Accumulation of uridine diphosphoglucose pyrophosphorylase in Dictyostelium discoideum via preferential synthesis

81, 173-185

J. MoZ. Biol. (1973)

Accumulation of Uridine diphosphoglucose Pyrophosphorylase in Dictyostelium discoideum via Preferential Synthesis JAKOB FRANKE AND MTAURICESUSSMAN~

Department of Biology, Brandeis

University

Waltham, Mass. 02154, U.S.A. (Received 17 April 1973) At the end of the exponential growth phase, the enzyme UDP-glucose pyrophosphorylase is present in the vegetative cells of Dictyostelium discoideum NC4 (haploid) at a low level (about 0.05% of total protein). During the initial stages of fruiting body construction, while the cells are entering into multicellular aggregates, the enzyme level remains constant, but increases dramatically thereafter reaching a peak (about 0.5% of total protein) at the end of fruiting body construction, and then partially decreasing. Previous studies have shown that both the accumulation and disappearance are keyed to the flow of morphogenetic events. In this study, cells were labeled with amino acids for different periods throughout the sequence. The enzyme was quantitatively immune-precipitated from crude cell extracts, the precipitate was washed and redissolved, and the enzyme protein separated by acrylamide gel electrophoresis in order to estimate the differential incorporation ratio, i.e. disints/min disints/min

in enzyme in total

protein protein

per 1Oe cells per lo* cells

x

100:

for each labeling period. During the initial stages, when the enzyme level remained relatively constant, this ratio was about 0.03 to 0.04%. As the enzyme began to accumulate it rose progressively, attaining levels of 0.6 to O3o/o toward the end of fruiting body construction before declining. The data are not consistent with the theory of Gustafson & Wright (1973) that differential turnover controls the level of this enzyme during the development of D. discoideum. They are consistent with the conclusion that directed changes in the differential rate of synthesis of UDP-glucose pyropbosphorylase is the controlling element. The estimates of enzyme content are based on a value for the specific enzyme activity of 100,000 units/mg enzyme, which had been determined previously using samples of the enzyme purified to apparent physical homogeneity. This figure has been confhmed in the present study by quantitative immuneprecipitation of the enzyme from crude extracts of homogeneously labeled cells. The method can be generally used to determine if a specific biological activity estimate obtained with a purified protein is consistent with its activity when measured before or during purification.

1. Introduction During

fruiting

body

construction

of several polysaccharides

by Dictyostelium discoideum, considerable

and one disaccha,ride accumulate

-i-Present address: The Hebrew requests should be addressed.

University

of Jerusalem,

amounts

(Gezelius & Ranby,

Jerusalem,

195’7 ;

Israel. To whom reprint’

174

J. FRANKE

AND

M. SUSSMAN

Clegg & Filosa, 1961; White & Sussman, 1963qb). The starting material for these syntheses is UDP-glucose. Hence the enzyme UDP-glucose pyrophosphorylase (EC 2.7.7.9) probably has an important role. Previous papers from this laboratory (Ashworth & Sussman, 1967; Newell & Sussman, 1969; Newell eE al., 1969,1972; Franke & Sussman, 1971) have reported that ; (a) UDP-glucose pyrophosphorylase increases about tenfold in specific activity during particular stages of fruiting body construction, and then declines to about half that value; (b) in mutants displaying morphogenetic aberrations and deficiencies, this pattern of accumulation and disappearance is disturbed in a manner consistent with the nature and timing of the morphogenetic derangement; (c) environmentally induced changes in the flow of morphogenetio events help to regulate this pattern; (d) the increase in enzyme activity is paralleled by a corresponding increase in antigenic activity solely attributable to UDP-glucose pyrophosphorylase; and (e) most if not all of the enzyme protein that accumulates is newly synthesized. Although subject to conflicting reports in the past (Wright, 1960,1966; Wright & Dahlberg, 1968 ; Wright & Pannbacker, 1967) there now appears to be a general agreement with respect to the above conclusions (Gustafson & Wright, 1972). However Gustafson & Wright (1973) recently reported that, at an early stage of fruiting body construction after the cessation of exponential growth but before UDP-glucose pyrophosphorylase activity had begun to increase, the cells incorporated appreciable amounts of exogenously added 14C-labeled amino acids into the enzyme protein. They concluded that UDP-glucose pyrophosphorylase is synthesized at a constant absolute rate throughout the life-cycle of this organism and that it is the rate of degradation, not the rate of synthesis that determines the level of this enzyme within the cells. Because UDP-glucose pyrophosphorylase has served as the paradigm for a large number of enzyme activities which accumulate and disappear during fruiting body construction and as a marker with which to dissect the pattern of gene expression during D. discoideum development, it was of particular importance to assess exhaustively the validity of the conclusion reached by Gustafson & Wright (1973). The results described herein do not appear to support this conclusion. Instead, they suggest that during fruiting body construction UDP-glucose pyrophosphorylase is synthesized at an appreciable differential rate only over the period when it actually does accumulate, and that its rate of degradation throughout development is certainly no more, and quite possibly less, than that of total cell protein.

