Turnover Rates of Phosphoryl Groups in Ribosomal Proteins of Physarm polycephalum Evidence for Two Different Mechanisms

Gilles BELANGER, C,laude GODIN, and Gerald LEMIEUX

Department of Biochemistry, Faculty of Sciences, Lava1 University, Quebec

(Received April 27/July 14, 1981)

The rate of phosphate exchange in individual ribosomal proteins of Physurum polyc~eplzaluni was determined in v i w . It was observed that the phosphoryl groups ofS3. lhc major plio~pliopi-oicili. had ;I turnover r a ~ c 01' 1 .S ' I , ,

per minute. The phosphoryl groups of proteins L1, L20 and L24 were stable. These results show that the phos- phorylation of ribosomal proteins is regulated by at least two different mechanisms. The rapid turnover of phos- phoryl groups of the major phosphoprotein is in agreement with the general observation that the phosphate content of this protein is modulated by the physiological state of the cells and possibly involved in the regulation of ribosome activity. The absence of phosphate exchange in acidic proteins suggests that these groups could play a structural role in the ribosome functions.

The phosphorylation of ribosomal proteins was initially obscrved in rabbit reticulocytes and rat liver [1,2]. This phenomenon is common to all eukaryotic cells [I-l9]. In all systems, one basic protein of the small subunit, S6, or its equivalent, is the major phosphoprotein. I t has also been generally observed that the phosphate content of this protein is related to the general physiology of the cells. Inhibition [3,20 - 241, or stimulation of protein synthesis [25,26], addi- tion of hormones [12,27-341, starvation [18-351, viral infection [5,24-361, growth conditions [37-401 as well a s other factors [23,34,41,42] alter the phosphate content of this protein.

In inany systems, the presence of additional ribosomal phosphoproteins has been reported. Although the exact number of ribosomal phosphoproteins is not yet clearly established, it is now generally accepted that the eukaryotic ribosome contains more than one phosphoprotein and that the phosphate content of these proteins is not altered by con- ditions known to modify the extent of phosphorylation of S6 11,3,11,14-16,31,32,43~51]. The available results sug- gest that the phosphorylation of ribosomal proteins is not controlled by a sole mechanism and may have different roles in ribosome activity and regulation.

The precise determination of the phosphoryl groups of ribosomal proteins is one way to analyse in more detail these mechanisms. In order to study this aspect of ribosomal pro- tein phosphorylation we have chosen to use Pliysarurn poly- ceplzalunz as a model system for the investigation of events occurring during the cell cycle and differentiation [52]. We have determined precisely the rate of phosphate incorporation in each ribosomal phosphoprotein. The turnover rate of phosphoryl groups in S3 , the majbr phosphoprotein was 1.5 '?i per minute. The phosphoryl groups of acidic proteins L l , L20 and L24 were stable.

These results confirm the existence of at least two different mechanisms involved in the phosphorylation of ribosomal

f k j w w . Firefly luciferase (EC 1.13.12.7).

proteins. One controls the phosphate content of S3, and could therefore selectively alter its phosphate content in rcsponse to various stimulus. The other mechanism regulates thc phos- phorylation of acidic proteins. These phosphoryl groups are stable and should not be involved in the regulation of ribosome functions, but more likely play a structural role.

MATERIALS AND METHODS

Isolarion o f ' Ribosomes

Cultures of Plzysarum polycepkalum, strain M3C, were grown under agitation at 26°C in 500-ml baffled flasks con- taining 200 ml of semi-defined media from which KHzP04 was omitted [16]. Ribosomes were prepared as prcviously described [53]. Briefly. microplasmodia were collected by a gentle centrifugation for 2 min at 200 xg. The cells were suspended in 2.5 vol. of cold buffer TB (0.2 M sucrose, 0.1 M KCI, 0.1 M MgC12, 0.025 M EDTA, 0.05 M Tris/HCI, pH 7.2) and were broken with four strokes in a Pottcr homog- enizer equipped with a Teflon pestle. The hoinogenate was centrifuged for 10 min at 15000 x g and the post-mitochon- drial supernatant was then transferred into a 50.2 Ti rotor (Beckman) and spun for 3 11 at 49000 rev./min. The pellet, containing ribosomes and large amounts of polysaccharide, was resuspended in buffer TB and centrifuged for 30 min at 30000 rev./min. The supernatant, containing most of thc ribosomes, was collected and centrifuged for 2.5 h at 49000 rev./min.

