Metal Ion-Dependence of a-Mannosidase-1 from Dictyostelium discoideum DAVID C. KILPATRICK Department of Microbiology, Queen Elizabeth College, London W8 7AH, U.K.

The cellular slime mould Dictyostelium discoideum has many advantages as a model system for studying the biochemistry of eukaryotic development and differentiation (Loomis, 1975). One approach has been to measure variations in enzyme activities as cells undergo synchronous development. Loomis (1970) first reported that the specific activity of a-mannosidase increased manyfold during development, reaching a maximum during terminal differentiation. A minor isoenzymic form (a-mannosidase-2) has now been discovered (Free & Loomis, 1974; Free et al., 1976) and the major form has been renamed a-mannosidase-1 . The purification and properties of a-mannosidase-1 have been described by Every & Ashworth (1973), and it is curious that the pH-activity profileobtained by themdifferedmarkedlyfrom that published by Loomis (1970). a-Mannosidases from human liver (Phillips et al., 1974) and human kidney (Marinkovic & Marinkovic, 1976) are sensitive to various metal ions, and since the inconsistency in slime-mould pH-activity profiles might be due to traces of metal ions contaminating the buffers used, it was decided to investigate the effect of metal ions on this developmentally important enzyme more fully. D. discoideum wild-type NC-4 was grown on Sussman’s (1966) nutrient agar plates in association with Escherichia coli B/r, and allowed to develop until most of the cells were present as ‘fingers’ on the agar surface. The cells were homogenized by sonication, and cell debris removed by centrifugation at 9000g for l0min. Centrifugation would also remove most of the a-mannosidase-2, which is membrane-bound (Free etal., 1976). The enzyme was assayed as a routine with 4-methylumbelliferyl-a-mannosideas substrate (Carroll et al., 1972), but p-nitrophenyl-a-mannosidewas also used to verify that similar results were obtained. The soluble homogenate was assayed for a-mannosidase over a pH range in the presence of either ZnS0, or EDTA (disodium salt). ZnS04 activated the enzyme at pH values below 4.5, whereas EDTA (disodium salt) had an inhibitory effect. Neither had much effect above pH4.5. ZnS0, could overcome the EDTAmediated inhibition, and it is likely that EDTA acted by chelating zinc and/or other metal ions already present in the enzyme preparation. One possible explanation for the pH-dependence of the zinc activation is that a-mannosidase-1 consists of two forms with overlapping pH-activity profiles, of which the form with the lower pH optimum alone is activated by zinc. To investigate this possibility, the soluble homogenate of D. discoideum was fractionated on DEAE-cellulose and a-mannosidase was assayed both at pH2.75 in the presence of ZnSO, (1 mM) and at pH4.5 in the presence of EDTA (1 mM). Two peaks of a-mannosidase were obtained (Fig. 1) under both conditions of assay. I have called these two peaks a-mannosidase-1A and a-mannosidase-lB, the former being the first to be eluted. a-Mannosidases-1 A and -1 B had very similar characteristics, including the same affinity for synthetic substrates. At pH4.5 the K , for 4-methylumbelliferyl-a-mannoside was 1.O, and that for p-nitrophenyl-a-mannosidewas 2.0. Both had similar pH-activity profiles (Fig. 2), with a pH optimum of 4-4.5, which could be shifted towards pH4.0 in the presence of zinc or towards pH4.5 in the presence of EDTA. The effect of several metal sulphates (1 mM) was examined at both pH2.75 and 4.5, and the pattern of inhibition was similar for the two forms. At pH2.75, both forms had only 60% of the control activity in the presence of cobalt, 50 % in the presence of cadmium, and 10 % in the presence of copper. At pH4.5, around 95 % of the control activity was measured in the presence of all three. Manganese brought about an 8% inhibition at both pH2.75 and 4.5. Na2S04had little effect at either of the pH values used. Both forms were thermally inactivated in two stages. About 30% of the activity was lost within a few minutes when preincubated at 55°C (pH4.9, but the remaining activity

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Fraction number Fig. 1. DEAE-cellulose chromatographyof a-mannosiduse-1 A soluble homogenate of D. discoideum ‘fingers’ was applied to a column of DEAEcellulose equilibrated with l0mM-sodium acetate, pH5.1, containing 0.25~-NaCI. After elution with a linear NaCl gradient, the fractions were assayed for a-mannosidase by using 4-methylumbelliferyl-a-mannosideeither buffered at pH2.75 with ZnS04 (1 m ~added ) (o), or buffered at pH4.5 with EDTA (1 mM) added (0).

