Capture of glycerol by cells of Escherichia coli

BIOCHIMICA ET BIOPHYSICA ACTA

479

BBA 45116

C A P T U R E OF G L Y C E R O L BY CELLS OF E S C H E R I C H I A COLI S. HAYASH]: AND E. C. C. L I N

Department of Biological Chemistry, Harvard Medical School, Boston, Mass. (U.S.A.) (Received J u n e 5th, I964)

SUMMARY*

I. Cells of five independent mutants of Escherichia coli K I 2 which lack glycerol kinase (EC 2.7.1.3o ) were all unable to accumulate radioactive material when incubated with [14CJglycerol, whereas wild-type cells were able to do so. 2. Cells of a m u t a n t lacking L-a-glycerophosphate dehydrogenase (EC 1.1.1.8) but possessing glycerol kinase retained the ability to accumulate radioactive material when exposed to labeled glycerol. Upon analysis of an extract from such cells, virtually all of the radioactive substance was found to be L-c¢-glycerophosphate and none to be :free glycerol. 3. A ,~ensitive method has been developed for the assay of glycerol kinase at low substrate concentrations. I t was shown that the Km of glycerol kinase (1.3 ~M) corresponded fairly closely to the "Kin" of intact cells (0. 9/~M). 4. Results obtained in this study showed that these cells do not possess an active tran,;port system for glycerol, that even at very low concentrations the entry of glycerol b y free diffusion is not rate-limiting for growth and that the kinase is responsible for the capture of the compound from the growth medium.

INTRODUCTION

Studies of its osmotic effects have shown that glycerol readily penetrates bacterial cells. As early as I9o 3, FISCHER1 observed that hypertonic solutions of glycerol did not produce plasmolysis in such organisms. Later MITCHELLAND MOYLE ~, and more recently BOVELL et al), showed that when glycerol was suddenly added to suspensions of Escherichia coli, osmotic equilibrium across the cell membrane was so quickly established that no significant change in cell volume could be detected b y measuring light scattering. BRITTEN AND McCLURE4 found that sudden exposure to an isotonic solution of glycerol was equally effective as that to distilled water in leaching sm all molecules from cells of E. coli by osmotic shock, indicating that glycerol entered too rapidly to act as an osmotic protector. Although free diffusion seems to account for the rapid entrance of glycerol * The t e r m active t r a n s p o r t s y s t e m is used to designate a stereospecific m e c h a n i s m which enables the cell to accumulate a c o m p o u n d a g a i n s t electrochemical gradient a t least u n d e r certain conditions. The t e r m facilitated diffusion is used to designate a catalyzed stereospecific p r o c e s s which leads only to equilibration of a c o m p o u n d across the cell m e m b r a n e . E x p r e s s i o n s such as. free diffusion or to diffuse freely are used to describe the r a p i d equilibration across the cell m e m b r a n e w i t h o u t catalysis.

Biochim. Biophys. Acta, 94 (1965) 479-48T

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s. HAYASHI, E. C. C. LIN

observed in the above experiments with high concentrations of the compound, the intervention of a carrier-mediated mechanism, i.e. active transport or facilitated diffusion, might still be necessary for the utilization of this compound at growthlimiting concentrations. The primary aim of the present study is to determine whether cells of E. coli can actively transport glycerol, and if not, how these cells can effectively capture glycerol at low concentrations. MATERIALS AND METHODS

Chemicals Glycerol was obtained from Merck and Co.; di-(cyclohexyl)ammonium-L-~glycerophosphate from Calbiochem, Inc.; DL-e-glycerophosphate and ATP from the Sigma Chemical Co. ; bovine serum albumin from Pentex Inc.; and vitamin-free casein acid hydrolysate from the Nutritional Biochemical Corp. [I,3-14C21Gly cer°l (4 C/mole) was purchased from the New England Nuclear Corp. L-cz-[I,3-14C21Glycerophosphate was synthesized enzymatically by incubation of labeled glycerol with ATP and glycerol kinase (EC 2.7.1.3o) from rat liver and was purified by means of a Dowex-I-formate column 5. The yield, as determined with L-~-glycerophosphate dehydrogenase (EC 1.1.1.8) from rabbit muscle 6, was found to be 7 ° % of the initial glycerol. Bacteria The phenotypic characteristics and the lineage of the four strains used in this study are summarized in Table I. Strain I came from Dr. C. LEVINTHAL'Slaboratory (referred to there as strain EI5). I t has a deletion in its structural gene for alkaline phosphatase (EC 3.I.3.I), and was obtained from E. coli KIO (refs. 7, 8) which, in turn, was derived from E. coli K I 2 Hfr CAVALLI-SFORZA9. The remaining three strains were all derived from Strain I by procedures which have been previously described 10-12. TABLE I CHARACTERIZATION

