Involvement of Glutathione in the Inhibition of Sea Urchin Egg Mitosis by Phenyl Glyoxal CHRISTOPHER M. AMY * AND LIONEL I. R E B H U N Department of Biology,Gilmer Hall, Uniuersity of Virginia, Charlottesuille, Virginia 22903

ABSTRACT Cell division in fertilized sea urchin eggs was reversibly inhibited when the ketoaldehyde phenyl glyoxal (PG) a t a concentration of 0.1 mM was added to eggs for ten minutes prior to the formation of the mitotic spindle. We investigated whether inhibition of mitosis was due to PG binding t o the cell surface (as previously suggested by Stein and Berestecky, '74) or to some intracellular effect. When 14C-PGwas added to eggs, label was readily taken up into the egg cytoplasm; very little label was associated with the egg surface. In the cytoplasm PG combined with equimolar amounts of reduced glutathione (GSH), decreasing the levels of cellular GSH to less than 15% of normal and accounting for a t least 50%of the PG taken up by eggs. The concentrations of oxidized and protein-bound glutathione were unaffected by PG treatment. We showed that glyoxalase enzymes were present in sea urchin eggs and were capable of metabolizing the PG-GSH complex, thereby restoring GSH to normal levels after PG was removed from the sea water. Though some other effect of PG cannot be ruled out, the major fate of PG in eggs was to combine with GSH, and the transient decrease in GSH which resulted could lead to inhibition of mitosis. While other reports (Nath and Rebhun, '76; Oliver et al., '76) have shown that reagents which oxidize GSH disrupt microtubule-related events, our results showed that such inhibition could be caused by decreased GSH levels alone. Phenyl glyoxal (PG) is a ketoaldehyde which inhibits mitosis when applied to a variety of cells. Gregg ('68) observed that several different glyoxals inhibited cell division in mammalian tissue culture cells and attributed this inhibition to decreased protein turnover. Klamerth ('68) believed that inhibitory effects of glyoxal on fibroblast growth were due to blocking of DNA and protein synthesis. Stein and Berestecky ('74, '75) found that PG inhibits colony growth and cell proliferation when added to HeLa cell cultures. Their results also suggested that PG binds to cell surface sites susceptible to pronase digestion. Since PG binds to arginine residues of isolated enzymes (Takahashi, '68), Stein and Berestecky ('75) proposed that PG binds to arginine groups of certain key proteins a t the cell surface. They reported that these surface sites were exposed in greater number during S, G, and M phases of the cell cycle and concluded that the surface proteins which bind PG play a crucial role in the control of cell J. CELL. PHYSIOL. (1979) 100: 187-198.

division in HeLa cells. Oliver et al. ('75) also reported that PG inhibits phagocytosis and other surface-related processes in leukocytes in a reversible manner. However, since there is no evidence that glyoxals are excluded from cells, and since they are highly reactive with sulfhydryl groups (Schubert, '351, it is possible that the effects reported by Stein and Berestecky ('74, '75) and Oliver et al. ('75) are due to the interaction of phenyl glyoxal with some intracellular sulfhydryl compoundk). In fact, glyoxals are metabolized by many cells to the corresponding a-hydroxy acids by the glyoxalase system using glutathione as a cofactor (Kosower and Kosower, '69). Thus, glyoxals could lead to a temporary and reversible decrease in glutathione levels. In light of this Received Dec. 5, '78. Accepted Feh. 13, '79. ' A preliminary report on this work was presented at the Seventeenth Annual Meeting of the American Society for Cell Biology (Amy and Rehhun, '77). Present addreas: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.

187

188

CHRISTOPHER M. AMY AND LIONEL I. REBHUN

possibility, we examined the effect of PG on mitosis in fertilized sea urchin eggs to distinguish between the possible involvement of protein surface sites and intracellular sites such as GSH. Our results showed that PG entered sea urchin eggs readily, attached to few if any surface sites, and was metabolized by the glyoxalase system. In the process of metabolism, normal cellular levels of GSH were dramatically reduced. We propose that the inhibition of cell division in these eggs is related to the removal of GSH from the intracellular pool during metabolism of PG. Since eggs were able to cleave after exogenous PG was removed, the effects of PG and the resulting decreased GSH levels are reversible. Thus, our results are consistent with the proposal that inhibition of cell division involves GSH. Though we cannot eliminate the possibility that PG may bind to some other cellular sites crucial for cell division, we also found no support for the idea that PG binding sites on the cell surface control cell division in sea urchin eggs. MATERIALS AND METHODS

