least with respect to their location within the 

 cell. Now, the enzyme we've been talking about 

 until now is the one found in the cell husk 

 fraction of the sorocarp and the acceptor for 

 the radioactive UDPG is in the cell wall; the 

 enzyme is bound to the acceptor. The product 

 is cell wall. That's the alkali-insoluble com- 

 plex of cellulose and glycogen. 



Also, we have been studying for some time 

 an enzyme in the 100,000 x g pellet. This is 

 the typical glycogen synthetase using UDPG and 

 it depends upon glycogen as primer. Now, if 

 this enzyme preparation is coaxed, it will use 

 alkali-insoluble cell wall material as acceptor. 

 Furthermore, cell wall primer is a competitive 

 inhibitor of glycogen synthesis. Thus, the same 

 enzyme catalyzes both reactions. In Table X 

 this enzyme is described. Here we see it is 

 possible to use an enzyme in the cytoplasm of 

 the amoeba to synthesize alkali -insoluble cell 

 wall polysaccharides. This enzyme is in the 

 100,000 X g pellet and, as you can see, it's 

 completely dependent upon G-6-P and primer, 

 the primer being alkali- and cellulase-treated 

 cell wall material. Finally, we have detected 

 an enzyme in the amoeba cell membrane. This 

 enzyme will use glycogen as an acceptor but 

 is unable to use alkali-insoluble primer. It 

 responds to EDTA in a manner similar to the 

 cell wall enzyme (Table XI). Perhaps prolonged 

 incubation of this amoeba cell membrane frac- 

 tion with partially soluble acceptors, such as 

 cellodextrins, will reveal a capacity to synthe- 

 size an insoluble product. It's our hope to 

 determine if these 4 enzymes (Table IX) are 

 all different or, perhaps, all the same except 

 for their localization in the cell and the primer 

 to which they are bound. 



In summary, we've seen that no single 

 event could possibly trigger cell wall synthesis 

 since a complex array of primer, substrates, 

 activators and enzymes are not only limiting 

 but must interact to bring about the accumula- 

 tion of cell wall material. The relative contribu- 

 tion of these factors and of RNA and genetic 

 control as well as the time at which each acts 

 relative to the differentiation process are ques- 

 tions for the future. The probable interaction 

 and interdependence of all of these mechanisms 

 presents a challenging problem, to say the 

 least. 



PAPACONSTANTINOU: Are the glycogen 

 enzymes the ones responsible for the linkage 

 of glycogen and cellulose later? 



B. WRIGHT: Right. 



Enzyme Source 



TABLE IX 



Acceptor of UDPG-^^C 



Sorocarp cell wall Cell wall (bound) 



Amoeba pellet Cell wall (added) 



Amoeba pellet Glycogen (bound and added) 



Amoeba cell membrane Glycogen (added) 



TABLE X 



100,000 X g Pellet Enzyme Donating to AlkaU-Treated 

 CeU WaU Primer 



Condit ion cpm 



0.2 mg primer 1,395 



0.1 mg primer 589 



No primer 8 



No G-6-P 11 



TABLE XI 



Amoebae Membrane Preparation Catalyzing Incorporation 

 of UDPG-i^C into Glycogen. 



EDTA 



Absent 

 Present 



Total cpm 

 Day 1 Day 2 



14 

 555 





 223 



PAPACONSTANTINOU: So, if it' s the same 

 enzyme, you're going to have to postulate some 

 mechanism for the change in function? 



B. WRIGHT: By the same enzyme I mean 

 we may only be hooking on glucose in alpha- 1, 

 4 linkages to the cell wall material. We're 

 looking at an artificial system; in the cell the 

 ratio is about 1:1 of cellulose to glycogen in 

 cell wall material, but in vitro we get 80% of it 

 in the glycogen fraction. So that when I' m talking 

 about this 100,000 x g pellet enzyme, it may 

 just be adding to the glycogen moiety of the cell 

 wall material. However, that is an alkali- 

 insoluble material because it is intimately 

 associated with the cellulose. Now, there is a 

 big problem about the origin of the insoluble 



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