OF BACTERIAL

STRAINS

All s t r a i n s are a l k a l i n e p h o s p h a t a s e n e g a t i v e . E n z y m e a c t i v i t i e s were a s s a y e d on cell-free e x t r a c t s a n d L - ~ - g l y c e r o p h o s p h a t e t r a n s p o r t was m e a s u r e d in s u s p e n d e d w hol e cells.

Strain

Parent strain

Mutagenesis

Glycerol kinase

L-~-glycerophosphate L-~-glycerophosphate dehydrogenase transport

i 4" 7 8

KIo i i 7

X-ray EMS** EMS EMS

Inducible Negative Constitutive Constitutive

Inducible Inducible Constitutive Negative

Inducible Inducible Constitutive Constitutive

* R e f e r r e d to as s t r a i n T3 in a p r e v i o u s p a p e r n . ** E t h y l m e t h a n e s u l f o n a t e .

General conditions employed for the growth of bacteria The basal medium contained inorganic ingredients and Tris-HC1 buffer (pH 7.5) as described by GAREN AND LEVINTHAL7. Inorganic phosphate was present at 0.6 raM. All the cultures were incubated at 37 ° on a rotary shaker operated at about 200 ~Biochim. Biophys. Acta, 94 (1965) 479-487

C A P T U R E OF GLYCEROL B Y

E. co~

481

cycles/min. ,Growth was monitored turbidimetrically in a Klett colorimeter with a 42o-m/~ filter. The concentration of bacterial suspensions was calculated from their turbidity and was expressed as ~g of dry wt. per ml.

Measurement of growth rates Growth rates on glycerol and on DL-a-glycerophosphate* as a function of substrate concentration were determined by counting viable cells instead of by measuring turbidity, since the low concentrations of carbon sources which had to be used permitted very limited ranges of growth. Cells of Strain I growing logarithmically in o.o2 M glycerol or in 0.04 M DL-a-glycerophosphate were washed and reinoculated at a level of 500 per ml in io ml of basal medium (pH 6.5) containing various concentrations of the carbon source (each in duplicate) on which the cells had been pregrown. The cultures were incubated in 2 × 15 cm tubes, and at hourly intervals aliquots were withdrawn, appropriately diluted, and plated in triplicate on rich agar. Because of the small inoculum of cells used it was possible to obtain several doublings in each medium without significant change in the concentration of the carbon source. This method permits the measurement of growth rate in concentrations of glycerol as low as o.i ~M.

Assay of upt,ake of [14C]glycerol by intact cells Cells which had been grown for about 6 generations on i % casein hydrolysate in the presence of 0.02 M glycerol and 0.04 M DL-a-glycerophosphate were collected while in logarithmic phase, washed and suspended at a concentration of 4 ° ~g dry wt. per ml in basal medium. The suspension was brought to 37 ° and [14Clglycerol was added 1:o give a concentration of 5/~M. At various intervals i-ml aliquots of this incubation :mixture were withdrawn. The cells were collected on Millipore filter (0.65 /, pore size and 25 mm diameter) and washed with IO ml of basal medium with suction. The filtration and washing were completed within IO sec. The filter discs were attached to planchets and dried, and the radioactivity on them was measured in an end-window gas-flow counter. Selfabsorption was negligible for the quantity of cells used.

Assay o[ L-,z-glycerophosphate transport Cells grown on 1% casein hydrolysate were collected by centrifugation when the culture reached 200-250 Klett units. They were washed, suspended in basal medium at 40 ~g dry wt. per ml and incubated in various concentrations of L-a-E14Clglycerophosphate at 15 °. After I rain an aliquot of I ml was withdrawn, and the cells were prepared for the measurement of radioactivity as described above.