Sea urchin egg treatment with PG Gametes were collected from Stronglyocentrotus purpuratus and Lytechinus pictus adults by intracoelomic injection of 0.5 M KCl, and the eggs were washed in several changes of sea water. The sea water used throughout these studies was the artificial M.B.L. formulation described by Cavanaugh (’64) adjusted to pH 7.8. Dejellied eggs were fertilized in a concentrated suspension, then diluted 10fold with sea water to prevent polyspermy and kept at 18°C with gentle stirring. Five 20-pl aliquots of the egg suspension were placed on a slide and counted under a microscope. These results were used to adjust the volume to a final concentration of 5,000 eggs per ml of sea water. The effects of PG on cleavage were examined by adding various amounts of PG to samples of 5,000 eggs per ml between 10 and 90 minutes after fertilization. Treatment was stopped by adding 15 ml of the suspension to a conical plastic tube and pelleting the eggs in a hand centrifuge. The eggs were then gently resuspended and washed in two changes of 15 ml of fresh sea water before being resuspended to their original concentration with sea water and allowed to develop further. The uptake of I4C-PG into eggs was measured using 5,000 eggs per ml unless specified otherwise. At the

end of the treatment period, a sample containing 75,000 eggs was removed and diluted to 15 ml with sea water and washed as described above. The washed egg pellets were then dissolved in 2 ml of 1 N NaOH at 70°C for two to four hours and assayed for protein by the method of Lowry et al. (‘51). Aliquots (100 and 2OOpl) of the digested egg samples were added to 5 ml of Biosolv cocktail (Humphreys, ’69) and counted in a Beckman liquid scintillation counter to determine the amount of label associated with the eggs. The amount of label in aliquots of the sea water supernatant separated from the eggs by the hand centrifugation steps was used to calculate the total radioactivity per minute per sample.

Purification of PG Radioactive PG (2-14C-phenylglyoxal monohydrate) was obtained from New England Nuclear and purified by a modification of the procedure of Takahashi (‘68) t o remove high molecular weight PG polymers. Approximately 100 pCi of 14C-PGwere dissolved in 2 ml of water and applied to a Sephadex G-10 column (1.0 X 77.5 cm) equilibrated with water. I4CPG was collected after elution with about 110 ml of water, and its purity was analyzed by thin layer chromatography as described below. Assay for glutathione Total glutathione plus oxidized and proteinbound glutathione were extracted from sea urchin eggs using the methods of Fahey et a\. (‘76). Eggs (75,000 per sample) were rapidly washed by hand centrifugation and the supernatant sea water was removed by aspiration. Egg pellets were mixed with 2 ml of 86%ethanol in 0.1 M phosphate buffer (pH 7.6) containing 5 mM ethyenediamine tetraacetate a t 70°C to extract total glutathione for a total of three minutes. The samples were cooled on ice and centrifuged in a clinical centrifuge, The glutathione in the supernatant was measured by the method of Fahey et al. (‘75) using a Beckman model 25 recording spectrophotometer. Each sample was assayed in triplicate along with an internal standard containing a known amount of GSSG. N-ethyl maleimide (100mM) was added to the extraction medium for determination of oxidized and proteinbound glutathione. Thin layer chromatography I4C-PG and its metabolites were separated

INHIBITION OF MITOSIS BY PHENYL CLYOXAL

by chromatography on silica gel 60 thin layer plates (Brinkmann) developed with n-butanol/pyridine/acetic acid/water (30:20:6:24, v/v) as described by States and Segal ('69). Standards for the identification of PG metabolite migration on thin layer chromatograms were prepared by incubating 14C-PG (0.5 mmole, 0.1 pCi) with equimolar GSH in 0.1 M phosphate buffer (pH 7.6). After a 20-minute incubation a t room temperature, 20 units of glyoxalase I were added to an aliquot of this mixture and 20 units glyoxalase I plus 200 units of glyoxalase I1 were added to another aliquot of the PG-GSH mixture. After another 20-minute incubation, the product of the three reaction tubes were chromatographed in separate channels along with '*C-PG alone. After the samples were separated and the plates were dried, radioactive compounds were located by autoradiography for one to four days with Kodak No-Screen X-ray Film. The regions containing 14C label were scraped from specific regions of the chromatogram and counted in 5 ml of toluene-based cocktail with no added solubilizer; the remaining segments of each chromatogram were also counted to obtain the total counts per minute per sample. To examine the metabolism of 14C-PGby sea urchin egg homogenates, 1ml of packed L. pictus eggs was suspended in 9 ml of 0.1 M phosphate buffer (pH 7.6) and thoroughly homogenized a t 4°C in a Dual1 glass homogenizer

189

(Kontes Glass Company). After centrifugation of the homogenate a t 20,000 g for 60 minutes at 4"C, 5O-pl aliquots of the supernat a n t were added to 50 pl of an equimolar mixture of 14C-PG and GSH (0.6 mM of each) which had been preincubated together for ten minutes. The reactions were stopped at appropriate times by addition of hot 86%ethanol as described for the glutathione assay. After centrifugation to remove precipitated protein, aliquots of the supernatants were chromatographed on silica gel plates and measured as described above.