Extraction, Reductivr Meihylaf ion and Electrophoresis of Ribosomal Proteins

Proteins were extracted with acetic acid and precipitated with 5 vol. of cold acetone [54]. Reductive methylation was performed under denaturing conditions [55]. Proteins (1 - 2.5 mg) were dissolved in 400 p1 of 8 h4 guanidinium chloride before the addition of 400 p1 of 0.04 M sodium

144

borate, pH 8.6; 80 pl of a 0.04 M formaldehyde solution was then added, followed at 30-s intervals by four successive additions of 16 p1 of NaB3H4 ( 5 mg/ml, with a specific radio- activity of 22Ci/mol). After 1 min, the treatment with formal- dehyde and sodium borohydride was repeated. After standing 10 min at 0"C, the reaction was stopped by the addition of 50 p1 of glacial acetic acid. A volume of 10 p1 of isoamyl alcohol was also added to prevent excessive foaming. This reaction was always performed in a well-ventilated hood. The solution was then desalted on a column of Sephadex G-25 (1.2 x 30 cm) equilibrated and eluted with 2 acetic acid. The fractions containing the proteins were pooled and lyophilized. In these conditions, a specific activity of 2.0 x 10' dis. min-' mg-' was routinely obtained.

The proteins were separated by two-dimensional gel electrophoresis according to Kaltschmidt and Wittmann [56], using thin gel slabs as described by Howard and Traut [57].

Incorporation qf Phosphate into Ribosomal Proteins

Cells were grown in the semi-defined media containing 100 pCi of 32P04/ml. Ribosomes were purified and the pro- teins were extracted and methylated. After two-dimensional gel electrophoresis, individual spots were cut out of the gels and digested with 0.4 ml of H 2 0 2 by incubation overnight at 50 ' C in tightly capped vials. After cooling for at least 1 h at 4 "C, 10 ml of Aquasol (New England Nuclear) was added. Vials were stored overnight in the dark at room temperature in order to eliminate chemiluminescence. Samples were then counted for 3H and 32P in a Beckman LS-350 liquid scintilla- tion counter. Results were corrected for quenching. The ratio of 32P/3H was also corrected for the specific activity of the tritium incorporated in the ribosomal proteins.

Determinacion of the Specific Activity ~f Inorganic Phosphaie and ATP

Cells were labeled with 32P04 as described in the pre- ceding section. Samples were drawn at various times and extracted with 8 % perchloric acid in 40% ethanol [58]. The supernatant was then extracted three times with chloroform/ isoamyl alcohol (24: 1, v/v) and clarified by centrifugation for 30 min at 130000 x g. The supernatant was lyophylized, and the residue was dissolved in water and assayed for ATP and inorganic phosphate.

The ATP was separated by thin-layer chromatography on poly(ethy1eneimine)-cellulose. The plates were previously washed with 0.1 M NaCI, and dried after rinsing with meth- anol. Chromatography was carried out in 1.5 M LiCl in the first dimension, and in 0.75 M KH2P04, pH 3.4 in the second dimension. The plates were washed in methanol and dried before the chromatography in the second dimension. The radioactivity was located by autoradiography. The ATP spots were cut out and counted in a scintillation counter. The amount of ATP present in the extracts was determined enzy- matically with the luciferin/luciferase system [59]. The assay was done in 50 m M Tris/acetate, pH 7.75, 1.5 mM EDTA, 0.075 'x bovine serum albumin, 10 mM magnesium acetate, 35 pM D-luciferin, 5000 U/ml luciferase. D-Luciferin and luciferase were obtained from Boehringer Mannheim Bio- chemicals. The assay was done in an integrating photometer, model 3000 from SAI Technology Co. ; 15 s after the addition of the extract, the emitted light was quantified for 1 min. In these conditions, a linear relationship was obtained for ATP concentration ranging between 1 pM and 1 nM.

