Polyribonucleotide phosphorylase fromDictyostelium discoideum

EXPERIMENTAL

MYCOLOGY

4, 181-185

olyribonucleotide

(1980)

Phosphorylase

from ~i~t~oste~i~~ A. KILLICK

KATHLEEN Department of Developmental

Biology, Boston Biomedical Accepted for publication

Research Institute, Boston, Massachseits

021i4

April 10, 1980

KILLICK, K. A. 1980. Polyribonucleotide phosphorylase from Dictyostelium discoideum. Ex:xperimental Mycology 4, 181- 185. Polyribonucleotide phosphorylase activity was detectable in cell-free extracts prepared from stationary-phase myxamoebae and young sorocarps of the cehular slime mold, Dictyostelium discoideum. Following electrophoresis, enzymatic activity was locahzed and assayed in polyacrylamide gels in the direction of polyribonucleotide formation. Polymer synthesis was: (1) detectable by staining with either acridine orange or methylene blue; (2) dependent on the presence of nucleoside diphosphate (either UDP or ADP); (3) a linear function of time; aud (4) independent of the presence of a primer, e.g., adenyladenosine. With myxamoebal extracts. the major band of enzymatic activity had an R, identical to that (i.e., 0.37) for the purified Escherichia coli phosphorylase, while the major isozymic forms of the enzyme from young sorocarps had R,, values of 0.27 and 0.37. INDEX DESCRIPTORS: Dictyostelium discoidearn; RNA degradation; polyanion activation of trehalose-6-phosphate synthase; trehalose; dormant spore; development.

The nonreducing disaccharide, CX,CX’trehalose represents a major carbohydrate reserve in the dormant spores of the cellular slime mold, Dictyostelium discoideum, where this sugar serves as a major carbon and energy source during germination. As such, regulation of trehalose synthesis during morphogenesis is critical to species survival. Since trehalose-6-phosphate (trehalose-6-P) synthase (UDPglucose:Dglucose-6-phosphate 1-glucosyl transferase, EC 2.4.1.15) catalyzes the rate-limiting step in trehalose synthesis, regulation in vivo of the catalytic properties of this enzyme must serve an important function in regulating the rate of trehalose accumulation. The in vitro alteration of the catalytic rqperties of the synthase by heparin and other glycosaminoglycans has been demonstrated for the enzyme from bacteria (Liu et al., 1969; Elbein and Mitchell, 1975; Elbein, 1974a, b) and Dictyostelium (Killick, 1979). In addition to the latter polyanions, ’ Abbreviations used: PNPase, polyribonucleotide phosphorylase; ApA, adenyladenosine.

low-molecular-weight polyrib~~~c effecters of the enzyme have been i from ~ycob~~teri~m smegmatis tuberculosis (Liu et al., 1969; Go Lornitzo, 1962; Lornitzo and 1964). Liu et al. (1969) demo purified po~yr~bo~~cleotide from matis altered the kinetic and phy erties of the synthase in a m to that effected by heparin and sulfate and involvement of cleotide phosphorylase (E PNPase)l in the synthesis oft

1964) demonstrated

that a

trehalose-6-P synthase c of a complex between oligoribo~u~leotide inhibitor. Although activation of the ~~~~~o~~e~~~~~ trehalose-6-P synthase arirs and chondroitin sulfate might to be of little p~~s~~~ogi~al significance since neither polysacc~aride is produce slime mold, ~Q~y~~o~~~~eoti

181 0147-59751~0/02Q18!-05$~~.~~i~ Copyright All rights

@I 1980 by Academic Press, Inc. of rqxodnction in my form reserved.

182

KATHLEEN

A.

