Abstract
The properties of Golgi-associated enzymes dephosphorylating thiamine pyrophosphate and several nucleoside diphosphates have been investigated by cytochemical methods under two different conditions of localization. These conditions were: (A) 33 mM Tris, 15 mM calcium chloride, 4 mM substrate material, pH 9.5 and (B) 80 mM Tris-maleate buffer pH 7.0, 5 mM manganous chloride, 3.6 mM lead nitrate, and 4 mM substrate material. Cryostat sections, 4 to 6 µ, postfixed in acetone for 1 hour at 0°C, were used in most studies. Golgi associated activity demonstrable under condition A hydrolyzed thiamine pyrophosphate at a rapid rate but inosine diphosphate and uridine diphosphate were hydrolyzed slowly. Under condition B, these substrate materials were hydrolyzed at a rapid and equal rate. Several points of difference were noted when adjuvants were included in these reaction systems. Under condition A in the presence of thiamine pyrophosphate, activity was enhanced by 50 mM potassium cyanide, was inhibited by 25 mM sodium fluoride, and was unaffected by 10 mM uranyl nitrate. Under condition B in the presence of either thiamine pyrophosphate or inosine diphosphate, activity was unaffected by 50 mM potassium cyanide and 50 mM sodium fluoride but was totally inhibited by 10 mM uranyl nitrate. Investigation of the distribution of Golgi associated phosphatase activity among cells failed to uncover any clear example of activity demonstrable with one substrate system and not with the other. It was concluded that different enzymes may be demonstrated by the two localizing systems but, in the absence of distribution differences, final evaluation of this point cannot be made. In most cases, these enzymes were associated with the lamellar components of the Golgi complex.