The inorganic phosphate was assayed by reduction as a phosphomolybdate complex, after extraction with an organic phase [60]. An aliquot of the same extract was also used for radioactivity determination.

RESULTS

In order to study quantitatively the rate of entry of radio- active phosphate in ribosomal proteins, it was necessary to determine if exchange of phosphate could occur in the course of purification of ribosomes. Significantly higher values would be obtained if phosphate exchange took place, partic- ularly in the initial period of labeling, where the specific radio- activity of the inorganic phosphate is much greater than the specific activity of protein-bound phosphoryl groups. On the other hand, lower values would be obtained if, in the course of purification, significant dephosphorylation occurred.

The following experiment was done to determine the relative contribution of these exchange reactions. A culture was grown in presence of radioactive phosphate for six gener- ations (48 h). The ribosomes were purified and the final post- ribosomal supernatant was saved. Ribosomes were also prepared from unlabeled cells and added to the radioactive supernatant. After an incubation period of 30 min at 0 "C, these ribosomes were pelleted. Another control experiment was done by homogenizing unlabeled cells in the radioactive post-ribosomal supernatant, followed by centrifugation of the ribosomes. The experimental set-up and results aresum- marizcd in Fig.1. It can be seen that there is no important exchange or loss of phosphoryl groups present in ribosomal proteins. However, in order to check further if these small variations could result from an important variation of a single phosphoprotein, we have studied each phosphoprotein indi- vidually, after separation by two-dimensional electrophoresis. The results are presented in Table 1 and showed that there was no specific dephosphorylation of any protein.

The rate of entry of radioactive phosphate into ribosomal protcins was studied over a period of 48 h. Preliminary experi- ments showed that such a long period was necessary to obtain the steady state. Since the proteins were analysed in the two- dimensional gel system of Kaltschmidt and Wittmann [56], it was necessary to take into account the variables losses that occur by aggregation in the sample gel and the incomplete transfer of the proteins from the first-dimension to the second- dimension gel. By reductive methylation of proteins with radioactive borohydride, it was possible to quantify the amount of phosphate present in each protein. The turnover rate of the phosphoryl groups can be expressed as the frac- tion being exchanged per minute [44], if the rate of increase of specific activity of the donor is known. We have therefore determined the specific activity of 32P in ATP, the most general donor for protein kinases. The increase of the specific radioactivity of intracellular inorganic phosphate has also been determined.

The results are presented in Fig.2. It can be seen that the uptake of inorganic phosphate is relatively slow, but that its subsequent incorporation in ATP is very fast. In fact, the experimental values of Pi uptake and incorporation in ATP are similar. These results show that there is no need to study specifically the rate of entry of radioactive phosphate in the y position of the ATP molecule, the limiting step being the rate of uptake of inorganic phosphate from the growth media. Knowing the rate of entry of phosphate in ATP and in ribo- somal proteins, one can calculate the turnover rate of the

145

C e l l s t 3 2 ~ ~ 4 C e l l s

J. 4

El I Omo s" a e

homogenate

Post mi tochondr ia l supernatant J(

Post mitochondr ia1 supernatan t

C e l l s

4 supernatant (a)

JI homogenate

Post mi tochondr ia1 supernatant I

h i g h speed supernatant

Ribosor (4.1

Ribosomes "Ca" (2.1%)

h i g h speed ---* supernatant

s "Ba" 1

h i g h speed supernatant 1

Ribosomes "Ab" (90%)

Fig. 1. Diagram of',~perimenralprocedure used to determine the degree ofpho.sphaic Pscliange in rihosomol prorei~ls cl~rrir~g illcir purificuiion. Ribosomes were prepared from labeled and unlabeled cells and resuspended in different conditions, as shown. After centrifugation, the degree of phosphorylation of the individual ribosomal proteins was determined. The relative specific activity of the total ribosomal proteins is indicated under each preparation. The ribosomes prepared from labeled cells were used as a reference