Materials Adult S. purpuratus and L. pictus sea urchins were obtained from Pacific Biomarine, Venice, California. Unlabeled phenyl glyoxal was from K and K Laboratories. Pronase (grade B) came from Calbiochem. Glyoxalase I and 11, glutathione reductase, GSH, GSSG, Sephadex G-10, phenylmethyl sulfonylfluoride, ovomucoid and mandelic acid were from Sigma. RESULTS

Conditions for cleavage inhibition When PG was added to suspensions of L. pictus or S. purpuratus eggs at concentrations of 100 p M in sea water immediately after fertilization, mitosis was arrested before first cleavage, At concentrations of PG below 100

Fig. 1 Phenyl glyoxal effect on L.pictus eggs with mitotic spindles. Eggs about to enter second cleavage were placed under a cover slip and observed with a Zeiss Photomicroscope equipped with polarization optics. Perfusion of sea water containing 400 WMPC was begun 76 minutes postfertilization (0 time frame). Photographs taken at 0.5 minute intervals show the gradual disappearance of spindles in each of two eggs in unison over a 2-minute period. With continued exposure to PG, spindle birefringence did not reappear, and the eggs did not cleave over the next 12 minutes. Bar: 50 fim.

190

CHRISTOPHER M. AMY AND LIONEL I. REBHUN

100

80

6 hrs

a,

60 m a, u 2

3 hrs 40 20 100 min

0 20

30

40

50

60

70

80

90

minutes after f e r t i I i z a t ion Fig. 2 The effect of adding 100 rM PG toS. purpuratus eggs for 10-minute periods. The duration of treatment is represented by t h e width of each bar, and t h e first treatment was begun 20 minutes after fertilization. At the end of each 10-minute treatment, the eggs were washed with sea water and allowed to develop in sea water a t 18°C. The percentage of eggs which had completed cleavage when observed a t 100 minutes postfertilization is represented by the cross-hatched part of each bar; the first three treatments had 0%cleavage a t t h a t point. Likewise, the white (unhatched) part of each bar represents t h e percent cleavage when the eggs were observed a t three hours postfertilization. and the hatched segment shows the percentage which had cleaved six hours after the eggs were fertilized. The results graphed are the mean percentages from three experiments.

p M , the percentage of eggs which cleaved in-

creased as the concentrations were lowered. For example, no effects on cleavage were seen with 10-25p M PG, about 50%of the eggs were delayed in cleavage a t 50 p M , and 80%of the eggs did not cleave a t all a t 75 p M PG. On the other hand, when eggs were allowed to develop to the mitotic spindle stage before being exposed to 100 p M PG, about 25% of the eggs cleaved while the rest lost their mitotic apparatuses and did not form cleavage furrows. PG concentrations had to be raised to 400 p M PG in order for complete inhibition of cleavage to occur if eggs were treated a t these later stages. At this higher concentration, spindle birefringence as seen with the polarization microscope disappeared within two minutes (fig. 1). These observations suggested the possibility that some stages of the first cleavage cycle were more sensitive to PG than others. To investigate this, 100 W MPG was added to S. purpuratus eggs for ten minutes starting a t different times after fertilization and was then washed out (fig. 2).At 100 minutes postfertilization (the time when 80%of the control eggs had completed cleavage), none of the eggs

treated with any 10-minute pulse prior to 50 minutes after fertilization had cleaved. By contrast up to 25% of eggs treated for 10minute intervals between 60 and 90 minutes after fertilization cleaved (cross-hatched portion of bars, fig. 2). The period of 60 to 90 minutes postfertilization corresponds to the earliest beginning of aster formation around the zygote nucleus to late anaphase. Thus, treatment during the period when some part of the spindle apparatus was already assembled was less effective in arresting cleavage than application prior to this period. On the other hand, eggs treated before spindle assembly had occurred (i.e., prior to 50 minutes after fertilization) recovered more quickly. This can be seen in figure 2, where the percentage of eggs which had cleaved by three hours postfertilization is indicated by the height of the white or white plus cross-hatched portion of each bar. By six hours, however, a t least 90% of the eggs had cleaved (total height of the bars) although most of these had an unequal distribution of cytoplasm between the two blastomeres. Thus inhibition of cleavage could be reversed when eggs were limited to 10minute pulses of PG, although cleavage was

191

INHIBITION OF MITOSIS BY PHENYL GLYOXAL

O

L

0

10

20

i I 30

I

1

I

I

I

I

10

20

30

40

50

60

minutes Fig. 3 The uptake of labeled PG by eggs, and the loss of label from eggs treated with and without pronase after removal of labeled PG. A 30-ml suspension of L.pictus eggs (75,000eggs per ml) was exposed to ’ E- P G (40 /AM,2 pCi) for 30 minutes and 1-ml aliquots were taken at intervals, washed with sea water and prepared for counting as described in MATERIALS AND METHODS. After 30 minutes (point marked by the arrow), the remaining 24-ml egg suspension was washed and divided into two 12-ml groups. One milligram per milliliter of BSA was added to one batch (0) and an equal amount of pronase was added to the other ( X 1. Samples of eggs were removed as before for determination of radioactive label associated with eggs over the next 60 minutes. Each point is the mean of duplicate determinations.