2. Materials and Methods (a) Organism D. discoideum (haploid), strain NC4, was grown in association with Aerobacter aerogenes (Sussman, 1966). The cells were harvested at the end of the exponential phase in cold water and washed free of bacteria by repeated centrifugations (5 min at 270 g). The cells were resuspended in LPS medium (buffer/salts/streptomycin), which was sterilized by filtration through a 0.45-pm Nalge membrane unit and stored frozen. Samples containing lo* cells were deposited on 4.25~cm Whatman no. 50 discs resting on pads (4*7-cm Whatman 17 discs) saturated with LPS medium. Under these conditions, the IO8 cells form about 10” equal sized aggregates and these develop synchronously into fruiting bodies, The entire sequence, starting from cell deposition, is accomplished in 24 h at 22°C.

UDP-GLUCOSE

175

PYROPHOSPHORYLASE (b) Chemicals

Narz51 from New England Nuclear (NEZ-033H); [14C]glucose from ICN (11050); mixture of 15 l&C-labeled amino acids from New England Nuclear (NEC-445); [3H]leucine additional 14C and 3H-labeled n-amino acids from New England Nuclear (NET-135H); were purchased from New England Nuclear, ICN and Schwarz-Mann; n-amino acids 1251-labeled ovalbumin and anti(A grade) from Calbiochem; crystalline ovalbumin, ovalbumin rabbit serum were a gift from Dr L. Levine. Reagents for the assay of UDPglucose pyrophosphorylase are listed elsewhere (Franke & Sussman, 19’71).

(c) Labeling

of the cells with radioactive

amino acids

For labeling periods longer than 3 h, the filters bearing the developing cell populations were switched to fresh pads saturated with 1.8 ml of LPS medium containing the radioactive amino acid(s). For labeling periods less than 3 h, each alter was placed directly on the Petri dish surface over 0.2 ml of undiluted labeling solution. This permitted an enhanced incorporation, and despite the reduced reservoir for metabolic waste products, the cells continued to develop and to accumulate UDP-glucose pyrophosphorylase normally for at least 3 h. After the labeling period the cells were harvested in cold LPS medium, centrifuged 5 min at 270 g, resuspended in 0.1 M-Tricine/NaOH (pH 7.6) containing 5 mm-UTP and stored at - 20°C. Four different labeling solutions were used. (1) An equimolar mixture of seven l&C-labeled amino acids (Asp, Glu, Leu, Lys, Phe, Pro, Thr) in 10 mM-NaCl was diluted fivefold with LPS medium. Final conditions: 20 &i/ml; 7.4 ~Ci/~mol for each amino acid; pH 6.4. (2) 15 14C-labeled amino acids (NEC-445) (3) A mixture of six 3H-labeled medium containing 3.5 mM-Nacl. activity 48 mCi/cc.mol; pH 6.4.

in 40 mivr-Nacl, pH 6.4, containing

ammo acids (Ala, Ile, Leu, 380 &i/ml; Final conditions:

(4) [3H]leucine in a mixture of LPS conditions: 370 &i/ml; 38.5 mCi/pmol;

medium and 0.1 M-NaCl pH 6.4.

(d) Enzyme

40 &i/ml.

Lys, Pro, Val) in LPS average specific radio(vol. ratio

10:7).

Final

assays

UDP-glucose pyrophosphorylase activity was measured spectrophotometrically in the direction of glucose l-phosphate formation. The method of cell lysis, assay conditions, and measurement of protein are described in detail in a previous paper (Franke 85Sussman, 1971). 1 unit: 1 nmol of glucose l-phosphate per min at 37°C. (e) Preparation

of antiserum

Purified UDP-glucose pyrophosphorylase (Franke & Sussman, 1971) was repeatedly injected with Freund’s adjuvant into the toepad of a rabbit. The antiserum yielded one band with samples of the antigen in agar double diffusion assays. A y-globulin fraction was prepared by the addition of 0.8 vol. of saturated ammonium sulfate (pH 75) to 1 vol. of antiserum. The precipitate was redissolved and dialyzed against 0.1 M-NaCl 0-02’3o sodium azide, 0.02 M-sodium phosphate (pH 7). The procedure ‘was repeated once. The resulting y-globulin preparation was heat-inactivated for 20 min at 60°C and clarified by centrifugation (30 min at 100,000 g). The preparation (referred to as a-yG in the following text) contained 62 mg protein per ml and the final titre was 1: 10,000 for 50% complement fixation (Wasserman & Levine, 1961). (f) Measurement