Table 1, Drphosphorylution of ribosomal proteins in ihe course o f ribo- .some purification Ribosomes were prepared from cells grown in presence of 32P04 and resuspended in the high-speed supernatant obtained from cells grown in the absence of "PO4. After an incubation period of 30 min at 0"C, the radioactive ribosomes were rcisolated by centrifugation. The proteins were extracted, mcthylated, separated by two-dimensional gel electro- phoresis, and the specific radioactivity of the various phosphoproteins was determined. A more comprehensive description of this cxperiinent is presented in Fig. 1

Protein Specific rddlOdCtlvlty Of ~ ~

ribosome A ribosome Ab (Ab cf A)

dia 32P min pg-' ( %)

s3 231 209 (91) L1 2x1 243 (86) L20 504 415 (82) L24 522 490 (94)

~ -~ ~- -~

phosphoryl groups in the various phosphoproteins, by using the following equation [44] :

(ke-"' - ae-k') k - a

p = 1

where P is the specific activity at a time t , divided by its ultimate specific activity. The constant u is related to the specific activity of ATP by the following equation :

A = 1 -

Time ( h )

Fig. 2. Uptake of inorganic phosplzute I@) by cells unil its incorporation inio ATP (w), The curve was calculated for ii turnover ratc of 1 ":, per minute

where A is the specific activity of the ?-PO4 of ATP divided by its ultimate specific activity. The factor k is a rate constant equal to the fraction of phosphoryl groups turning over per minute.

Since the rate of incorporation of radioactive phosphate in acidic protein was found to be slow, the growth of the cells during the experiment had to be taken into account. A factor C can be calculated according to the following equation:

c = 1 - e-"

where C is the fraction of new cells relative to the total cell population at a time t . The constant factor c is a growth rate

146

Time ( h )

b - protein LI I I I I

c m

a

- - 2

I 48

Time (h )

c .- 3 L a

,. > Y 0

.- .- .- I

.- F " Y

._ + .-

l%

M)Od Protein L24'

I I I . . 450 - -

-

-

1 12 24 36 48

Time ( h )

constant equal to the fraction of newly stable components synthesized per minute.

The results obtained by these calculations are presented in Fig. 3. It can be seen that the rate of entry of inorganic phos- phate in protein S3 was much faster relative to the proteins L1, L20 and L24. The turnover rate was found to be 1.5 ?< per minute. The turnover rates of phosphoryl groups in acidic proteins were similar and much slower; a value of 0.1 5 was obtained. However, if one takes into account the contribution of cell growth, a growth rate constant of 0 .14x is found, based on a division period of 8 h [52]. This value is equal, within experimental error, to the turnover rates of the phos- phoryl groups in acidic proteins L1, L20 and L24, suggesting that there is no turnover of phosphoryl groups in the acidic ribosomal proteins.

S3 is the major phosphoprotein in Physaruvn polycepha- lum [16]; however, in Fig.3 it can be seen that the phosphate content of L20 and L24 was higher than that of S3. There is no direct relationship between the phosphate content of a given protein and the relative contribution of this phospho- protein to the amount of phosphoryl groups present in total ribosomal protein. Various factors may explain this apparent contradiction. The phosphate content of an individual pro- tein was expressed relative to its lysine content and the pro- portion of this amino acid may be different in the various phosphoproteins. The stoichiometry of each protein in the two-dimensional gels may not be identical and the different phosphoproteins have different molecular weights.

The turnover of phosphoryl groups of protein S9 has not been determined. This phosphoprotein contains only 3 "/, of the total phosphate present in ribosomal protein and its posi- tion in two-dimension gel does not permit a precise determi-

nation of the 32P/3H ratio since it cannot be completely separated from other proteins [I 6,531.

DISCUSSION

Before undertaking a precise determination of the turn- over rates of the phosphoryl groups in ribosomal protein, it was a prerequisite to exclude any significant exchange of these phosphoryl groups in the course of ribosome purifica- tion. The control experiments (Fig. 1 and Table 1) showed that, since no phosphate exchange occurred, the results obtained were representative of the situation prevailing in vivo.

The uptake of inorganic phosphate by the cells was found to be much slower than its exchange with ATP inside the cell (Fig. 2). However, once inside the cell, the exchange with ATP was so rapid that experimental results fit on the same curve. It was therefore no1 necessary to determine the increase in specific activity of the ultimate potent donor, the y P 0 4 of the ATP, since no lag period could be observed between the increase in specific activity of ATP, and inorganic phosphate inside the cells.