Preparatio~'~ of cell-free extract As a source of glycerol kinase and L-a-glycerophosphate dehydrogenase for kinetic studies, cells of Strain 7, which constitutively produce high levels of these enzymes, were grown in a 2-1 erlenmeyer flask containing 500 ml of basal medium supplemented with 1% casein hydrolysate. When the turbidity of the culture reached * P r e v i o u s s t u d i e s s h o w e d t h a t D - ~ - g l y c e r o p h o s p h a t e c a n s e r v e n e i t h e r a s a s u b s t r a t e for t h e L - a - g l y c e r o p h o s p h a t e t r a n s p o r t s y s t e m n o r a s a s o u r c e of c a r b o n a n d e n e r g y for cells of E. coli K I O (refs. I:~, 12).

Biochim. Biophys. Acta, 94 (1965) 4 7 9 - 4 8 7

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S. HAYASHI, E. C. C. LIN

200 I(lett units, the cells were collected, washed with i % NaC1, suspended in 5 ml of o.I M Tris-HC1 buffer (pH 7.5) and disrupted in a model-6oW MSE ultrasonic disintegrator while chilled in a --IO ° bath. The resulting suspension was centrifuged at 35000 × g for 15 min and the supernatant cell-free extract was used for enzyme assays.

Enzyme assays The following assay for glycerol kinase was devised in order to obtain a sufficiently sensitive method for accurate measurement of the Km for glycerol. The reaction was carried out at a temperature of 37 ° and at a pH of 6. 5 (probably close to intracellular pH) with [~4Clglycerol at the desired concentration, 0.2 mM ATP, I mM MgC12 and o.I mg bovine serum albumin per ml. The last component was employed to stabilize glycerol kinase. In order to avoid excessive precipitation of salts at the step of isopropanol treatment, additional buffer was not employed. The reaction was initiated by the addition of the cell extract which had been diluted to a convenient concentration with a solution of bovine serum albumin (o.I mg/ml at pH 6.5). At various intervals I-ml aliquots were withdrawn and mixed with 3 ml of cold 5 ° % isopropanol saturated with lead acetate. Immediately afterwards 0.05 ml of o.I M carrier I)L-a-glycerophosphate was added. The mixture was kept at o ° for 30 rain after which time it was again thoroughly shaken and an aliquot of 2 ml was filtered through Millipore paper (0.65 /~ pore size and 25 mm diameter). The retained precipitate was washed with I ml of the isopropano] solution. The filter paper was fastened firmly to a planchet by a piece of Scotch tape with adhesive on both surfaces, dried under a heating lamp, and the radioactivity on it was measured in an endwindow gas-flow counter. The recovery of L-a-glycerophosphate by this method was found to be 7 ° % when tested by adding L-~-[l*C]glycerophosphate to the assay mixture in place of labeled glycerol. Under the standard conditions employed for the assay, formation of L-a-glycerophosphate proceeded linearly for at least 4 ° rain (Fig. IA) and was proportional to enzyme concentration over a I5-fold range (Fig. IB). .o.

3

0.2 ~I

/

~o

,~,

2'0

INCUBATION

/o TIME

(rain)

4'o

o o. 0.1 tic ~o

0.5 BACTERIAL

LO ' Ii 5 PROTEIN (.,ug/ml)

Fig. I. Glycerol kinase. (A) Kinetics. (13) A c t i v i t y as a f u n c t i o n of e n z y m e c o n c e n t r a t i o n .

The L-~-glycerophosphate dehydrogenase, which is not DPN-linked, was measured by a method which depends on the reduction of a tetrazolium dye to its formazan. This method has been described previously 11 except that in the present study L-~-glycerophosphate was employed as the substrate instead of the racemic mixture. Biochim. Biophys. Acta, 94 (1965) 4 7 9 - 4 8 7