of total acid-insoluble

protein

radioactivity

Samples (25 ~1) of labeled extracts were applied to 12 mm x 28 mm strips of Whatman 3 MM paper, fixed and extracted as described by Rosenbaum et al. (1969). The air-dried samples were counted in 15 mm x 45 mm vials with 4 ml toluene containing 4 g PPO and 0~05 g POPOP per liter. Counting efficiencies: 16% for 3H; 70% for 14C.

176

J. FRANKE

AND

M. SUSSMAN

(g) Immune precipitation and recovery of antigen Frozen cell samples were thawed and lysed by the addition of Cemulsol NPTl2 detergent (Melle Bezons Inc. France) to a concentration of 0.15%. Portions were reserved for enzyme assays and determination of total acid-insoluble radioactivity and protein content. Sodium azide was added (0.02%) to another portion of the extract, which was centrifuged 30 m in at 100,000g. The supernatant fluid containing more than 90% of the enzyme activity was mixed with a-rG in the proper ratio and the total volume (see Results) incubated 30 mm at 37°C and stored overnight at 4°C. The immune precipitate was collected by centrifugation in calibrated McNaught tubes at 1500 g for 1 h and washed twice with cold sterile 0.14 M-NaCl, 0.02 m-Trioine/NaOH at pH 7.6. The pooled supernatant and washings contained no detectable enzyme activity. The pellet was suspended in 25 ~1 solution containing 12% sodium dodecyl sulfate and 12% mercaptoethanol, adjusted with water to a final vol. of 150 ~1, and boiled 5 to 15 min until all the material dissolved, after which sucrose was added to a final concentration of 10%. A measured portion of the final solution, usually 100 ~1, was added to a 6 mm x 100 mm 7.5% polyacrylamide gel in O*l”h sodium dodecyl sulfate, 0.1 M-sodium phosphate (pH 7) and subjected to electrophoresis at 10 mA per gel for 9 h at room temperature. The gel was stained 1 h in 0.2% Coomassie brilliant blue dissolved in water/methanol/acetic acid (5 : 5: 1) at 37°C and destained in 7% acetic aoid-O.B”h methanol at 50°C. After recording the staining pattern, the gel was frozen and cut into l-3-mm slices. These were hydrolyzed with 0.4 ml of 30% H,Oz at 60 to 70°C for 3 h and counted with 3.5 ml Aquasol (New England Nuclear) in 15 mm x 45 mm vials. Counting efficiencies: 20% for 3H; 83% for 14C. (h) Iodination of UDP-glucose pyrophosphorylase A purified preparation of the enzyme was labeled with lZ51 according to a published procedure (Work & Work, 1970). The iodinized antigen was shown to be quantitatively removed from solution by the above immune precipitation procedure (see Table 1) and to migrate in sodium dodecyl sulfate-polyacrylamide gels together with sodium dodecyl sulfate-treated samples of the unlabeled enzyme.

3. Results (a) Evaluation of the procedure for immune precipitation

and recovery

of the uricline diphosphoglucose pyrophosphorylase

The procedure described in Materials and Methods was tested with two purified, ls51-labeled antigens : UDP-glucose pyrophosphorylase from D. discoideum, purified to apparent physical homogeneity (Franke & Sussman, 1971) and crystalline ovalbumin. In both oases the antigen was quantitatively removed from solution. After the immune precipitate had been dissolved in sodium dodecyl sulfate and separated by acrylamide gel electrophoresis, all of the radioactivity appeared in a single peak whose position coincided with that of the corresponding monomeric polypeptide visualized with a protein stain. The recoveries of the radioactivity originally present in the reaction mixtures were 76% and 66%, respectively (Table 1). The prooedure was tested further by using crude extracts of cells that had been uniformly labeled during exponential growth with l*C (see legend to Table 1). The cells were harvested at two stages of fruiting body construction; at an early stage when the UDP-glucose pyrophosphorylase activity was still at a low level and at a later stage after the peak level of activity had been attained. The recoveries, as shown in Table 1, ranged between 60 to 80% of the expected levels. Trial experiments with the purified lz51-labeled antigens showed that 10% of the loss is incurred during gel electrophoresis and preparation of the gel slices for counting. The remaining 20% is

UDP-GLUCOSE

177

PYROPHOSPHORYLASE TABLE

1

Recovery of antiyens from immune precipitates

Total units of

Experiment sample

Total protein (disints,min,mg) enzymeprecipitated with antiserum

Radioactivity in antigen protein recovered after immune precipitation and eel electrophoresis (disints/:min) Expected