generally not normal and reversal was delayed.

group of eggs exposed to pronase lost 30%of the initial label and thus contained about 83% of the label of control eggs incubated for the Uptake and distribution of PG same time. Examination of the pronaseWe next examined the fate of labeled PG treated eggs showed that a small number of added to suspensions of sea urchin eggs. Fer- eggs had lysed. Thus, a t least part of the label tilized L. pictus eggs were exposed to I4C-la- was released by egg breakdown or leakage. In beled PG, and the I4C-label which remained three other experiments in which labeled eggs associated with aliquots of eggs after they were treated with pronase for 30 minutes, 86 were washed with two 15-ml changes of sea 6% of the label remained associated with water was measured. As shown in figure 3, the the eggs compared to 93 -+ 5% of the label amount of label taken up by eggs reached an bound to control eggs incubated for the same apparent maximum 15 minutes after the label period without pronase; thus, pronase-treated was added. After an additional 15-minute ex- eggs lost 7%more label than control eggs durposure to labeled PG, the eggs were washed, ing a 30-minute period. resuspended in sea water, and divided into two To examine whether PG entered the egg cygroups. One group was exposed to pronase (1 toplasm, labeled eggs from these same three mg per ml) under conditions which Johnson experiments were frozen and thawed twice and Epel (‘75) have shown removes 88-92%of and centrifuged a t 27,000g for one hour. This the lZ5Ilabel added to proteins on the sea treatment completely lysed the eggs and 83 -+ urchin egg surface by lactoperoxidase iodina- 2% of the radioactive label was recovered in tion. Bovine serum albumin (BSA) was added the supernatant. Another sample of labeled to a control group. The eggs which were incu- eggs was resuspended in 100 mM PG in disbated in sea water containing BSA lost about tilled water a t pH 3.5 to duplicate the condi15%of the 14C-labelover the next hour. About tions which Stein and Berestecky (‘75) used to the same amount of label was lost when the la- demonstrate that PG was bound to HeLa cell beled eggs were washed and resuspended in surfaces. After freezing and thawing the eggs sea water containing unlabeled 100 pM PG three times and centrifuging as before, 82%of (data not shown). At the end of one hour the the total radioactivity was recovered in the

*

192

CHRISTOPHER M. AMY AND LIONEL I. REBHUN

supernatant. Stein and Berestecky (‘75) found that HeLa cells treated in this manner released only 8% of the radioactivity. Thus most of the label associated with sea urchin eggs was released when eggs were lysed, suggesting that this label was part of the soluble cytoplasm. To determine how much, if any, of the I4CPG taken up by eggs combined with protein, a homogenate of labeled eggs was dialyzed against 50 mM PIPES buffer (pH 7.0) supplemented with 0.4 mM phenylmethyl sulfonylfluoride and 0.1 mg per milliliter ovomucoid t o inhibit proteolytic enzymes. The dialyzed protein sample retained fewer than 3% of the total radioactivity after 22 hours of dialysis a t 4°C. After centrifugation of the dialysate at 100,000 g for 90 minutes, 0.7% of the total label was in the membrane pellet. Another batch of labeled S. purpuratus eggs was treated with cold 5% trichloroacetic acid to precipitate protein. After two washes of the protein pellet with additional 5% TCA, no more than 2% of the total radioactive label was recovered in the protein precipitate. Thus, W - P G did not appear to be bound to egg protein t o a significant extent either after dialysis to remove unbound label or after TCA precipitation of egg protein. We also examined the rate of uptake of labeled PG as a function of time after fertilization. When eggs were exposed to 5-minUte pulses of I4C-PG (200 p M ) a t intervals through first cleavage in two separate experiments, the rate of uptake of label in the samples remained constant, varying by less than 7% from the mean value of 6 pmoles of PG taken up per milligram protein per minute through first cleavage (data not shown). These results suggested that PG entered sea urchin eggs readily and that the lowered sensitivity t o PG during mitosis was not due to changes in the rate of uptake of the inhibitor. Glutathione levels and PG Since glyoxals are known to be metabolized by glyoxalase enzymes (Racker, ’511, we examined egg cytoplasm for this enzyme system. If it were present, then each PG molecule transformed to mandelic acid (the a-keto acid corresponding to phenyl glyoxal) by these enzymes would require one molecule of GSH as a cofactor. The GSH would then be released unchanged a t the completion of these enzymatic steps as shown in figure 4. We therefore assayed the GSH levels in treated eggs to