483

CAPTURE OF GLYCEROL BY E. coli

Identification of the radioactive substance in cells incubated with [lffSlglycerol Paper chromatography was used to analyze the chemical nature of the radioactive material accumulated by cells of Strain 8 during their brief incubation with Et4C~glycerol. Cells grown on 1% casein hydrolysate to stationary phase were suspended in 5° ml of basal medium at a concentration of 40 t~g dry wt. per ml. To this suspension [laC~glycerol was added to give a final concentration of IO /zM. After 5 rain of incubation at 3°0 the cells were collected on a Millipore filter (0.65/* pore size and 47 mm diameter) and washed with 20 ml of basal medium with suction. The filter disc assembly was transferred to a clean suction flask and the cells on the filter were washed with 20 ml of cold distilled water. This procedure has been reported to leach small molecules from cells of E. coli ~ and has been observed to release more than 95 % of the cold-trichloroacetic acid-soluble fraction of the total radioactive material in our system. The filtrate was concentrated in vacuo and applied on Whatman No. I paper along with the standards: ~t4C~glycerol and L-a-I14Clglycerophosphate. The chromatography was carried out at 25 ° with a solvent system composed of methyl cellosolve-rnethyl ethyl ketone-3 N NHaOH (7 : 2 : 3, v/v) ~3. The paper was dried and cut into strips perpendicular to the solvent front and the radioactive spots were located by a Nuclear Chicago automatic chromatogram scanner. RESULTS

Test for accumulation of [14C~glycerol The p~ssible existence of an active transport system for glycerol was first tested in Strain 4, a mutant which lacks the kinase for glycerol and therefore cannot metabolize this compound. To ensure that such a transport system would be formed, if it did exist, the cells were grown on casein hydrolysate in the presence of both glycerol and DL-a-glycerophosphate. The last was included because L-a-glycerophosphate is believed to be the inducer of glycerol kinase ~4. When these cells were incubated with [14Clglycerol no significant retention of radioactive material occurred (Fig. 2). The

j/ .

5

3

.

.

.

i

.

.

.

.

.

i

B

[14C]GLYCEROL

L-oc-O%IGP

tx 0 . . . .

i 5

INCUBATION

. . . .

i

I0 TIME

(n'lin)

Fig. 2. Comparison of accumulation of radioactive material by cells incubated with ~14C~glycerol. • - - O , Stra.in i (kinase positive); O - - - O , Strain 4 (kinase negative). Fig. 3. Paper-chromatographic analysis of an extract of cells of Strain 8 incubated with [14C]glycerol. (A) Cell extract. (B) Standard compounds. The solvent front moved from right to left. The cross-h~ttched areas represent radioactivity. GP, glycerophosphate.

Biochim. Biophys. Acta, 94 (I965) 479-487

484

s. HAYASHI, E. C. C. LIN

few counts which were detected in these cells after the incubation (too few to register in the figure) were shown to be due to impurity in the sample of labeled glycerol, since the addition of IO mM carrier glycerol has no dilution effect. In contrast to the inertness of these m u t a n t cells, wild-type cells possessing glycerol kinase accumulated radioactive material rapidly during the course of a similar incubation, and the addition of carrier glycerol as used above virtually abolished this uptake. Thus there is a correlation between the ability to retain labeled material and the possession of the kinase. In order to make sure that the above correlation is not coincidental, four other kinase~negative mutants, each obtained from a separate clone, were examined. In no case could significant uptake of glycerol be demonstrated. Accumulation of glycerol was also sought in a m u t a n t which is blocked at the step beyond the phosphorylation. For this purpose we used Strain 8, which lacks L-a-glycerophosphate dehydrogenase but possesses glycerol kinase. Cells of this mutant took up radioactive material as rapidly as those of the wild-type strain during brief incubation with labeled glycerol. As shown in Fig. 3 virtually all of the radioactive material recovered from these m u t a n t cells was chromatographically indistinguishable from L-~-glycerophosphate. If 1 % of the total radioactive material were glycerol, it would have been detected; yet none was found.

Correlation of the ability of cells to capture glycerol with the Km of glycerol kinase Further evidence indicating that glycerol can not be actively transported was obtained b y comparing the avidity of whole cells for this compound with the apparent affinity of glycerol kinase. When the growth rates of wild-type cells were measured as a function of glycerol concentration and plotted according to the method of L1NEWEAVER AND BVRK, a linear relationship was obtained for [glycerol]/growth rate versus [glycerol]. The "Kin" (usage adopted here for the sake of comparison) has a value of 0. 9 /~M, which is reasonably close to the value of 1. 3 FM for t h e / £ m of the kinase measured with cell-free extracts (Fig. 4). This agreement suggests that even at very low concentrations, glycerol equilibrates across the cell membrane so rapidly that its intracellular and extracellular levels are always the same. To confirm the validity of this approach, we also investigated the utilization

A CELL GROWTH ,

,

,

,

B. GLYCEROLKJNASE

,

Km= ~ ,

Km=~

#

CE ~ o

o

/ GLYCEROLOJM)

.J

/ GLYCEROL(.uM)

Fig. 4. Lineweaver-Burk plots of cell growth (A) and glycerol kinase activity (13).