1Z51-labeled ovalbumin ?C-labeled enzyme

-

purified

Recovery (%)

Observed

-

22801_

1510

66

330

1970t

1500

76

A B C

860,000 960,000 780,000

810 460 308

69605 4420 2390

4550 2660 1650

65 60 65

Extracts of cells from A the same cultures as B above but at a later C stage of morphogenesis

740,000 830,000 650,000

330 480 570

2440 3980 3700

1920 2600 2880

79 66 78

Extracts of homogeneously labeled cells at early stage of morphogenesis$

t The expected radioactivity of the 1251-labeled antigen was determined by sodium dodecyl sulfate gel electrophoresis of known amounts of 1251-labded antigen. The expected value is alculeted from the amount of radioactivity originally added to the serological reaction mixture and corrected for the fraction of the mixture actually applied to the gel. The gels were stained, sliced and counted as described in Materials and Methods. $ E. COG, strain C, was grown to the stationary phase (3 x lo9 cells/ml) in a medium (pH 7) containing per liter: 7 g KH2P04, 3 g K,HP04, 0.1 g MgS04.7Hz0, 1 g (NH&SO+ 2 g glucose and uniformly labeled [14C]glucose. The bacteria were pelleted, washed once with sterile 0.04 w-potas-

sium/disodium phosphate (pH 6.4) and suspended in this buffer at a density of lOlo cells/ml. A sample of 40 ml in a 500-ml Erlenmeyer flask was inoculated with D. discoideum spores and incubated at 22°C on a wrist shaker. After a 15 to 20-h germination and lag phase the amoebae grew exponentially to a density of I.2 x lO’/ml with a doubling time of 3.5 to 4 h. The amoebae were harvested and freed of remaining bacteria by repeated centrifugation and deposited on filter paper discs as described in Materi& and Methods. Three separate experiments designated A, B and C were done. 5 disintslmin expected =

units of enzyme precipitated 100,000

X

disintalmin mg

total protein

(100,000 units/mg enzyme).

presumably

incurred by mechanical

precipitate. The above evaluation

is particularly

losses during the manipulation important

of the immune

since the enzyme was precipitated

directly from crude extracts without prior purification, and therefore under conditions identical with those of the actual experiments to be carried out. It should be noted that in this last test the radioactivity of the separated enzyme protein represented only about 20% or less of the total radioactivity in the washed immune precipitate. The remainder included non-specifically absorbed or cross-reacting materials scattered

178

J. FRANKE

AND

M. SUSSMAN

throughout the gel, but fortunately not near the position of the enzyme monomer. Granner et al. (1970) reported the same level of contamination when they precipitated tyrosine transaminase from crude extracts of hepatoma cells. Two parameters of the immune precipitation procedure that strongly affected the recovery of radioactivity were: (a) the ratio of antigen to antibody in the reaction mixture, and (b) the absolute concentration of antigen (Fig. 1). In all the experiments described later, these parameters were held at the levels indicated by the arrows. Wherever tested, the &s/mm recovered were directly proportional to the amount of antigen originally precipitated with antibody; mock precipitations using normal rabbit serum did not yield detectable counts at the position of the enzyme monomer.

*:I 500

1500

2500

Enzyme concn (units/ml)

(a)

20 Ratio (enzyme

60 units/pi (b)

100 a-yG)

Fm. 1. Recoveries of r4C-labeled enzyme after immune precipitation at varying concentrations of enzyme and varying ratios of enzyme to a-yG. (a) Samples of homogeneously 1%labeled extract (see legend to Table 1) with a specific enzyme activity of 500 units/mg protein were mixed with a solution of purified enzyme (5600 units/ml) or with early extract (156 units/mg protein; 530 units/ml) in order to provide the desired concentration of enzyme and at an approximately constant protein concentration of 1.8 mg/ml in 0.1 M-Trioine/NaOH (pH 7.6) containing 5 m&r-UTP. 1 ~1 of a-yG was added per 32 units of enzyme and the immune precipitates were collected and treated as described in Materials and Methods. (b) Samples of homogeneously r4C-labeled extract prepared as above (1790 units/ml; 2.4 mg protein/ml) were treated with varying amounts of a-yG and the immune precipitates were analyzed as before.

