The stability of uridine diphosphoglucose pyrophosphorylase was examined in extracts prepared at different stages of development in Dictyostelium discoideum.
OF BACTERIOLOGY, Mar. 1968, p. 983-985 Copyright © 1968 American Society for Microbiology
JOURNAL
Vol. 95, No. 3 Printed in U.S.A.
Stability In Vitro of Uridine Diphosphoglucose Pyrophosphorylase in Dictyostelium discoideum BARBARA E. WRIGHT AND DONNA DAHLBERG Department of Developmental Biology, Institute of Biological and Medical Sciences, Retina Foundation, Boston, Massachusetts 02114
Received for publication 4 January 1967
The stability of uridine diphosphoglucose pyrophosphorylase was examined in extracts prepared at different stages of development in Dictyostelium discoideum. In the early stages, the kinetics of inactivation were nonlinear, and, therefore, it was not possible to determine the specific enzyme activity. In the later stages of development, the enzyme was stable, but it could be rapidly inactivated by a heat-labile inhibitor present in extracts prepared at an early stage.
Previous reports from this laboratory have in- 0.01 M phosphate buffer (pH 6.5) and 0.001 M ethylenedicated that, in extracts prepared at the early diaminetetraacetic acid, disodium salt (EDTA) (1). At stages of differentiation, uridine diphosphoglucose the desired stage of development, cells were harvested in 0.01 M tris(hydroxymethyl)aminomethane (Tris), (UDPG) pyrophosphorylase is unstable (3, 6). pH 8.5, frozen and thawed (or passed through a When prepared at the later stages of develop- French pressure cell at 30,000 psi), and centrifuged ment, however, the enzyme is stable. Such differ- for 5 min at 12,000 X g; the supernatant fluid was ential effects in vitro, as a function of the stage of assayed immediately for UDPG pyrophosphorylase morphogenesis, can be very misleading with activity in the direction of glucose-i-phosphate forrespect to extrapolations concerning enzyme mation from UDPG (2). Into a quartz microcell of levels in vivo. Differential enzyme inactivation in 1-cm light path was pipetted 0.575 ml of a solution of the cellular slime mold has now been found for 4 X 10-2 M Tris buffer (pH 8.5), 1.2 X 10-2 M MgC12, six enzymes: isocitric dehydrogenase, glucose-6- and 2 X 10-8 M cysteine (freshly neutralized). To this was added 10 ,uliters of 0.05 M NADP, 5 ,uliters of gluphosphate dehydrogenase (5), pyruvate kinase cose-6-phosphate (160 enzyme units/ (Cleland and Coe, Biochim. Biophys. Acta, in ml), 10 ,uliters ofdehydrogenase phosphoglucomutase (20 enzyme press), cell wall glycogen synthetase (7), cellulase units/ml), 10 ,uiters of 0.1 M UDPG, and 2 to 10 (Rosness, unpublished data), and UDPG pyro- ,Aliters of extract. The reaction was started by the adphosphorylase. This report deals with the dition of 10 ,diters of 0.1 M inorganic pyrophosphate, kinetics of the inactivation of UDPG pyro- and the increase in absorbancy at 340 m,u (A340) was phosphorylase caused by the presence of a heat- followed on a Gilford recorder (Gilford Laboratolabile inhibitor in the early stages of differentia- ries, Inc., Oberlin, Ohio). The increase in A340 was with time and enzyme concentration at 23 C. tion. Other enzymes studied in the cellular slime linear Protein was determined as previously described (9). mold are not subject to differential inactivation. One unit of enzyme catalyzes the reaction of 1 ,umole These enzymes include glutamic acid dehydro- of substrate per min. Specific activity is expressed as genase, alanine-a-ketoglutaric transaminase, lactic micromoles of reduced NADP (NADPH2) formed acid dehydrogenase, an alkaline phosphatase, and per minute per milligram of protein. glycogen synthetase (5, 6, 9). RESULTS MATERIALS AND METHODS The change in specific activity of UDPG pyroUridine-5'-triphosphate (UTP) and UDPG were phosphorylase was examined as a function of obtained from Sigma Chemical Co., St Louis, Mo. developmental stage. The extracts prepared were Glucose-6-phosphate dehydrogenase (from yeast), assayed at 23 C: (i) immediately and (ii) after phosphoglucomutase (from rabbit skeletal muscle), and nicotinamide adenine dinucleotide phosphate incubation at 35 C for 30 min. These two assay (NADP) were obtained from Calbiochem, Los procedures are compared in Table 1. It can be seen that the apparent increase in specific activity Angeles, Calif. Preparation of enzyme. Dictyostelium discoideum during differentiation was 4.5 times greater in the was grown on a rich medium in the presence of preincubated series. This observation is consistent Escherichia coli and transferred at the amoeba or with a greater instability of the enzyme when preaggregation stage to sheets of 2% agar containing pared from earlier as compared to later stages of 983