Biochim. Biophys. Acta, 94 (1965) 479-487

485

CAPTURE OF GLYCEROL BY E. coli

of L-a-glycerophosphate, which does not cross the cell membrane freely but is concentrated by an active transport system ~*. In this case we would expect the growth behavior to reflect the Km of the active transport system and not t h a t of L-a-glycerophosphate dehydrogenase, the first intracellular enzyme in the catabolic pathway.

A.

LO

CELL

GROWTH

L-o~-GP TRANSPORT

B. ,

,

,

•

C.

"

•

Km=4~M

5

;/ L-o{.-GP OJM)

L-a-GP DEHYOROGENASE

,

L-a-GP OJM)

L-c[-GP

(raM)

Fig. 5. Lineweaver-Burk plots of cell growth (A), L-~-glycerophosphate transport activity (B), and L-a-glycerophosphate dehydrogenase activity (C). GP, glycerophosphate.

Indeed this was found to be true. The Km of intact cells was relatively close to t h a t of the transport system but much lower than that of the first enzyme (Fig. 5)- The much lower value for the Km of an active transport system relative to t h a t of the first enzyme in the same pathway is not an unusual phenomenon. Similar results have been found in the cases of lactose15, and galactosel~, TM. It might be significant, however, that both in the cases of glycerol and L-aglyceropho,~phate, the Km for growth is somewhat lower than that for the first catalyzed reaction. These variances suggest that cells growing in different concentrations of substrate do not possess a constant amount of the kinase or of the transport system, a condition required for proper measurement of the Km. Restriction of the source of carbon and energy is probably accompanied b y gradual lifting of catabolite repression, thus permitting the cells to augment the synthesis of the gene productsl4,19. Such a compensatory mechanism would, of course, improve the scavenging power of tl~Lecells and thereby distort the measurement of the Km for growth, giving it a value which is lower than expected.

TM

DISCUSSION

Results of the present study eliminated active transport as a means of entry of glycerol into cells of E. coli, but the question still remains as to whether the rapid entrance ot: this compound is the result of free or facilitated diffusion. Although the latter has beeu shown to be responsible for the passage of glycerol through erythrocyte membranes of certain vertebrate species *°-~2, there are reasons for believing that this is not true for bacterial cells. First, in every case where a special mechanism has been evolw.'d for the uptake of nutrient molecules, a system capable of active transport Biochim. Biophys. Acta, 94 (~965) 479-487