The turnover rate of phosphate in ATP was found to be 1 per minute. Kabat [44] reported a turnover rate of 97;) per minute for ATP in reticulocytes. This difference means that the whole process of uptake and exchange is faster in reticulocytes than in Physarum polycephalurn. It is very likely that the uptake of the inorganic phosphate is faster in reti- culocytes.

Thc rate of exchange of phosphoryl groups in protein S3 was found to be IS'Z per minute. Kabat calculated a turn-

147

over rate of 3'j: for the phosphoryl groups of rabbit reticulo- cyte ribosomes [44]. It must be noted, however, that the opti- mal growth temperature of P. polycephalum is 26 "C, and that Kabat incubated the reticulocytes at 37 "C. This difference accounts at least partially for the higher turnover rate of phosphoryl groups in reticulocyte ribosomal proteins.

The phosphoryl groups of acidic ribosomal proteins in P. polwphalum were found to have no significant turnover. This finding is in contrast with the results of Kabat who observed a similar turnover rate of phosphoryl groups in all ribosomal phosphoproteins of reticulocyte ribosomes [44]. However, this work had been done before the generalized use of two-dimensional gel electrophoresis [56] and, therefore, the identity of the phosphoproteins was not clearly estab- lished. It is likely that some of the phosphoproteins were not truly ribosomal because their apparent molecular weight, deduced from their mobilities in dodecyl sulfate gels, was much higher than the values obtained with proteins extracted from high-salt-washed ribosomes [45]. It is also ofinterest to note that the conclusion about the similarity of turnover rates of phos- phoryl groups in the various proteins was reached by visual inspection of the autoradiogram of the one-dimensional gels. A more recent report by Traugh and Porter [45] showed that 99 :{ of the phosphate incorporated in reticulocyte ribosomal proteins was associated with the major phosphoprotein. These workers used a two-dimensional gel analysis of proteins extracted from high-salt-washed ribosomes. However, their results do not exclude the presence of additional ribosomal phosphoproteins that would have a low turnover rate or that would be phosphorylated permanently, since the labeling period was relatively short (2 h) and reticulocytes do not synthesize ribosomes.

The presence of two different mechanisms involved in the phosphorylation of ribosomal proteins is consistent with the general observation that the phosphate content of the major phosphoprotein is related to the general physiological state of the cells and can be altered rapidly in response to various stimuli. The relatively high turnover rate of phos- phoryl groups in S3 of P. polycephulum is in agreement with these observations. On the other hand, the stability of the phosphoryl groups in the other ribosomal proteins is much greater. These findings are also in agreement with many obser- vations that the major ribosomal phosphoprotein is the only one whose phosphate content is significantly modified in relation to the physiological state of the cell, and imply that the exchange of phosphoryl groups of this protein takes place on the ribosome itself. It is therefore likely that the phosphoryl groups of acidic proteins play a structural role in the ribosome functions rather than regulate its activity. If, effectively, these phosphoryl groups have no turnover, their addition to the proteins must occur at some stage after their synthesis or in the course of ribosome assembly.

It has been observed in yeast [3] that the phosphorylation of ribosomal proteins is not associated with the synthesis or assembly of the ribosome; also, the phosphorylation of the major phosphoprotein is not associated with protein syn- thesis, but the phosphorylation of the other ribosomal pro- teins is markedly reduced when protein synthesis is inhibited.

These results are consistent with a model in which the phosphorylation of the major phosphoprotein would be regulated by a mechanism functioning at the ribosome level. This phosphorylation may control some ribosome functions. The other proteins would be phosphorylated by a different mechanism after their synthesis, and retain their phosphoryl groups permamently. This absence of turnover suggests a

structural role in thc activity of the ribosome, rather than a mechanism involved in the regulation of protein synthesis.

This work has been aupported by a grant from Natural Sciences and Engineering Research Council of Canada. We wish to thank Anne Barden and Gilles Paradis for expert technical assistance.