(b) Incorporation

of amino acids into enzyme protein during fruiting

body construction

Washed cells harvested at the end of the exponential phase were deposited on filter paper circles (see Materials and Methods) so that they could form multicellular aggregates and construct fruiting bodies. Radioactive amino acids were added exogenously for periods of different duration throughout the morphogenetic sequence. At the end of a labeling period the cells were harvested, washed and frozen as described. Replicate measurements were made of (a) UDP-glucose pyrophosphorylase activity and (b) the specific radioactivity of the total protein in the cell extracts. In addition, measurements of enzyme activity and total protein were made using unlabeled sister cells harvested at the beginning of, and during, each labeling period.

UDP-GLUCOSE

i-

0

4

179

PYROPHOSPHORYLASE

8

12

16

20

24

Time(h)

FIG. 2. Changes in the level of UDP-glucose pyrophosphorylase and in total protein content during fruiting body construction. As described in the text, measurements of specific enzyme activity obtained with orude cell extracts were converted into pg of enzyme by using the value of 100,000 units/mg enzyme protein, the specific activity of the purified enzyme (see also Results, section (a)). The chronology of the morphogenetio sequence is indicated by drawings below the abscissa. ---_O-, pg enzyme/lO* cells; -_O-_O-, total protein content/IO* cells.

(Fig. 2 shows the course of enzyme accumulation and the decrease in total protein content during the entire period of fruiting body construction.) The enzyme was immune-precipitated from samples of the labeled extracts in order to measure the radioactivity associated with the monomeric polypeptide. The differential incorporation ratio, disints/min in enzyme protein/108 cells disints/min in total protein/lo8

cells

x 100,

was taken to indicate the relative extent to which nascent enzyme accumulated during a labeling period as compared with the accumulation of all other nascent proteins. The data and a sample calculation of this ratio for two different labeling periods are given in Figure 3 and a summary of the values for all the labeling periods is given in Figure 4. Some cells were harvested from the filters after 17 hours of development, disaggregated to a single cell suspension and redeposited on fresh filters at the original cell density. As reported previously (Newell et al., 1972), these cells immediately reaggregated and within three hours recapitulated the morphogenetic sequence that had taken them 17 hours to accomplish the first time. Then they went on at the normal rate to complete fruiting body construction. During this time they proceeded through a second complete round of UDP-glucose pyrophosphorylase accumulation. Figure 4 shows the differential incorporation ratio during this second round.

180

J. FRANKE

AND

M. SUSSMAN T-Globulin

BSA dlmer

ESA monomer

20

40

& UDPG PPose

60

80

H-chain Carboxy peptidose

100

Millimettes

FIG. 3. Determination of the differential incorporation ratio. The experimental procedures are described in Materials and Methods. The graphs show the distribution of radioactivity in the gels for two different labeling periods. The calculations of the differential incorporation ratios are reproduced below. It should be noted that the differential incorporation ratios are calculated exclusively from direct measurement of enzyme activity/ml of extract and the radioactivities/ml of extract. They are independent of estimates of specific enzyme activities either of the purified enzyme or crude cell extracts. BSA, bovine serum rtlbumin; UDPG PPase, UDP-glucose pyrophosphorylase. Labeling period in [sH]leucine Units/ml of enzyme activity in the crude cell extract at t, Total enzyme units precipitated with a-yG Disintslmin (over background) in enzyme protein in the gel Corrected for mean recovery of 70% (see Table 1) Disintslmin in enzyme protein/ml extract Disintslmin in total cell protein/ml extract Differential incorporation ratio

t0 t, 2.5 to 6.5 h 400 620 900 1290 830 2,710,000 0.031 y.

to tn

18 to 20.5 h 720 1000 5100 7300 5250 940,000 0.56%

The data in Figure 4 would appear to permit the following conclusions. (1) During any period of fruiting body construction in which the level of UDPglucose pyrophosphorylase activity was increasing, a significant proportion of the radioactive amino acids incorporated was associated with this protein. The highest differential incorporation ratio observed was 0.8 to 1.1%. (2) During the period when UDP-glucose pyrophosphorylase activity increased very slightly (1 to 6 h), the radioactivity incorporated into this enzyme was barely above the background level (Figs 3 and 4). In five repetitions of this experiment the mean differential incorporation ratio was 0.03 to 0.04% or about 0.05 of the highest level. At the last stage of fruiting body construction, after the level of pyrophosphorylase activity had begun to decrease rapidly, the differential incorporation ratio fell to about a fifth of the peak value. (This is probably too high an estimate since the population of developing fruiting bodies was slightly asynchronous at this stage.)