WRIGHT AND DAHLBERG
984
J. BACrERIOL.
TABLE 1. Enzyme specific activity as a function of developmental stage Stage
Imme-
diate
asaaya
Amoeba (4 hr) ................. 0.067 Pseudoplasmodium............. 0.39 Preculmination ................ 0.54 0.63 Late culmination............ ....
Assay after 30 min at 35 Cb
0.013 0.32 0.55 0.53
N
.\sa A Agg.( 12 hrs)
60 60-
Agg.
(to hrs)
Fold increase (amoeba-culmina-
tion) ........................ 9.4
42
a Enzyme was prepared in 0.01 M Tris (pH 8.5) containing 0.01 M UDPG. b Enzyme was prepared in 0.01 M Tris (pH 8.5) without UDPG. Cells were ruptured by freezing and thawing, and the supernatant fluid was assayed according to the procedure described in Materials and Methods. The reaction time was 5 min at 23 C.
development. UDPG has a slight stabilizing effect on the enzyme and was added to the samples that were assayed immediately; complete stabilization could not be achieved. Whether the enzyme was prepared by freezing the cells or rupturing them by passage through a French pressure cell at 30,000 psi, the overall results were the same. The stability of the enzyme was next examined at five successive stages, from amoeba to preculmination. The data in Fig. 1 clearly indicate a progressive and striking change in enzyme stability as differentiation progressed. Attempts were then made to obtain clear evidence for the existence of an inhibitor in the extracts prepared from cells in the earlier stages of development. A typical experiment is shown in Fig. 2. Again, extreme instability was observed for the enzyme prepared from amoebae, and complete stability for the enzyme present at preculmination. After inactivation of the amoeba enzyme (30 min at 35 C), a sample was mixed with the preculmination enzyme. The latter enzyme, which had previously shown complete stability at 35 C, now followed an inactivation curve similar to that of the enzyme from amoebae. Incubation of the amoebae extract at 100 C for 5 min destroyed the inhibitor. Figure 2 also demonstrates that a similar kinetics of enzyme inactivation occurs at 25 C. One curve is plotted on a logarithmic scale (see insert). DIscussION Since the kinetics of enzyme inactivation are nonlinear, even in the presence of UDPG, it is
i
(
Amoeba and 6,0 hrs )
O
10
20
3, v
20
0
30
MiI/nutes at 350 C FIG. 1. Differenitiation took place at 17 C, and thze enzymes were prepared and assayed according to the description in Materials and Methods. An immediate assay was performed, and the enzyme was then incutbated at 35 C. Samples were removed and assayed at thle stated time intervals. The reaction rates were determined over a 5-min interval at 23 C. The initial reaction rate was taken as 100% activity. Specific enzyme activities (micromoles per minute per milligram of protein) at the states indicated were: amoeba (3 hr), 0.056; amoeba (6 hr), 0.061; aggregation, 0.098; late aggregation, 0.170; preculmination, 0.610.