REFERENCES

I . Kabat, D. (1970) Biochemis/ry, Y , 4160-4175. 2. Loeh, J. E. & Blat, C. (1970) FEBS Left. 10, 105-108. 3. Zinker, S. & Warner, J. R. (1976) J . B i d . Chem. 251, 1799- 1807. 4. Becker-Ursic, D. & Davies, J. (1976) Biochemi,siry, 15, 2289-2296. 5. Kaerlcin, M. & Horak, I. (1976) Nururc, (Lond.) 2SY, 150-151. 6. Lastick, S . M., Nielsen, P. J . & McConkey, E. H. (1977) M o l . Gerz.

7. Grc5sncr. A. M. & Wool, I . G . (1974) Biochcm. Biophj1.s. RCT. Cum-

8. Bitlc. L. & Kabat, D. (1972) J . Biol. C'hem. 247. 5345-5350. 9. Rankine, A. D. & Leader, D. P. (1975) FEBS Lett. 52, 284-287.

Genei. 1.52, 223-230.

miin. 60. 1482- 1490.

10. Leader, D. P., Rankine, A. D. & Coia, A. A. (1976) Biochcm. Bio-

1 1 . Rupp, R. G., Humphrey, R. M. & Shaeffer, J . R. (1976) Biochinz.

12. Barden, N. CQ Labrie, F. (1973) Biochen7is/ry, 12, 3096 - 3102. 13. Trewavas, A. (1973) Plan1 Physiol. 51, 760-767. 14. Van Agthoven, A. J . , Madsen, J . A. & Moller, W. (1977) Biochcw.

15. Van Agthovcn, A. J., Kriek, J . , Amom, R . & Moller, W. (1978)

16. Belanger, G., Bellemare, G. & Lemicux, G . (1979) Biochem. Bio-

17. Kristiansen, K., Plesner, P. & Kruger, A. (1978) Eur. J . Biochem. 83,

18. Kristinnsen, K. ,Q Kruger, A. (1978) Biochim. Biopli.v.s. A w . 521,

19. Larsen, A. & Sypherd. P. S. (1979) Mol. Gm. Gcrzel. 175, 99- 109. 20. Treolar, M . A,, Treolar, M. E. & Kisilevsky. R. (1977) J . Rid .

21. Gressner, A. M. & Wool. 1. G. (1976) Nurure iLun(1.l 2.59, 148-150. 22. Gressner, A. M . & Wool, 1. G. (1974) Biochem. Biuphjs. Re.7. C'om-

23. Gressner, A. M. 'Pr Grciling, H. (1977) FEB'BS Lctr. 74, 77-81. 24. Kaerlein, M. & Horak, I. (1978) Eur. J . Biochem. YO, 463-469. 25. Gressner, A. M. & Wool, I . G. (1974) J . Biol. Chem. 24Y. 6917-

26. Ziv, E. C% Stratman, F. W. (1976) FEBS Lert. 68, 86-88. 27. Schubart, U . K., Shapiro, S., Fleischer, N . & Rosen, 0. M. (1977)

28. Blat, C. NL Loeb, J . E. (1971) FEBS L e / f . I K , 124- 126. 29. Gressner, A. M. & Wool, I. G. (1976) J . Bid. Chem. 251, 1500-

30. Correze, C., Pinell, P. & Nunez, J . (1972) FEBS Leit. 23. 87-91, 31. Cawthon, M. L., Bitte, L. F., Krystosek, A. & Kabat. D. (1974) .I.

32. Roberts. S. & Ashby, C. D. (1978) J . Biol. Chem. 253, 288-296. 33. Lastick, S. M. & McConkey, E. H. (1980) Biochem. Biop/zI..s. Rc.s.

34. Hasclbacher, G. K.. Humbel, R . E. & Thomas. G. (1979) FEBS

35. Kristiansen, K. & Kruger, A. (1979) E , Y ~ . CeN Res. 118, 159- 169. 36. Marvaldi, J . & Lucas-Lenard, J. (1977) Biochemisrry, 16, 4320-

37. Ixndcr, D. P. LQ Coia, A. A. (1978) Bioc,him. Rioph~. .~. Acirr, Sly,

38. Leader, D. P., Coia, A. A. & Fahmy, L. H. (1978) Bioc,hem. Bio-

39. Thomas, G., Seigmann. M., Kubler, A. M., Gordon, J. & De Ama,

plij's. Res. Commun. 71, 966 - 974.