KILLICK

and/or inhibition of this enzyme could be of (Peacock and Dingman, 1967) or to a solupotentially greater metabolic importance in tion composed of 1% (w/v) acridine orange terms of (1) prevention of product inhibition plus 1.03% (w/v) lanthanum chloride in 25% by UDP and (2) feedforward activation of acetic acid (Richards et al., 1965). Gels the synthase by accumulated poly(UMP) were incubated in either staining solution (or poly(XMP)). Because of these consid- for 18 h at 23°C. Gels stained with methyerations studies were undertaken, as de- lene blue were destained by diffusion with scribed in the present report, to determine distilled water, while those stained with acwhether Dictyostelium produces a polyriridine orange were destained electrophobonucleotide phosphorylase-like activity. retically with 7.5% acetic acid. Destained D. discoideum (NC-4) ATCC 24697 was gels were scanned in the linear transporter grown on nutrient agar with Escherichia of a’ Gilford 240 spectrophotometer. Maincoli as the bacterial associate. After con- taining minimal slit-width, full-scale absumption of the bacteria, myxamoebae sorption was 3.0, the scanning rate was 1 were harvested, washed, and spread onto cm/min, and the recorder speed was 1 nonnutrient agar sheets and incubated at inlmin. Scans were recorded on a Gilford 22°C for either 2 h (myxamoebae) or 23 h 6050 recorder. Electrophoresis of aliquots of crude (young sorocarps). Cells were harvested cell-free extracts from myxamoebae of D. with 50 mM potassium phosphate (pH 8.0) discoideum followed by incubation of the buffer that contained 5 mM 2-mercaptopolyacrylamide gels in the standard phosethanol and extracts were prepared by a phorylase incubation medium resulted in freeze-thaw cycle of the cells. Following prodcentrifugation of the homogenate (39,OOOg), the formation of a polyribonucleotide the resultant supernatant liquid served as the uct, detectable by staining the gels with either acridine orange or methylene blue. source of phosphorylase activity. Electrophoresis was performed at 4°C using the As shown in Fig. 1, one major band of activity was detectTris -glycine discontinuous buffer system PNPase polymerizing of Ornstein (1964) and Davis (1964). Sep- able after a 24-h incubation of the gels in the assay medium. Formation of this band was arating gels were cast at acrylamide monomer and bisacrylamide concentrations of dependent on the presence of substrate 7..5%T and 0.8%C, respectively, while stack- (i.e., ADP plus ApA). In agreement with ing gels consisted of 2.5%T and 2O%C. Elec- the observations of Fitt et al. (1968), the prodtrophoresis was initiated and maintained at sensitivity of the polyribonucleotide a constant current of 1 mA/gel until the dye uct to detection was significantly greater blue than with acridine front was about 0.5 cm from the end of the with methylene orange staining. Band III had an R, value gel. Following electrophoresis, phosphorylase activity was localized by incubating the equal to 0.37 and represented, based on relgels at 37°C in a mixture that contained 20 ative peak area sizes, about 90-95% of the mM ADP, 5 mM MgCl,, 0.2 mM EDTA, 0.2 stainable enzymatic activity. The other two mg/ml adenyladenosine (ApA) in 75 mM bands, i.e., I and II (Fig. l), had R, values Tris-HCl (pH 9.0) buffer (Fitt et al., 1968). equal to 0.20 and 0.27 and together com5- 10% of the total Control gels were incubated in the above so- prised approximately stainable phosphorylase activity. Polymer lution minus ADP and ApA. The incubation was terminated at timed intervals and the gels formation occurred as a function of time were rinsed with a solution of 7% acetic (Fig. 2) and the distribution of enzymatic acid and were then transferred to either a activity between bands II and III was not solution of methylene blue (0.2% w/v) in a affected by the length of the incubation 0.4 M sodium acetate (pH 4.7) buffer time. Parallel studies on homogenous

Dicryosrelium

POLYRIBONUCLEOTIDE

Fro. 1. Polyacrylamide gel electrophoretograms of polyribonucleotide phosphorylase from D. discoideum. Enzyme samples prepared from stationaryphase myxamoebae (A) and young sorocarps (B) were subjected to electrophoresis for 2 h at 1 mAlgel(4”C). Polyribonucleotide phosphorylase activity was assayed in the direction of polymerization (22 h; 37°C) and polyadenyhc acid was detected on the gels with methylene blue as described in the text. The arrows indicate the positions of the tracking dye on the two gels

PNPase from E. coli demonstrated that the enzyme was 0.37. m for the bacterial reeze-thaw threatment of the enzyme generated an additional form of the phosorylase having an R, equal to about 0.39. th prolonged gel incubation (i.e., 24 h) major and minor bands of deposited product merged together. Analysis of the primer dependency of the enzyme by densitometry allel gels incubated in the presence or rice of ApA indicated comparable s of deposited polyribonucleotide on o gels, indicating that the enzyme reparation was primer independent. Comparable studies on the enzyme from young sorocarps indicated the presence of two major bands of enzymatic activity having R, values equal to 0.27 and 0.37 for bands II and III, respectively (Fig. 1). ased on relative peak area sizes, bands II and III represented most of the recoverable activity and comprised about 30 and 70%, respectively, of the total stainable enzymatic activity. In view of the fact that this apparent distribution of enzymatic activity

P~OS~~O~~L~SE

FIG. 2. Polyadenylic acid synthesis as a f~nc~~~~ of time with polyribonucleotide pbospbo~y~as~ from stationary-phase myxamoebae ofQ. discoideum. After subjecting samples of cell-free extracts from myxamoebae to electrophoresis, polyr~bonucl~oti~e phosphorylase activity was localized in situ in tbe direction of polymerization as described in the text. Polyadenylic acid was detected with metby~e~e blue following incubation of the gels for 22, 7, 4.5, and 2 R (i.e., gels A to D, respectively). The arrows indicate tbe positions of poly(A) deposition on the gels.