UDP-GLUCOSE

181

PYROPHOSPHORYLASE

I-24-

v) = E “0 Y

redeposited 12

i--j<0

04 (Mean

of 5 measurements

)I

I

I

I

8

I

I

4

I

12

16

20

24

-

a

Tcme( h)

FIG. 4. The differential incorporation rstios measured over periods of labeling before, during and after the accumulation of UDP-glucose pyrophosphorylase. Solid lines: these represent the accumuletion of enzyme activity/lO* cells (a) during the normal morphogenetio sequence, and (b) when the cells were disaggregated and allowed to reaggregate and develop further. These curves were fitted to points obtained by enzyme assays of crude extracts, as described in the text. Some of the points are shown in Fig. 2. Bars: these represent the labeling periods, i.e. the left end of each bar designates the time at which the radioactive amino acids were added to the cells and the right end designates the time at which the cells were harvested. (The relative heights of the bars have no significance.) The numbers represent the differential incorporation ratios calculated as described in the text. 1%7aa, 7 r4C-labeled amino acids; 14C-1Saa, 16 14C-labeled amino acids.

(c) Sources of ambiguity (i) Non-speci$cally absorbed and cross-reacting materials As mentioned previously, in cell. extracts from the later stages of fruiting body construction the radioactivity associated with the enzyme accounted for only about 200/, of the total radioactivity of the washed immune precipitate and, in extracts from early stages (2 to 6 h), less than 40/,. As Figure 4 indicates, the contaminating proteins were generally heterogeneous. However, in the early extracts a considerable level of radioactivity appeared to be associated with one sharp peak (see Fig. 2). It is very doubtful that this protein was cross-reacting with the anti-UDP-glucose pyrophosphorylase y-globulin since extensive experiments, both by immune precipitation and quantitative complement fixation, have failed to demonstrate the presence of any antigenically cross-reactive

182

J. FRANKE

AND

M. SUSSMAN

material lacking catalytic activity (Franke & Sussman, 1971). Furthermore, addition of purified unlabeled enzyme to these labeled extracts did not reduce the level of the contaminating peak. It is possible that the protein in question was reacting with another y-globulin species not present at high enough titre to yield a visible band in agar double-diffusion assays. The presence of such contaminants emphasizes the need for rigorous proof that a given level of radioactivity in the immune precipitate is associated with the enzyme at any stage of development. (ii) Bacterial contamination LPS medium stored in large volumes at 4°C rather than in the frozen state, and not sterilized by filtration before use, was found to contain metabolically active, viable bacteria which could incorporate significant amounts of radioactive amino acids during the labeling experiments described above. The immune precipitates from such contaminated cell samples contained abnormally high levels of radioactivity compared with controls prepared under sterile conditions. When such precipitates were separated on sodium dodecyl sulfate-acrylamide gels, several radioactive peaks not previously encountered were observed including one close to the normal position of the enzyme subunit. This ambiguity was particularly serious in cell samples labeled during the early stages of fruiting body construction, since without careful attention to the positions of the stained bands in the gel the contaminating peak might be wrongly identified. The addition of sodium azide to the serological reaction mixture as a preservative was also found to be crit,ical since incubation for 30 minutes at 37°C and overnight in the cold in the absence of azide, resulted in the appearance of extraneous radioactivity in the pellets after centrifugation.

4. Discussion

As previously described (Franke & Sussman, 1971) and confirmed by the data shown in Figure 2, D. discoideum NC4 (haploid) amoebae growing exponentially in association with A. aeroyenes contain about 2.5 pg of UDP-glucose pyrophosphorylase/108 cells. After the cessation of growth and while the amoebae are still in the process of forming multicellular aggregates, the enzyme remains at a relatively constant level, but accumulates thereafter at a progressively increasing rate to reach a peak of about 16 pg/108 cells toward the end of fruiting body construction. The level ultimately declines to about half this value in the mature fruiting body, the loss coming exclusively from the terminally differentiated, necrotized cells in the stalk and basal disc (Ashworth & Sussman, 1967). The experiments reported here with purified lz51-labeled enzyme and with homogeneously labeled cells have demonstrated that the enzyme can be quantitatively precipitated from crude cell extracts with rabbit y-globulin, separated from the antibody by boiling with sodium dodecyl sulfate and recovered after acrylamide gel electrophoresis with an efficiency of 70%. This has permitted us to compare the level of incorporation of labeled amino acids into this enzyme protein, relative to the incorporation into all other proteins during the entire developmental sequence. As shown in Figures 3 and 4 the differential incorporation ratio was about 0.6% during the period of most active enzyme accumulation (12 to 20 h), rising to peak levels of 0.8 to 1.1%. In contrast, this ratio was very much lower during the early stage of

