Exercise V 



ENZYMES 31 



toward polysaccharide synthesis: the system 

 tends to synthesize rather than degrade poly- 

 saccharides. 



This synthesis, however, can occur only if two 

 conditions are realized: (1) It proceeds, not from 

 glucose, but from glucose- 1 -phosphate. The 

 organism must begin by spending considerable 

 energy in forming the initial glucose-phosphoric 

 acid bonds. (2) The synthesis requires the 

 presence of some polysaccharide on which to 

 build ("primer"). The reaction adds glucose 

 units to the end of already existing polysac- 

 charide chains. The phosphorylase reaction 

 builds up and degrades only straight polysac- 

 charide chains. The branching of such chains, 

 or the attack upon branched chains, requires 

 another type of enzyme, which exchanges glu- 

 cose- 1,6-glucose links at branch points for the 

 glucose- 1,4-glucose links of straight chains. 

 Unaided phosphorylase, therefore, synthesizes 

 only straight-chain polysaccharides, or straight- 

 chain projections from highly branched polysac- 

 charides. In such reactions the molar concentra- 

 tion of polysaccharide does not change. One 

 starts with the concentration of the primer, and 

 all that happens during the synthesis is that the 

 primer grows bigger as glucose units are added 

 to it: 



phosphorylase 



glucose- 1 -phosphate + primer =^ 



primer-glucose + H3PO4 



Phosphorylases occur in many animal and 

 higher plant tissues and in yeast. Today we will 

 extract phosphorylase from the potato tuber. 

 In Exercise I (p. 3) you examined starch grains 

 in potato cells, staining them with the lo-KI 

 reagent. Try this again today if you like. 



Experiment 



Prepare the following test tubes: 



(1) 3 ml of 0.01 M glucose 



(2) 3 ml of 0.01 M glucose- 1 -phosphate 



(3) 3 ml of 0.01 M glucose- 1 -phosphate 



(4) 3 ml of 0.01 A/ glucose- 1 -phosphate 



(5) 3 ml of 0.01 M glucose- 1 -phosphate + 

 1 ml of0.2MKH2PO4 



(6) 3 ml of 0.2% soluble starch + 

 1 mlof0.2MKH2PO4 



(7) 3 ml of 0.2% soluble starch + 

 1 ml of 0.2 A/ KH2PO4 



Add a very small drop of the 0.2% starch 

 solution to Tubes 1, 2, 4, and 5 to act as primer. 

 There should be so little starch present in these 

 tubes that the I2-KI test is negative — check it. 



With a paring knife, peel a small potato and 

 cut it into small cubes. Place these in a Waring 

 blendor, add 40 ml of 0.01 N sodium fluoride, 

 and grind for 30 sec. {Note: FLUORIDE IS A 

 POISON! We use it here to inhibit potato 

 phosphatase, which would otherwise hydrolyze 

 glucose- 1 -phosphate to glucose and phosphoric 

 acid.) 



Filter the homogenate through a double layer 

 of cheesecloth into a beaker. Squeeze out as 

 much of the liquid as you can. Centrifuge the 

 suspension for 3 min, then decant and keep the 

 supernatant. Test this extract to see that it is 

 negative to the I2-KI reagent. Transfer approxi- 

 mately 10 ml of the extract to a test tube, and 

 heat for 5 min in a boiling water bath. 



Now add 3 ml of the enzyme preparation to 

 Tubes 1, 2, 3, 5 and 6; and 3 ml of the boiled 

 enzyme preparation to Tubes 4 and 7. {Note: Use 

 the enzyme as soon as you have finished prepar- 

 ing it, since it deteriorates rapidly.) 



Test each of these mixtures at once and at 

 3-min intervals thereafter with lo-KI. The re- 

 action should be completed within about 30 

 min. Record and explain your results. 



What was the purpose of each component in 

 the mixtures you prepared ? What was the point 

 of each mixture? What might have happened 

 had you left the fluoride out of the enzyme 

 preparation? If you have time, try doing that. 

 How do you account for the fact that though the 

 number of polysaccharide molecules has not 

 changed (see above), you now obtain a test 

 with I2-KI whereas initially you didn't? 