might have been an artifact, partly ~a~s~d by long-term incubation of the study of polymer formation was (Fig. 3). Average value III, after incubation for the phosphorylase assay me and 68% of the total ret spectively, thus demonstrative t distribution of p tween bands II and III these in situ methods was neith dent upon, nor a consequence of, of the incubation time of the assay medium. As was also t with the enzyme preparation amoebae (1) freeze-thaw treatrn~~~ of prepared crude, generated an ad nucleotide phospho rate of ~~ectro~h~retic mob activities of the isozymes sorocarps were primer inde

184

KATHLEEN

FIG. 3. Polyadenylic acid synthesis as a function of time with polyribonucleotide phosphorylase from young sorocarps of D. discoideum. After subjecting samples of cell-free extracts from young sorocarps to electrophoresis, polyribonucleotide phosphorylase activity was localized in situ in the direction of polymerization as described in the text. Polyadenylic acid was detected with methylene blue following incubation of the gels for 3, 6.5, 22, and 48 h (i.e., gels A to D, respectively) at 37°C. The arrows indicate the positions of the two major bands of sorocarp PNPase activity. The lowest band depicted on the gels is the tracking dye.

(3) the peak area of the deposited polyribonucleotide product obtained with UDP as substrate was two or three times greater than that obtained with ADP as the substrate. The physiological function of PNPase in Dictyostelium is unknown. Although the phosphorylase-catalyzed reaction is reversible, the sensitivity of the polymerization reaction to phosphate inhibition, coupled with the lack of template specificity by the enzyme, strongly suggest that in vivo, the enzyme functions in the direction of phos-’ phorolysis. Thus, it may be speculated that this enzyme together with RNase II plays an important role in regulating not only the rate of degradation of ribosomal RNA but also that of mRNA following transcription. In addition to its involvement with RNA turnover, it is possible that PNPase functions in the activation in vivo of trehalose-6-P synthase during development. Such a situation might be visualized

A. KILLICK

as occurring as the consequence of either the degradation of a polyribonucleotide inhibitor or the synthesis of a polyribonucleotide activator. Additionally, coupling between trehalose-6-P synthase and PNPase activities via UDP could prevent product inhibition of the synthase by nucleoside diphosphate. Activation of both the synthase and PNPase by accumulated poly(U) could, moreover, evoke a type of cascade amplification, which might underly, at least in part, the activation of trehalose synthesis during morphogenesis in Dictyostelium. ACKNOWLEDGMENT This investigation was supported by research Grants AGO0922 and GM25534 from the National Institutes of Health. REFERENCES DAVIS, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. ELBEIN, A. D. 1974a. The metabolism of 01-a’trehalose. Advan. Carbohyd. Chem. Biochem. 30: 227-256. ELBEIN, A. D. 1974b. Interactions of polynucleotides and other polyelectrolytes with enzymes and other proteins. Advan. Enzymol. 40: 29-64. ELBEIN, A. D., AND MITCHELL, M. 1975. Protein: Polyanion interaction. The effect of heparin on the trehalose-phosphate synthetase of Mycobacterium smegmatis. Arch. Biochem. Biophys. 168: 369-377. FITT, P. S., FITT, E. A., AND WILLE, H. 1968. A study by polyacrylamide-gel electrophoresis of the effect of proteolysis on Micrococcus lysodeikticus polynucleotide phosphorylase. Biochem J. 110: 475-479. GOLDMAN, D. S., AND LORNITZO, F. A. 1962. Enzyme systems in the mycobacteria. XII. The inhibition of the transglycosidase-catalyzed formation of trehalose 6-phosphate. J. Biol. Chem. 237: 3332-3338. KILLICK, K. A. 1979. Trehalose 6-phosphate synthase from Dictyostelium discoideum. Partial purification and characterization of the enzyme from young sorocarps. Arch. Biochem. Biophys. 196: 121- 133. KILLICK, K. A., AND WR&iT, B. E. 1974. Regulation of enzyme activity during differentiation in Dictyostelium

discoideum.

Anna.

Rev.

Microbial.

28:

139- 166. LIU, C., PATTERSON, B. W., LAPP, D., AND ELBEIN,

Dictyostelium

POLYRIBONUCLEOTIDE

A. D. 1969. Properties of a trehalose phosphate synthetase from Mycobacterium smegmatis: Activation of the enzyme by polynucleotides and other polyanions. J. Biol. Chem. 246: 4561-4579. LORNITZO,

F. A.,

AND GOLDMAN,

D. S. 1964. Purifi-

cation and properties of the transglucosylase inhibitor of Mycobacterium tuberculosis. J. Biol. Chem. 239: 2730-2734. QR~~STEIN, L. 1964. Disc electrophoresis. I.

~HO~~H~~YL~~E

Background and theory. Ann. N. Y. Acnd. Sci. 12%: 321-349. PEACOCK,

A. C., AND DINGMAN,

C. W. 196‘7’9.

tion of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Bioche~nisrq~ Q: 181% I927. RICHARDS, E. G., COLL, 1. A., AND GF~ATZHI, W. B. 1965. Disc electrophoresis of ribonwleic acid in polyacrylamide gels. Anal. Biochem. 12: 4X-471.