UDP-GLUCOSE

PYROPHOSPHORYLASE

183

fruiting body construction (1 to 8 h), ranging between O-016 to 067% in five separate experiments with a mean of 0.03 to 0.04%. The variation is intrinsic to the experiment, because at low levels of differential incorporation the subtraction of background counts from the enzyme protein peak after gel electrophoresis becomes an increasingly arbitrary exercise (see Fig. 3). The differential incorporation ratio is a measure of the accumulatiolz of UDPglucose pyrophosphorylase versus all other proteins and hence represents the result of four separate activities: the rate of synthesis of the enzyme and the rate of its degradation; the combined rate of synthesis of all the other proteins and the rate of their degradation. The turnover theory of Gustafson & Wright (1973) must be tested in this context. On the basis of the data, their theory appears to be untenable. Let us compare two different periods of fruiting body construction: 16 to 18 hours during which lo8 cells accumulated 3 pg of UDP-glucose pyrophosphorylase, and 4 to 6 hours during which the enzyme level remained virtually constant. From the measurements shown in Figure 4, the differential incorporation ratio between 16 and 18 hours appears to have been about 0.65%. Hence a total of 3 pg/ O-0065 = 460 pg of nascent protein must have accumulated over this period. At the same time the total protein content of the cells fell slightly from 3.5 mg to 3.4 mg. Hence the accumulation of 460 pg of new protein must have been accompanied by the disappearance of 560 pg of old protein. This represents a turnover of about 8% per hour. Wright & Anderson (1960a,b) studied the disappearance during fruiting body construction in the presence of unlabeled methionine of pre-existing cell protein labeled with [35S]methionine. They concluded that old protein turned over at a constant rate of 7% per hour throughout the sequence. Thus the two estimates based on quite different techniques agree very closely. A comparison of the differential incorporation ratios, for very short and very long labeling periods, obtained while the enzyme was accumulating most rapidly, indicates that the turnover of the pyrophosphorylase was not significantly different from the turnover of total cell protein. Thus if the former were turning over more rapidly, one would expect that, during a short labeling period in which the labeled products comprised a very small proportion of the protein population, the differential incorporation ratio would be very much higher than for a long labeling period in which a substantial proportion of both the pyrophosphorylase and other proteins had become labeled, and would themselves be subject to significant degradation. But as Figure 4 indicates, the ratios for pulses as short as two hours and as long as 6 to 11 hours did not differ markedly. This being so we must conclude that the incorporation ratio of O*65o/oaccurately reflects the differential rate of pyrophosphorylase synthesis over this period. Between four and six hours the differential incorporation ratio was about 0.035%. Suppose we assume with Gustafson & Wright (1973) that the pyrophosphorylase was being synthesized at the maximum rate, but was degraded so rapidly that the net level remained virtually unchanged. Then the cells would have synthesized at least 3 pg of enzyme during the two hours and destroyed almost 3 pg. Unless one invokes an unspecified mechanism by which the old, unlabeled enzyme was retained and only the new, labeled enzyme was disposed of, it must be concluded that at least 60% of the 3 ,ug of pyrophosphorylase .present when the cells were harvested at six hours was newly made, labeled enzyme. If one assumes that the rest of the new protein made during the two-hour period was turning over at the rate of 7 to 8%

184

J. FRANKE

AND

M. SUSSMAN

per hour, the differential incorporation ratio should have been at least O-65 x O-6= O*4o/o,i.e. tenfold greater than the level actually measured. If one argues that during the early stages, the differential rate of UDP-glucose pyrophosphorylase synthesis remained constant but the over-all rate of protein synthesis was greatly reduced, the differential incorporation ratio should have been even closer to 0.65% since less of the newly made, labeled enzyme would have been available for degradation. Nor can one argue that during the period between four and six hours the rate of pyrophosphorylase synthesis remained unchanged but that the rate of synthesis and degradation of all other proteins was tenfold higher. This would mean that over the two-hour period lo8 cells would have had to have synthesized at least 4.6 mg (460 pg x 10) of cell protein. But the mass doubling time of this organism under optimal growth conditions is about four hours (Sussman, 1966). It is not likely that stationary phase cells incubated at high population density in a medium devoid of all exogenous nutrients could synthesize protein at a rate that would correspond to a log phase cell doubling time of about 2.5 hours. Moreover the recent findings of Gustafson & Wright (1973) do not appear to be in accord with their own model. These workers deposited cells on filters to begin fruiting body construction. After 1.5 hours the cells were exposed to mixtures of 14C-labeled amino acids. Samples were harvested after 3, 4, 5 and 7 hours incubation. Using essentially the same techniques of immune precipitation and sodium dodecyl sulfateacrylamide gel separation, they measured the specific sadioactivity of the pyrophosphorylase and of total cell protein. In the four determinations the ratios of these two specific radioactivities were 0.85, 1.24, 0.93 and l-23, respectively. Suppose, as Gustafson & Wright argue, the enzyme were being synthesized at the rate characteristic of the later stages of fruiting body construction and was being turned over so rapidly as to maintain a constant level while total cell protein was turning over at the steady 70/ per hour rate reported previously (Wright & Anderson, 1960a,b). One would expect the ratio for the three-hour labeling period to be the highest and to decrease progressively during longer and longer labeling periods since, as previously noted, a short labeling period in which a relatively small amount of labeled products had accumulated would more accurately reflect the real differential rate of synthesis than would a long labeling period in which labeled pyrophosphorylase would provide a substantial proportion of the enzyme undergoing degradation. In another experiment (using [35S]methionine), labeling periods of 3, 5, 7, 10 and 11 hours were used (Gustafson & Wright, 1973). The specific radioactivity ratios obtained for the shorter labeling periods actually doubled during the longer periods. This was accompanied by a slight rise in enzyme content, comparable to the one shown in Figure 2. Assuming an over-all protein turnover of 7 to 8% per hour, the net accumulation of enzyme would in fact require the observed doubling in the specific radioactivity ratio. The following interpretation is consistent with the data presented here and with the results of Gustafson & Wright (1973). During growth, cells synthesize UDPglucose pyrophosphorylase at a rate amounting to about 0.05% of the total rate of protein synthesis. For the first six to eight hours of fruiting body construction the differential rate of synthesis decreases to a slightly lower level, which is balanced by the 7 to 8% per hour turnover of pre-existing protein. Starting at about eight hours, the differential rate of pyrophosphorylase synthesis rises rapidly to a peak level

