100- - 

 z 

 o 



< 



= 75-1- 



< 



< 

 Z 



X 50-- 



< 

 2 



2 25-t7 



X 



o 



0. 



a. 



4 



/ GIP 



AM AGG PS CULM SORO 



STAGE 



EXPERIMENT 



I 



TABLE I 

 Stability of Enzymatic Product ' 



From Wright, Barbara E.: Control of Carbohydrate 

 Synthesis in the Slime Mold. In Developmental and Meta- 

 bolic Control Mechanisms and Neoplasia (A Collection of 

 Papers Presented at the Nineteenth Annual Symposium 

 on Fundamental Cancer Research, 1965), p. 303. Balti- 

 more, The Williams and Wilkins Company, 1965. 



Fig. 5. 



TABLE II 

 Substrate Specificity 



the reaction stopped by boiling, carrier cellu- 

 lose added and the material washed repeatedly, 

 boiled in alkali, wash some more and finally 

 counted in a scintillation counter. 



Table I indicates the alkaline and acid .«!ta- 

 bility of the radioactive, alkali-insoluble prod- 

 uct. Gezelius and Ranby isolated comparable 

 material from D. discoideum, and the most rig- 

 orous treatment in their purification was twenty 

 hours at 100°C in 1% alkali. They studied this 

 material very carefully by x-ray diffraction 

 and other types of analyses and concluded that 

 it was an amorphous form of cellulose (3). They 

 found only glucose on acid hydrolysis. In con- 

 firmation of this, we found only radioactive 

 glucose on acid hydrolysis of our radioactive 

 cell wall material. A substrate specificity study 

 revealed that UDPG was by far the preferred 

 substrate (Table II). GDPG, which has been 

 recently shown by Hassid's group to be a pre- 

 cursor to cellulose synthesis in plants (4), was 

 only about 1/10 as active. 



We were able to carry the purification of 

 cell wall material one step further than Gezelius 

 and Ranby, and separate it into two fractions, 

 4 and B, by solution in a cuprammonium hy- 

 droxide solution known as SchweizSr's reagent 

 (Table III). We will be talking now just about 

 fraction A and soluble fraction B, not insoluble 

 fraction B. After solubilization in the cupric 

 ammonium hydroxide solution, fractional pre- 

 cipitated out on neutralization and the addition 



EXPERIMENT SUBSTRATE juMOLES jiMOLES GLUCOSE INCORP. (x lO-*) 



39.0 

 

 

 



5.6 

 0.5 

 0.2 



of water. This material is not water soluble. 

 Fraction B precipitated out from the supernatant 

 following the addition of ethanol. Chemical, 

 enzymatic and chromatographic analyses of the 

 radioactive and nonradioactive fraction A and 

 fraction B have identified the latter as an alpha- 

 D-l,4-linked polymer and fraction^ as cellu- 

 lose. Some of the enzymatic analyses are sum- 

 marized in Table IV. Oyster glycogen was used 

 as a control. The expected limit dextrin was 

 made from nonradioactive, insoluble fraction B 

 by phosphorylase treatment. Complete degrada- 

 tion was achieved by further attack of amylo-1, 

 6 glucosidase. Analysis of radioactive material 

 revealed that most of the radioactivity is in- 

 corporated into fraction B and that fraction A is 

 contaminated with the alpha-D-1, 4- linked 

 polymer. Thus our studies have led to the con- 

 clusion that the alkali-insoluble cell wall ma- 



112 



