is induced to form de novo by bicarbonate, the 

 G6PDH is present in both OC and RS cells. We 

 have had some reasons to suspect, however, that 

 bicarbonate induction of morphogenesis is as- 

 sociated with a bicarbonate-induced compart- 

 mentation of G6PDH within the cell. We have set 

 up the hypothesis (Fig. 12) that (a) during the 

 development of an OC cell, intracellular G6PDH 

 is soluble, but that (b) during the exponential 

 development of an RS cell, bicarbonate induces 

 differential distribution and/or differential syn- 

 thesis of this enzyme in such a way that it 

 becomes localized on or near the cell wall or 

 the membranes associated with it, and that 

 (c) after the point of no return in RS develop- 

 ment, soluble enzyme once again appears in- 

 side the cell (either via release of wall-bound 

 enzyme into the soluble pool, or destruction of 

 the wall-bound enzyme and concomitant de novo 

 synthesis of soluble G6PDH, or some combina- 

 tion of these two). Some of the evidence follows. 



To begin with, if the notion has validity, 

 one might expect that during RS development the 

 exponential rate of synthesis of total intracel- 

 lular G6PDH would reflect (or at least be more 

 nearly similar to) the exponential rate of 

 deposition of the surface area of the cell rather 

 than its weight or volume. Conversely, for the 

 OC cell, one might expect the opposite to hold 

 true. The data available suggest that this is, 

 indeed, the case (Fig. 13). Thus, the in vivo evi- 

 dence, although it does not prove the point, is 

 consistent with the notion expressed in Fig. 12. 



With these results sufficiently suggestive. 

 Dr. Prem Pandhi and I have begun m vitro 

 studies of Blastocladiella's G6PDH. Although 

 attempts to purify it by conventional means 

 (fractionations with ammonium sulfate, acetone, 

 DEAE-cellulose, etc.) have only led, thus far, 

 to several-fold increases in specific activity, 

 experiments designed to test the hypothesis 

 in Fig. 12 are yielding evidence in its favor. 

 For example, when 36 hour RS cells are 

 homogenized in 0.005 M TRIS-HCl buffer con- 

 taining 0.001 M EDTA and then centrifuged at 

 112,000 x G, about 98% of alltheG6PDH activity 

 is in a soluble form (HSS in Fig. 14). When the 

 pellet is extracted three times in succession 

 with 0.005 M TRIS-HCl buffer, the remaining 

 1-2% of the G6PDH activity comes out - most of 

 it in the first wash (IW in Fig. 14); this is the 

 amount one would expect to find if it had simply 

 been trapped in the fluid volume held back by 

 the pellet. Only traces of activity are found in 

 the second and third washes (2W and 3W in Fig. 

 14). A final extraction of the pellet with 1 M TRIS- 



HCl (IM in Fig. 14) yields insignificant activity. 



However, similar analyses of RS cells 

 undergoing exponential growth - in this case 24 

 hour cells - yield quite different results (Fig. 

 14). Only about 40% of the total G6PDH activity 

 is directly soluble in 112,000 x G supernatants. 

 The first wash yields about half as much again 

 of the enzyme, a great deal more than one would 

 expect if it had simply been trapped in the pellet. 

 The second and third washes yield additional 

 quantities of G6PDH activity, and the final ex- 

 traction with 1.0 M TRIS-HCl yields another 

 20%. Note, too, that the specific activities of the 

 enzyme in the washes do not vary greatly from 

 one another (labeled "S.A." in the figure). Thus, 

 as seen in the insert in Fig. 14, essentially all 

 of the G6PDH in a 36 hour RS cell is soluble. 

 But in a 24 hour RS cell which is growing expo- 

 nentially and has not reached its point of no 

 return, less than half of the G6PDH is soluble; 

 more than half of it appears to be "bound" - 

 albeit loosely "bound," since it behaves as if it 

 were partitioning between two phases during 

 successive extractions. 



Dr. Pandhi and I are now in the process of 

 tracking the soluble and insoluble G6PDH 

 throughout the ontogeny of RS and OC cells; I 

 would like to show you some of the things we 



HCOJ - 



INDEPENDENT 

 OC PATH 



HCO3 -INDUCED 

 RS PATH 



EXPONENTIAL 

 GROWTH 



CELL 

 DIFFERENTIATION 



Fig. 12. 



Hypothesis regarding the effect of bicarbonate on glu- 

 cose- 6- phosphate dehydrogenease In Blastocladiella 

 emersonii. 



157 