Biopbys. Acra, 418, 81 -92.

Bioph,v.s. Rcs. Comniun. 77, 989 - 998.

Eirr. J . Biochem. Y I . 553 -556.

phys. Res. C'ommun. 86, 862-8868,

395 - 403.

43s -451,

Chem. 252, 621 7 - 6222.

~ Z U I Z . 60. 1482- 1490.

6925.

J . Biol. Chem. 252, 92 - 101.

1504.

Bid . Clicm. 24Y, 275 -278.

Commun. 95.911-922.

L ~ z I . 100, 185- 190.

4327.

224 - 232.

/?/t.\,s. Rcs. Commun. 83, SO - 58.

L. H. (1980) Cell. I Y , 1015-1023.

148

40. Thomas, G., Sicgmann, M. & Gordon, J. (1979) Prof . Nail Acad.

41. Larsen, A. & Sypherd, P. S. (1980) J . Bacteriol. 141, 20-25. 42. Martini, 0. H. W. & Kruppa, J. (1979) Ezir. J . Biochem. Y5, 349-

358. 43. Leader, D. P. (1980) in Recently Discovered Systems qf’ Enzyme

Regulation by Reversible Phosphurylalion (Cohen, P., ed.) pp. 203 - 233, Elsevier/North-Holland, Amsterdam.

44. Kabat, D. (1972) J . Biol. Chem. 247, 5338-5344. 45. Traugh, J. A. & Porter, G. G. (1976) Biochemislry, IS, 610-616. 46. Ashby, C. D. & Roberts, S. (1975) J . B id . Chcm. 250, 2546-2555. 47. Leader, D. P. & Coia, A. A. (1978) Biochim. Biophys. Acta, 519,

48. Rankine, A. D., Leader, D. P. & Coia, A. A. (1977) Biochim. Bio-

49. Horak, I. & Schiffman, D. (1977) Eur. J . Biochrm. 7Y, 375-3380. 50. Arpin, M., Madjar, J. J . & Reboud, J. P. (1978) Biochim. Biophys.

Sci. U S A , 76, 3952- 3956.

213 -223.

phys. Acta, 474, 293- 307.

Acia, 519, 537-541.

51. Tsurugi, K., Collatz, E., Todokoro, K., Ulbrich. N., Lightfoot, H. N. & Wool, I . G. (1978) J . B id . Chem. 253, 946-955.

52. Daniel, J . W. & Baldwin, H. M. (1964) in Methods in Cell Physiol- ogy (Prescot, D . M., ed.) pp. 9-14, Academic Press, New York.

53. Lemieux, G., Belanger, G., Nicole, L. & Bellemare, G. (1979) Bio- chin?. Biophys. Acta, 578, 351 -364.

54. Barritault, D., Expert-Bezanqon, A,, Guerin, M. F. & Hayes, 0. (1976) Eur. J . Biochem. 63, 131 - 135.

55. Reboud, A. M., Buisson, M., Rrpin, M. & Reboud, J. P. (1977) Biochim. Biophys. Acta, 474, 578-587.

56. Kaltschmidt, E. & Wittman, H. G. (1970) Anal. Biochem. 36, 401 - 412.

57. Howard, G. I. & Traut, R. R. (1973) FEBS Leti. 29, 177-180. 58. Bersier, D. & Braun, R. (1974) Biochim. Biophys. Acta, 340, 463-

59. Boehringer (Mannheim) GmbH (1979) Adenosine-S‘-Tripho.~phute;

60. Martin, J . B. & Doty, D. M. (1949) Anal. Chem. 21, 965-967.

471.

Bioluminescence Assay Using Lucijerase.

G. Belanger, C. Godin, and G. Lemieux, Departement de Biochimie, FacultC des Scienccs et de Genie de I’Universite Laval, Citt Universitaire, Quebec, Canada, G1K-7P4