not possible to predict the specific enzyme activity in the early stages of differentiation. The extent of inactivation between the time of cell rupture and immediate assay can be neither determined nor estimated. Furthermore, had the first assay been performed at 10 min, it would have appeared as though inactivation was linear and as though extrapolation to zero-time was possible. Had it been possible to assay at 1-min intervals during the first 5 min, the inactivation curve may well have been more strikingly nonlinear. After the late aggregation or early pseudoplasmodium stage of development, specific enzyme determinations can be made in the relative absence of artifacts in vitro. The stage at which such artifacts play a minor role can vary from one group of cells to another, depending upon the stage of the cells at the time of harvest or the temperature at which differentiation occurs, or both. In any event, the significance of changes in enzyme specific activity to reaction rates in vivo
STABILITY OF UDPG PYROPHOSPHORYLASE
VOL. 95, 1968
985
It has recently been demonstrated that enhanced enzyme levels cannot be responsible for this increased rate of synthesis, in view of the substrateKm ratios involved and the accumulation patterns during differentiation of glucose-l-phosphate and UDPG (Wright, Simon, and Walsh, unpublished data). ACKNOWLEDGEMENT
This investigation was supported by Public Health Service grant GM15938-01 from the National Institute of General Medical Sciences.
Minutes ot 35 10
Mefinutes FIG. 2. Amoeba and preculmination enzymes were prepared as described in Materials and Methods after rupture of the cells by freezing. The enzymes were incubated at 35 C (or 25 C as indicated), and samples were removed periodically for assay. A sample of the amoeba extract which was incubated for 30 min at 35 C was then added in a I: I ratio to the preculmination extract. Samples comparable to that of preculmination alone were assayed during incubation of the mixture at 35 C. The initial enzyme activity (before incubation) was taken as 100%. The insert shows a logarithmic plot of the lowest curve (amoeba, 35 C).
cannot be predicted. In fact, reactions in vivo have been shown to increase in the absence of changes in enzyme specific activity, as well as to decrease in spite of increases in enzyme concentration (8). The rate of synthesis of UDPG increases
about threefold in vivo from the aggrega-
tion to the culmination stage of development (4).
LITERATURE CITED 1. LIDDEL, G. U., AND B. E. WRIGHT. 1961. The effect of glucose on respiration of the differentiating slime mold. Develop. Biol. 3:265-276. 2. MUNCH-PETERSEN, A. 1955. Investigations of the properties and mechanism of the uridine diphosphate glucose pyrophosphorylase reaction. Acta Chem. Scand. 9:1523-1535. 3. PANNBACKER, R. G. 1967. Uridine diphosphoglucose biosynthesis during differentiation in the cellular slime mold. II. In vitro measurements. Biochemistry 6:1287-1293. 4. PANNBACKER, R. G. 1967. Uridine diphosphoglucose biosynthesis during differentiation in the cellular slime mold. I. In vivo measurements. Biochemistry 6:1283-1286. 5. WRIGHT, B. E. 1960. On enzyme-substrate re-
lationships during biochemical differentiation. Proc. Natl. Acad. Sci. 46:798-803. 6. WRIGHT, B. E. 1964. Biochemistry of Acrasiales, p. 341-381. In S. H. Hutner [ed.], Biochemistry and physiology of protozoa, vol. 3. Academic Press, Inc., New York. 7. WRIGHT, B. E. 1965. Control of carbohydrate synthesis in the slime mold, p. 296-316. In D. N. Ward [ed.], Developmental and metabolic control mechanisms and neoplasia. The Williams & Wilkins Co., Baltimore. 8. WRIGHT, B. E. 1966. Multiple causes and controls in differentiation. Science 153:830-837. 9. WRIGHT, B. E., AND D. DAHLBERG. 1967. Cell wall synthesis in Dictyostelium discoideum. II. Synthesis of soluble glycogen by a cytoplasmic enzyme. Biochemistry 6:2074-2079.