PH Fig. 2. pH-activityprofiles of a-mannosiduse-I A and -1B The dependence of activity on pH was determined by using glycine/HCl (pH2.0-2.5), citrate/phosphate (pH2.5-5.5) and sodium phosphate (pH 6.0-7.0) buffers for (a) amannosidase-1A and (6)a-mannosidase-1B. Measurements were made in the presence of ZnS04 ( 0 )or EDTA (0)and compared with controls (A) to which nothing had been added. 1976



decayed much more slowly. The relatively stable activity was rendered more labile by incubation with EDTA, but it was unaffected by zinc. If, however, enzyme samples were assayed at pH2.75 in the presence of Zn after preincubation at 55"C,no unstable activity was detected whether zinc had been present during preincubation or not. The similar characteristics of the two forms of a-mannosidase separated on DEAEcellulose suggests that they might be closely related and may differ as a result of posttranslational modification rather than from being products of different genes. Superficially at least, they appear to be related in the same way as the A and B forms of human a-mannosidase (Phillips et al., 1974). The thermal-stability experiments indicate that each form is itself heterogeneous, but the relationship between stability, pH-dependence and metal ion-dependencehas not been fully clarified. I thank Dr. B. G. Winchester for helpful discussion. I am also grateful to Dr. M. J. Bazin in whose laboratory this work was carried out. Carroll, M., Dance, N., Masson, P. K., Robinson, D. & Winchester, B. G. (1972)Biochem. Biophys. Res. Commun. 49,579-583 Every, D.& Ashworth, J. M. (1973)Biochem.J. 133,3747 Free, S.J. & Loomis, W. F. (1974)Biochimie 56,1525-1528 Free, S . J., Cockburn, A. & Loomis, W. F. (1976)Deu. Biol. 49,539-543 Loomis. W . F.(1970)J.Bacteriol. 103,375-381 Loomis, W. F. (1 975) Dictyostelium discoideurn : A Developmental System, Academic Press,

London and New York Marinkovic, D. V. & Marinkovic, J. N. (1976)Biochem.J. 155,217-223 Phillips, N.C.,Robinson, D. & Winchester, B. G. (1974)Clin. Chim. Acta 55,11-19 Sussman, M. (1966)Methods CellPhysiol. 2,397410

The Preparation of Coproporphyrin I11 and its Iron Complex from Rhohpseuhmonas spheroides HARIS HATZIKONSTANTINOU and STANLEY B. BROWN Department of Biochemistry, University of Lee&, 9 Hyde Terrace,

Leeds LS2 9LS, U.K. The biosynthesis of porphyrins by bacteria has been extensively studied in a number of systems including Corynebacterium diphtheriae (Hale et al., 1950) and particularly Rhodopseudomonas spheroides (Lascelles, 1955, 1956). Relatively large amounts of free porphyrin are formed when the latter organism is grown anaerobically in the light, on media deficient in iron salts (Lascelles, 1955). Further work by Lascelles (1956) showed that cell suspensions of Rhodopseudomonas spheroides were able to convert glycine and 2-oxoglutarate into porphyrins under similar conditions. Coproporphyrin I11 was the major component formed, along with traces of coproporphyrin I and uroporphyrins I and 111. Our aim in the present work was to develop the systems used by Lascelles to optimize the yield of coproporphyrin III and to devise a simple and rapid means of production of relatively large quantities of this material of the order of 1OOmg. Rhodopseudomonus spheroides (N.C.I.B. 8253) was obtained as a freeze-dried culture. A specially constructed apparatus (shown and described in Fig. 1) was used for photosynthetic anaerobic growth of cells and also for porphyrin production from resuspended cells. All culturesoriginated from singleall colonies grown aerobically on medium S of Lascelles (1956). Photosyntheticgrowthwasin twostagesof lOOmI and 1litre, thefinalstagerequiringabout12h until the end of exponential-phase growth. TWOmethods were used for porphyrin production from fully grown cell cultures. In method A the original procedure of Lascelles (1956) was followed. Cells were harvested by centrifugation, washed and resuspended in 1litre of medium I of Lascelles (1956). The suspension was placed in the light-irradiated apparatus as before. Coproporphyrin secretion into the medium was monitored by taking samples at regular intervals over the next 24h, after which time there was no

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Metal ion-dependence of alpha-mannosidase-1 from Dictyostelium discoideum.

565th MEETING, STIRLING 1083 Metal Ion-Dependence of a-Mannosidase-1 from Dictyostelium discoideum DAVID C. KILPATRICK Department of Microbiology, Q...
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