determine whether they were affected by PG. When PG was added to S. purpuratus eggs for 10 minutes beginning 60 minutes postfertilization at concentrations from 10-200 pM, total glutathione (oxidized plus reduced glutathione) levels were reduced by amounts which increased with increasing PG concentration (fig. 5 ) . Phenyl glyoxal levels as low as 25 pM decreased total glutathione by almost 20%of the amount in untreated eggs while 100 pM lowered cellular levels by 85%during the 10-minute exposure. Oxidized glutathione (GSSG) and protein-bound g l u t a t h i o n e (GSSP) levels were determined in untreated eggs and found t o be only one-hundredth and one-tenth as great as the total glutathione levels in untreated eggs respectively. These numbers agree with the results of Fahey et al. (‘76) for the same species of egg. Since the GSSG levels account for less than 1%of total glutathione, reduced glutathione (GSH) was essentially equal to the total glutathione value. Neither GSSG nor GSSP levels in eggs were altered by treatment with various concentrations of PG. Thus, PG caused a rapid and great decrease in GSH levels in these eggs, and the lower GSH levels were not accompanied by higher levels of GSSG or GSSP. Samples of eggs from this experiment were examined after 90% of control eggs had cleaved. As noted earlier, normal cleavage was observed with 10 p M and 25 p M concentrations while 50 pM PG delayed cleavage in about 50% of the eggs. Only 20% of the eggs treated with 75 pM PG had cleaved. None of the eggs treated continuously with 100 pM or higher had cleaved after several hours of incubation. Thus, 50 pM PG caused a decrease of about 40-60%in the GSH level in cells during the period when the mitotic apparatus was beginning to form and was accompanied by cleavage delay in half of the treated eggs. We next examined the effect of phenyl glyoxal on the GSH levels of eggs with time. Labeled PG (100 pM) was added to a suspension of sea urchin eggs, and aliquots of eggs were removed a t intervals before and after the cells were washed free of unbound PG (fig. 6). Untreated eggs maintained a constant level of GSH throughout a 2-hour period of about 24 pmoles of GSH per g protein. This GSH concentration was the equivalent of 3.0 mM GSH in the egg cytoplasm. GSH levels dropped steadily in eggs treated with PG, reaching a level of 2.2 pmoles per gram protein or 10%of the control value after 12 minutes. When the

193

INHIBITION OF MITOSIS BY PHENYL GLYOXAL

c = o GSH L , c=o

Gly

I

, . .~

-,

,

--n

Phenyl glyoxal

Mandelic acid

Fig. 4 Pathway of metabolism of phenyl glyoxal by glyoxalase enzymes.

GSSP

41

:;I-,

0' '

'

.-.-.-. ,

0

0

50

1

I

,

, 100

I

;>;

150

1

200

pM PG Fig. 5 The effects of phenyl glyoxal on glutathione concentrations in eggs. Concentrations of PG ranging from 10-200pM were added to samples of S.purpuratus eggs (75,000 eggs per 15 ml) 60 minutes after fertilization. At the end of the 10-minute treatment, each sample was washed and extracted with ethanol plus or minus NEM for determination of GSH, GSSG and GSSP aa described in MATERIALS AND METHODS. The results are expressed as pmoles of GSH, GSSG or GSSP per g of protein in the whole egg sample.

194

CHRISTOPHER M. AMY AND LIONEL I. REBHUN

30

I

Y

0

10

20

30 minutes

60

90

0

120

Fig. 6 The fate of labeled phenyl glyoxal added t o eggs and its effect on the GSH levels of eggs. Y - P G (100wM, 0.6 Fci) was added to a suspension of S. purpuratus eggs (5,000 eggs per ml, 200 ml) 60 minutes after fertilization. After 15 minutes (point marked by the arrow), the eggs were washed with sea water to remove unbound PG and resuspended in sea water. The amount of GSH (&moles per g protein) extracted from 15-ml aliquots of PG treated (0) and untreated ( 0 )eggs were determined at intervals. The radioactive label which was associated with the eggs ( x ) is expressed as the percent of the total cpm in each aliquot of eggs.

eggs were washed free of exogenous PG after 15 minutes, the GSH levels rose, reaching a level approximately equal to that of the untreated eggs after a 15-minute period without PG. When the amount of the 14C-labeledmaterial in eggs was determined a t each time point, there was a sharp rise in the amount of label extracted from eggs exposed to labeled PG. As can be seen, GSH decrease and 14Clabel uptake are essentially reciprocal events. After I4C-PGwas removed from the sea water medium, the amount of label which remained associated with t h e eggs was constant through the end of the experiment as was also seen in the experiment described in figure 2. During this period, the extractable radioactivity was about 8.1% of the total label originally added to the eggs and was equivalent to roughly 6 mM PG within the egg cytoplasm. Greater than 95%of the label associated with eggs was extracted with hot 86%ethanol in this experiment. Metabolism of FG ' These results supported the suggestion that PG was entering the egg cytoplasm, combin-

ing with GSH, and being metabolized. Although the nature of the metabolite (retained label) was not known, the most reasonable hypothesis was that the glyoxalase system was involved. To test this possibility, we first established that the products of the glyoxalase enzymes could be separated by thin layer chromatography. When purified 14C-PGwas tested in the solvent system described in MATERIALS AND METHODS, 79% of the total radioactivity recovered from the silica gel plate appeared in a single spot with a n Rf of 0.71. Two minor spots were also present which ran slightly slower than the major spot and accounted for the remaining 21%of the label. Since glyoxals can polymerize (Takahashi, '68), it is likely that the slower spots are small polymers of PG since these spots returned on rechromatography of the main spot. When equimolar GSH was incubated with 14C-PG, the major spot (36%of total cpm) was still at an Rf of 0.71, and a second spot appeared at a n Rf of 0.21 with 26%of the total cpm (fig. 7a). This is likely to be GSH-PG hemimercaptal, the spontaneous adduct of PG and GSH. When glyoxalase I was added to PG plus GSH, the major

