereas the latter produces it. this link between the activities of 1250-1256 . the two enzymes was considered consistent with a role for Shaw. C. R. & Koen. A. L. (1964) Biochi",. Biophys. Acta 92. aspartate dehydrogenase in ammonia assimilation. However. the 397-400 Wright. I. P. & Sundaram. T. K. (1979) Bioche", . .I. 177.441-448 apparent K m for ammonia (33 mM) was not.

Kinetics of ornithine carbamoyltransfcrase oQ

TOM R. C. BOYDE and MOHAMMED RAIIMATULLAII wide dilTerences. however. in the conclusions of various workers Departmel/t of Biochemistry. UI/iversity of lIollg KOl/g. as to the best conditions for assay: bulTer. concen• Hong Kong. trations and optimum pH (Snodgrass & Parry. 1969: Ceriotti. 1973: Vasser. 1978) .. Assay of ornithine carbamoyl in serum is important Fig. I shows that the apparent optimum pH for ornithine because a rise in the activity of the enzyme is a specific and carbamoyltransferase falls as the ornithine concentration is' sensitive indicator of liver damage (Reichard. 1962). There are increased (curves e to c). This explains much of the previous

1981 s 1 595th MEETING, MANCHESTER 455

10 disagreement. but the results obtained by Snodgrass & Parry a (1969) cannot be fully explained on this basis and may be attributable to their use of a maleate buffer at lower pH values. The apparent affinity of ornithine carbamoyltransferase for r ornithine rises with pH (K m 455. 286. ] 50. ] 17 and 103,11M at pH6.9. 7.3. 77. 8.1 and 8.5 respectively). Recalculation in n terms of ornithine zwitterion. the proportion of which rises with o 8 pH. gives Km values of 7.3. 11.0. 13.5. 28 and 40,llM respectively. i.e. a fall of affinity with rising pH. Curves a and b y :t (Fig. I) show that at constant concentration of ornithine n zwitterion the dependence of observed rate on pH approximates e that of Vmo •.• These observations may be held to reinforce the suggestion that the true substrate is ornithine zwitterion n b 64 d (Snodgrass & Parry. 1969: Marshall & Cohen. 1972). and e '--'"0 I ~~u '22-~ I indicate that assays should be at a pH close to 6.9 with a .~<::::ue :~ sufficiently high ornithine concentration and with avoidance of )- E inhibitory buffers ..

;e Ornithine inhibition occurs also. at high pH. and responds reciprocally to carbamoyl phosphate concentration. i.e. is .e a alleviated by carbamoyl phosphate. Our findings hold only for }t saturating concentrations of carbamoyl phosphate. Another problem with kinetic studies of ornithine carbamoyl• y n transferase has been non-linearity of double-reciprocal plots. a leading to the suggestion· of different K m values for the three d subunits (Marshall & Cohen. 1972) and making interpretation Ie of such effects as those outlined above extremely difficult. We have found. however. that plots are linear from 0.05 mM- to )• 7.5 mM-ornithine at pH 6.9. provided that full allowance or t: correction is made for ornithine and citrulline present in serum and for several non-specific reactions of carbamoyl phosphate. Ie e al 2 0.1 mM , Hoogenraad et al. (1980) found linear plots with ornithine •n carbamoyltransferase purified by affinity chromatography . We have used triethanolamine as buffer because the others te tested were either inhibitory or gave high background absor• 'y bance (Tris. imidazole. Veronal and glycylglycine). \ Ie :d to o This work forms part of the thesis to be submitted by M. R. to the {s 6.9 7.3 7.7 8.1 8.5 University of Hong Kong in partial fulfilment of the Ph.D. degree Iy pH requirements. M. R. thanks the Hong Kong Government for award of a of Fig. I. Variatioll of serllm omit hilie carbamoyl trails/erase Commonwealth Scholarship. activity with pH.

(a) V mIX. from double-reciprocal plots. (b) Observed activity at ;h constant ornithine zwitterion concentration of 100,11M. (c) to (e) Observed activity at constant total ornithine concentration as Boyde. T. R. C. & Rahmatullah. M. (1980) Allal. Biochelll. 107. 424-431 shown. Carbamoyl phosphate concentration was 5 mM Ceriotti. G. (1973) Clill. Chilli. Acta 47. 97-105 throughout, with 0.27 M-triethanolamine-HCI buffer. at 37°C. Hoogenraad. N. J .• Sutherland. T. M. & Howlett. G. J. (1980) Allal. Citrulline produced was assayed by the method of Boyde & Biochelll. 101.97-102 79 Rahmatullah (1980). Serum pools were prepared from indivi• Marshall. M. & Cohen. P. P. (1972)J. BioI. Chem. 247. 1654-1668 15. duals having 2-16 units. (pmol of citrulline formed/min) per Reichard. H. (1962) Acta Med. SCat/d. 172.723-738 litre. One pool was used for curve a. and a different pool for Snodgrass. P. J. & Parry. D. J. (1969) J. Lab. Clill. Med. 73.940-950 curves b to e. Vassef. A. A. (1978) Clill. Chem. 24. 101-107

12.

An efficient new method for the separation, from small volumes of human blood, of leucocyte samples particularly suitable for use in diagnostic ftuorogenic enzyme assays

K. FATIMA ZAIDI and DAVID H. LEA BACK The resultant mixture is admitted (avoiding air bubbles) to a ] ml Departmellt of Biochemistry. Illstitllte of Orthopaedics. Royal Luer-tipped syringe (S, Fig. ]a) and the latter attached to the :rs Natiollal Orthopaedic Hospital. Stallmore. Middx. HA 7 4LP. lower end (L) of 40cm of transparent silicone-rubber tubing :n• U.K. (2mm bore. 1mm wall; Esco Rubber Co.) supported at its upper :ti. end (U). The cell suspension is displaced upwards and the tubing In a typical example of the new procedure. a specimen of fresh clamped off at its lower end (C " Fig. ] b). After 45 min ,the ne human venous blood (500,111)is added with immediate mixing to erythrocytes sediment to a lower red zone separated by a is a polystyrene tube containing 200,111 of Plasma Gel solution clearly-defined boundary (B. Fig. Ie). Two further clamps (C2 us (Bailon. Neuilly-sur-seine. France) and lithium heparin (34 i.u.). and C), Fig. Id) are added and the tubing cut at the boundary

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