UDP-GLUCOSE

PYROPHOSPHORYLASE

185

10 to 12-fold higher. Thus a change in the differential rate of synthesis, and not a change in the differential rate of turnover, represents the controlling factor in the accumulation of this enzyme. Results summarized elsewhere (Sussman & Newell, 1972) suggest compellingly that, as in many other differentiating cells, this shift is the direct consequence of the differential transcription of the genome. This research was supported by National Institute

of Health grant no. GM18689.

REFERENCES Ashworth, J. M. & Sussman, M. (1967). J. Bid. Chem. 242, 1696-1700. Clegg, J. & Filosa, M. (1961). Nature (LolzcZon), 192,1077-1078. Franke, J. & Sussman, M. (1971). J. Biol. Chem. 246, 6381-6388. Gezelius, K. & Ranby, B. (1957). Equ. Cell Res. 12, 265-289. Granner, D. K., Thompson, E. B. & Tomkins, G. M. (1970). J. BioZ. Chem. 245, 1472. Gustafson, G. L. & Wright, B. E. (1972). Ci-iticul Reviews in. Microbiology, 1, 453-478. Gustafson, 0. L. & Wright, B. E. (1973). Biochem. Biophys. Res. Commun. 50, 438-442. Newell, P. C. & Sussman, M. (1969). J. BioZ. Chem. 244, 2990-2995. Newell, P. C. Ellingson, J. S. & Sussman, M. (1969). Biochim. Biophys. Acta, 177, 610-614. Newell, P. C., Franke, J. & Sussman, M. (1972). J. Mol. BioZ. 63, 373-382. Rosenbaum, J. L., Moulder, J. E. & Ringo, D. L. (1969). J. Cell BioZ. 41, 600. Sussman, M. (1966). In Methods in Cell Physiology (Prescott, D., ed.), vol. 2, pp. 387-409, Academic Press, New York. Sussman, M. & Newell, P. C. (1972). In Molecular Genetics and Developmental Biology (Sussman, M., ed.), pp. 275-203, Prentice Hall, New Jersey. Wasserman, E. & Levine, L. (1961). J. Immunol. 87, 290-300. White, G. J. & Sussman, M. (1963a). Biochim. Biophye. Acta, 74, 173-178. White, G. J. & Sussman, M. (19633). Biochim. Biophys. Acta, 74, 179-187. Work, T. S. & Work, E. (eds.) (1970). Laboratory Technigues in Biochemistry and MolecuZar Biology, vol. 1, p. 540, North Holland Publishing Company, Amsterdam. Wright, B. E. (1960). Proc. Nat. Acad. Sk., U.S.A. 46, 798-803. Wright, B. E. (1966). Science, 153, 830-837. Wright, B. E. & Anderson, M. L. (1960a). Biochim. Biophya. Acta, 43, 62-66. Wright, B. E. & Anderson, M. L. (1960b). Biochim. Biophys. Acta, 43, 67-78. Wright, B. E. & Dahlberg, D. (1968). J. Bacterial. 95, 983-985. Wright, B. E. & Pannbacker, R. (1967) J. Bacterial. 93, 1762-1764.