195

INHIBITION OF MITOSIS BY PHENYL GLYOXAL

OL'

0

'

2

I

I

5

10 minutes

20

Fig. 7 The metabolism of I C P G by t h e glyoxalase enzymes and by egg homogenates. a A trace of t h e autoradiograph prepared from a chromatogram of the various intermediates of glyoxalase metabolism prepared as described in MATERIALS AND METHODS. PG alone was run in the first channel followed by t h e PG-GSH mixture and the products of t h e glyoxalase I (gly I) reaction with the PGGSH complex. The last channel contained the product of glyoxalase I1 action on the mixture of the other components. Distinct spots are indicated by solid lines while weakly radioactive spots are surrounded by dotted lines; the origin ( 0 ) and t h e solvent front (f)are also indicated. b A graph of t h e progressive metabolism of ' C - P G by a n L. pictus egg homogenate as described in MATERIALS AND METHODS. Samples were taken after various periods a t 25°C. The radioactivity was expressed a8 t h e percent of t h e total radioactivity from each channel which appeared in the PG ( x ) spot and t h e spots characteristic of the glyoxalase I product ( 0 )and t h e glyoxalase I1 reaction (0).

spot appeared a t an Rf of 0.21 (58%of total cpm) along with a minor spot which co-migrated with PG indicating a catalytic effect of the enzyme on formation of GSH hemimercaptal (Friedman, '73). Finally, the addition of glyoxalase I1 to the other components produced a single major spot a t an Rf of 0.41. This labeled material co-migrated with authentic mandelic acid and contained 72% of the total label recovered from the sample added to the silica gel plate. This separation technique used in conjunction with the standards generated by the glyoxalase enzymes allowed us to analyze the products formed after PG was added to a cell homogenate preparation (fig. 7b). Though material extracted from the egg homogenate along with the labeled compounds interfered with the separation procedure to some extent, the percent of the total radioactivity present as PG a t each time point decreased over the 20-minute period from 36% to 5%.The spots which co-migrated with the products of the glyoxalase I and I1 reactions rose correspondingly over the same period. The remain-

ing radioactive label was located a t the origin and a t the region which contains the presumed PG polymers. The percentage of total cpm in each of these regions of the chromatogram remained constant throughout the experiment. The same thin layer chromatography system was used to analyze the material extracted from whole eggs treated with labeled PG. Though we were unable to separate the labeled compounds on thin layer plates as we had done for the egg homogenates because of extensive smearing, it was possible to show that less than 3%of the total label taken up by eggs was associated with the protein precipitated by the ethanol extraction procedure. DISCUSSION

In searching for the biological basis for the inhibition of cleavage by phenyl glyoxal, our first objective was to examine the possibility raised by a previous study that this ketoaldehyde affected cell division by altering proteins a t the cell surface. Unlike Stein and Berestecky ('741, we found that extracellular