486

s. HAYASHI, E. C. C. LIN

h a s been the solution of choice *. The absence of an active t r a n s p o r t s y s t e m in the case of glycerol therefore implies t h a t this molecule can diffuse freely into these cells. Secondly, a m o n g more t h a n 4 ° g l y c e r o l - n e g a t i v e m u t a n t s which have been screened in this l a b o r a t o r y none gave a n y evidence for a block in the p e r m e a t i o n step. If free diffusion is the case, then neither the acquisition of an active t r a n s p o r t s y s t e m , whose work would be d i s s i p a t e d in a sisyphean m a n n e r , nor the acquisition of a m e c h a n i s m for facilitated diffusion, whose function would be gratuitous, should y i e l d a n y biological benefit. Selective pressures for effective c a p t u r e of glycerol u n d e r such a circumstance would h a v e to b e a r u p o n the evolution of the s t r u c t u r e of glycerol kinase. The p h o s p h o r y l a t i o n of the s u b s t r a t e not only increases its b u l k b u t also a d d s t o it a charged group, b o t h of which should render escape t h r o u g h the m e m b r a n e more difficult 23. The action of this kinase is therefore o p e r a t i o n a l l y e q u i v a l e n t to a c t i v e t r a n s p o r t , a n d i n c r e m e n t s in the affinity of this e n z y m e for glycerol should e n h a n c e the scavenging power of the cell. Hence it is not surprising t h a t the Km of this kinase (1. 3 /zM) is more t h a n two orders of m a g n i t u d e lower t h a n t h a t of galactose kinase (EC 2.7.1.6 ) (0. 7 raM) is, whose s u b s t r a t e can be a c c u m u l a t e d in cells of E. coli b y an active t r a n s p o r t s y s t e m 17. On the other hand, it is n o t c o n t r a r y to e x p e c t a t i o n t h a t values for the K m ' s are r e l a t i v e l y high in the cases of glycerol kinases from a n i m a l cells which would not h a v e to c o m p e t e with each other for glycerol in order to survive. T h u s t h e Km of the e n z y m e from pigeon liver is 0. 4 mM "4 a n d t h a t of the e n z y m e from r a t liver is a b o u t 15 /~M (determined with a crude e x t r a c t b y the m e t h o d d e s c r i b e d in this paper). A different situation p r o b a b l y o b t a i n s for the evolution of L-~-glycerophosphate d e h y d r o g e n a s e . Since its s u b s t r a t e can be supplied at r e l a t i v e l y high c o n c e n t r a t i o n s b y the o p e r a t i o n of either glycerol kinase or the active t r a n s p o r t s y s t e m for L-ag l y c e r o p h o s p h a t e , there should be no selective pressure to m a x i m i z e its affinity for t h e substrate. I n fact, considering t h a t L-~-glycerophosphate is p r o b a b l y also an o b l i g a t o r y i n t e r m e d i a t e in t h e biosynthesis of lipids**, it should be a d v a n t a g e o u s not to h a v e this e n z y m e acquire too low a Kin, if t h e b i o s y n t h e t i c pool is to be safeguarded***. On the o t h e r hand, excessive a c c u m u l a t i o n of L-a-glycerophosphate can cause stasis of g r o w t h (an i n t r a c e l l u l a r c o n c e n t r a t i o n of 4 ° mM severely inhibits g r o w t h of a m u t a n t lacking z - ~ - g l y c e r o p h o s p h a t e dehydrogenase), similar to the p h e n o m e n a o b s e r v e d with several o t h e r p h o s p h o r y l a t e d i n t e r m e d i a t e s 25-29. P r o t e c t i o n from a u t o - i n t o x i c a t i o n , therefore, should require t h a t the Km of this e n z y m e not be too high. T h e value of 2 mM a c t u a l l y o b s e r v e d for t h e Km of this e n z y m e is in t h e It is not difficult to appreciate why facilitated diffusion can serve as a satisfactory means for the uptake of nutrient molecules in metazoan but not in protozoan systems. Most of the cells of the former are bathed in interstitial fluids well provided with substrates and intercellular competition for nutrients is not the important mode of existence for such organisms. In the case of unicellular organisms, facilitated diffusion is clearly inferior as a permeation mechanism, not only because it fails to provide means of accumulation against concentration gradient, but also that it can actually hasten the loss of the intraeellular pool whenever the extracellular concentration is lower. ** E. P. KENNEDY AND M. LUBIN, personal communications. *** Under most conditions of growth the L-a-glycerophosphate which is biosynthesized is protected from catabolism, because the dehydrogenase is not induced. However, a dangerous situation may occur when cells have to undergo transitions of growth on glycerol or L-a-glycerophosphate to growth on other carbon sources. Unless the Km is relatively high there is no simple mechanism which would curb the persisting dehydrogenase from attacking the L-a-glycerophosphate which must now be endogenously produced for purposes of biosynthesis. Biochim. Biophys. Mcta, 94 (1965) 479 487

CAPTURE OF GLYCEROL BY E . coli

487

range expectable from a compromise between these two opposing functional demands. In conclusion, it would seem that comparison of the values of the Km of whole cells and that of the intracellular enzyme mediating the first step in the dissimilation of the compound can provide presumptive evidence for the presence or absence of active transport, an approach which might be especially useful when satisfactory assays for transport are not available. I t would also seem that in the study of the evolution of enzymes, the characteristics of their Michaelis constants should not be overlooked in the search for clues of teleonomic significance. ACKNOWLEDGEMENTS

We a~e grateful to T. M. CHUSED for helping to explore the conditions for the glycerol kinase assay and to J. P. KOCH and N. R. COZZARELLIfor critical discussions during the preparation of our manuscript. This investigation was supported b y research grants from the American Cancer Society, the National Science Foundation (GB-722) and the U.S. Public Health Service (G~[-K3-~7,925 and RG-o8383). I~EFERENCES i 2 3 4 5 6 7 8 9 lO II 12 13 14 15 16 17 18 19 2o 21 22 23 24 25 26 27 28 29

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