196

CHRISTOPHER M. AMY AND LIONEL I. REBHUN

proteolytic enzyme digestion removed only a ated with eggs for a t least two hours after the small proportion of labeled PG associated with removal of labeled PG from the sea water reeggs even though Johnson and Epel ('75) have mained a t a high level. These facts were conshown that pronase digestion removed 90% of sistent with the hypothesis that the glyoxlZ5Iadded to the surface of sea urchin eggs by alase enzymes were converting the GSH-PG lactoperoxidase iodination. Also, proteolytic hemimercaptal to labeled mandelic acid, enzyme treatment has been shown to cause thereby restoring the normal intracellular other types of cells to become leaky to a va- GSH concentration. When we added up to 500 riety of materials such as cell actin (Rubin et K M mandelic acid to suspensions of fertilized al., '78). It is likely, therefore, that leakiness eggs, cleavage was unaffected (unpublished plus some egg lysis accounted for the pronase- results). An endogenous enzyme system thus induced loss of egg-associated label. Further, eliminated t.he potentially toxic ketoaldehyde lysing eggs by freezing and thawing caused from the eggs in exchange for the apparently the release of over 80%of the radioactive label innocuous a-hydroxy acid product, mandelic into the supernatant, suggesting that this la- acid. This occurred a t the expense of a subbel was soluble and not membrane bound. Ex- stantial temporary reduction in GSH levels periments with hot ethanol or TCA extraction with no significant change in GSSG of GSSP showed that less than 5% and probably less levels. We propose that i t is the decreased inthan 2%of the label in whole eggs was associ- tracellular concentration of GSH during early ated with macromolecules. While these re- development rather than a direct phenyl glysults cannot completely rule out the possi- oxal effect on proteins which led to the inhibility that the phenyl glyoxal effect on bition of cleavage. cleavage was due to its binding to a cell surSeveral observations support this contenface protein crucial for cell division, they ren- tion. First, eggs treated with PG for periods of dered it unlikely and led us to examine the in- 10 or 15 minutes could recover and cleave at a teractions between PG and non-protein cyto- later time, although considerable delay was plasmic components. In addition, since PG experienced. This demonstrated that phenyl reacts with sulfhydryl groups (Schubert, '35) glyoxal did not have an irreversible toxic efas well as with arginine (Takahashi, '681, we fect on the formation of the mitotic and focused attention on possible interactions cleavage apparatuses, a t least when it was apwith sulfhydryl groups. plied for short periods of time. Second, prepaThe major non-protein sulfhydryl compound rations of soluble components of egg homogein the cytoplasm of sea urchin eggs and other nates were able t o metabolize phenyl glyoxal animal cells is reduced glutathione (Kosower in vitro, confirming that the glyoxalase enand Kosower, '69). This led us to examine the zymes were capable of forming mandelic acid possibility that added PG was entering the and GSH from the GSH-PG hemimercaptal. Additional support for the proposal that eggs and reacting with GSH. We found that GSH levels were reduced by approximately 85- cleavage inhibition by PG may be due to a de90% when eggs were exposed to PG for ten crease in GSH levels comes from other investiminutes at concentrations which inhibited gations into the role of GSH in microtubulecleavage. When 14C-PGwas added to eggs, la- dependent processes, including cleavage. bel became associated with the cells at the Nath and Rebhun ('76) showed that diamide, same rate as GSH was lost from eggs as long as which oxidizes GSH to GSSG, inhibits cleavPG remained i n the sea water medium. De- age in sea urchin eggs. Oliver e t al. ('76) have pending on the experiment, between 50 and shown that both diamide and tertiary butyl 85% of the 14C-PGtaken up by eggs could be hydroperoxide (BHP) promote capping in accounted for by the decreased levels of GSH human neutrophil cells, apparently by causafter a 15-minute incubation assuming a one ing the disassembly of preformed cytoplasmic to one interaction between the two com- microtubules as well as preventing their pounds. Clearly, the major initial fate of PG assembly from tubulin subunits. We also obtaken up into sea urchin eggs was to form a served the disappearance of microtubule biGSH-PG complex. refringence within two minutes after the adAfter extracellular PG was removed from dition of PG to eggs containing mitotic spinthe sea water following a 15-minute incuba- dles. Though phenyl glyoxal, diamide and tion, the GSH levels gradually returned to BHP all lower GSH levels in cells, a crucial normal. However, the amount of label associ- difference between their effects is that both

INHIBITION OF MITOSIS BY PHENYL GLYOXAL

diamide and BHP oxidize GSH to GSSG, while phenyl glyoxal has no effect on either GSSG or protein-bound glutathione levels in cells. Thus, phenyl glyoxal is the only one of these compounds whose effects cannot be due to a rise in GSSG levels rather than a decrease in GSH levels. It is well established that GSH levels do not change appreciably throughout early sea urchin development (Fahey et al., '761, and it therefore seems logical that the glutathione status of developing eggs has no effect on the timing of cleavage. Though we have yet to learn how alterations in the glutathione status of eggs interferes with normal cleavage, the results reported here and the studies discussed above with GSH oxidizing agents suggest that the maintenance of a uniform level of GSH within cells is required for the polymerization of tubulin into microtubules as well as for the stability of existing microtubules. A model for this has been presented in previous papers (Rebhun, '78; Rebhun et al., '76) which suggested that appropriate target proteins containing sulfhydryl groups can be regulated in their sulfhydryl-disulfide status through interactions with a reductase and a source of reducing power. If the reductase is inhibited or the "hydrogen battery" (which may be GSH or NADPH) is depleted, the target protein can be changed in redox status. Since oxidation or blockage or the sulfhydryls of tubulin inhibits its polymerization (Mellon and Rebhun, '76; Kuryama and Sakai, '74; Ikeda and Steiner, '78), some tubulin sulfhydryl groups must be in reduced form for polymerization to occur. Thus, the PG-induced reduction in GSH could lead to a shift in equilibrium of tubulin to a more oxidized and thus less polymerized stage, resulting in spindle disassembly. This model differs somewhat from that proposed by Burchill et al. ('78) which suggests that in polymorphonuclear leukocytes disassembly of microtubules may be correlated with an increase in proteinbound glutathione. Since no increase in either GSSP or GSSG was observed in eggs treated with PG, this explanation cannot apply to the present case. Since, however, cells may use their ability to regulate redox state in many different ways (Rebhun, '781, the results with eggs compared with those with polymorphonuclear leukocytes are not necessarily contradictory. Though we have shown that PG dramatically alters the levels of GSH in sea urchin eggs,

197

it must be emphasized that this ketoaldehyde has the potential to react with many components of cells. We cannot yet say which of these potential targets for PG binding in cells leads to cleavage inhibition. LITERATURE CITED Amy, C. M., and L. I. Rebhun 1977 Inhibition of sea urchin egg cleavage by phenyl glyoxal. J. Cell Biol., 75: 34 (Abstract). Burchill, B. R., J. M. Oliver, C. B. Pearson, E. D. Leinbach and R. D. Berlin 1978 Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes. J. Cell Biol., 76: 439-447. Cavanaugh, G. M. 1964 Formulae and Methods of the Marine Biological Laboratory Chemical Room. Marine Biological Laboratory, Woods Hole, Massachusetts. Fahey, R. C., S. B r d y and S. D. Mikolajaczyk 1975 Changes in the glutathione thiol-disulfide status of Neurospora crassa conidia during germination and aging. J. Bact., 121: 144-151. Fahey, R. C., S.D. Mikolajczyk, P. G. Meier, D. Epel and E. J. Carroll 1976 The glutathione thiol-disulfide status in the sea urchin egg during fertilization and the first cell division cycle. Biochim. Biophys. Acta, 437: 445-453. Friedman, M. 1973 The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and Proteins. Pergamon Press, Oxford. Gregg, C. T. 1968 Inhibition of mammalian cell division by glyoxals. Exp. Cell Res., 50: 65-72. Humphreys, T. 1969 Efficiency of translation of mRNA before and after fertilization in sea urchins. Devel. Biol., 20: 435-458. Ikeda, Y., and M. Steiner 1978 Sulfhydryls of platelet tubulin: their role in polymerization and colchicine binding. Biochemistry, 17: 3454-3459. Johnson, J.,and D. Epel 1975 Relationship between release of surface proteins and metabolic activation of sea urchin eggs a t fertilization. Proc. Nat. Acad. Sci., 72: 4474-4478. Klamerth, 0. L. 1968 Influence of glyoxal on cell function. Biochim. Biophys. Acta, 155: 271-279. Kosower, E. M., and N. S. Kosower 1969 Lest I forget thee, glutathione.. . Nature, 224: 117-120. Kuryama, R., and H. Sakai 1974 Role of tubulin -SH groups in polymerization in microtubules. Functional -SH groups in tubulin for polymerization. J. Biochem., 76: 651-654. Lowry, 0. H., N. J. Rosebrough, A. B. Farr and R. J. Randell 1951 Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193: 265-275. Mellon, M. G., and L. I. Rebhun 1976 Sulfhydryls and the in vitro polymerization of tubulin. J. Cell Biol., 70: 226-238. Nath, J., and L. I. Rebhun 1976 Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs. Involvement of NADP' and glutathione. J. Cell Biol., 68: 440-450. Oliver, J. M., D. F. Albertini and R. D. Berlin 1976 Effects of glutathione oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils. J. Cell Biol., 71: 921-932. Oliver, J. M., H. H. Yin and R. D. Berlin 1975 Control of the lateral mobility of membrane proteins. In: Leukocyte Membrane Determinants Regulating Immune Reactivity. V. P. Eijsvoogel, ed. Academic Press, New York, pp. 3-14. Racker, E. 1951 The mechanism of action of glyoxalase. J. Biol. Chem., 190: 685-696. Rebhun, L. I. 1978 Sulfhydryl-disulfide status and state

198

CHRISTOPHER M. AMY AND LIONEL I. REBHUN

transitions in cells. In: Cell Reproduction. E. Dirksen, D. Prescott and L. Goldstein, eds. Academic Press, New York, pp. 547-556. Rebhun, L. I., M. Miller, T. C. Schnaitman, J. Nath and M. Mellon 1976 Cyclic nucleotides, thiodisulfide status of proteins, and cellular control processes. J. Supramol. Struct., 5: 199-219. Rubin, R. W., R. H. Warren, D. S. Lukeman and E. Clements 1978 Actin content and organization in normal and transformed cells in culture. J. Cell Biol., 78: 28-35. Schubert, M. P. 1935 Combination of thiol acids with methyl glyoxal. J. Biol. Chem., 111: 671-678.

States, B., and S. Segal 1969 Thin-layer chromatographic separation of cystine and the NEM adducts of crysteine and glutathione. Anal. Biochem., 27: 323-329. Stein, S. M., and J. M. Berestecky 1974 Inhibition of growth by masking of arginine moieties in protein a t the cell surface. Cancer Res., 34: 3112-3116. 1975 Exposure of a n arginine-rich protein at the surface of cells in S, G, and M phases of the cell cycle. J. Cell. Physiol., 85: 243-250. Takahashi, K. 1968 The reaction ofphenyl glyoxal with arginine residues in proteins. J. Biol. Chem., 243: 6171-6179.