Lois W. Tice

THE FINE STRUCTURAL LOCALIZATION OF GLUCOSE-6-PHOSPHATASE IN RAT LIVER

Glucose-6-phosphatase activity was demonstrated histochemically in rat liver using either the Wachstein-Meisel medium or a modified Chiquoine medium, and the characteristic properties of the enzyme activity were confirmed. The distribution of activity in both unfixed and hydroxyadipaldehyde-fixed material was demonstrated with the electron microscope. Activity was found in both smooth- and rough-surfaced elements of the endoplasmic reticulum of hepatic cells, including the nuclear envelope, but was absent from the plasma membrane. These findings further implicate the endoplasmic reticulum as an organelle of transport, and in addition suggest that the nuclear envelope has functional as well as morphological continuity with the endoplasmic reticulum.

LEAD-ADENOSINE TRIPHOSPHATE COMPLEXES IN ADENOSINE TRIPHOSPHATASE HISTOCHEMISTRY

Chelation of lead by adenosine triphosphate (ATP) and its consequences for adenosine triphosphatase histochemistry were examined. The formation constant of lead-ATP chelates was found by two methods to be 4.6-4.7 x 104. The characteristics of enzyme inhibition by lead were consistent with the predicted effects of lead-ATP chelation. Inhibition was overcome by increasing ATP concentrations. With the adenosine triphosphatase from liver microsomes, which retained some activity in the presence of 4mM Pb(NO3)2, substrate inhibition disappeared and increased MgCl2 was required for optimal activity. Increased solubility of lead phosphate was observed in the presence of increasing quantities of ATP in a manner predictable from lead-ATP chelation.

LEAD AND PHOSPHATE AS SOURCES OF ARTIFACT IN NUCLEOSIDE PHOSPHATASE HISTOCHEMISTRY

The contribution of lead and lead phosphate binding to tissue as a possible source of artifact in the Wachstein-Meisel histochemical procedure was considered. In addition, the relevance of lead-catalyzed substrate hydrolysis in tissue water and in the reaction media, as well as the efficacy of the enzyme inhibitors, para-mercurobenzoate and potassium fluoride were examined. Lead-catalyzed hydrolysis of substrate in tissue water does not play a significant role in reaction product deposition. Lead-catalyzed hydrolysis in the reaction media does produce significant amounts of lead phosphate. Furthermore, the enzyme inhibitors in addition to inhibiting enzyme activity alter lead binding to tissue, precipitate lead from solution and decrease tissue staining. Loss of tissue staining may result from a decrease in phosphate-trapping efficiency of lead when the free lead ion concentration is lowered in the reaction medium. Lead phosphate and lead localize as deposits of electron-dense material at tissue sites parallel to those observed with the complete Wachstein reaction mixture. It is concluded that tissue and chemical events other than enzyme activity may contribute to reaction product localization.

LEAD ION AND PHOSPHATASE HISTOCHEMISTRY III. THE EFFECTS OF LEAD AND ADENOSINE TRIPHOSPHATE CONCENTRATION ON THE INCORPORATION OF PHOSPHATE INTO FIXED TISSUE

This communication attempts to separate and define the relationships between lead inhibition of tissue adenosine triphosphatase activity, lead. catalyzed adenosine triphosphate hydrolysis and reaction product localization when the Wachstein-Meisel reaction is applied to kidney. Using a radiochemical assay of adenosine triphosphatase activity and varying the concentration of lead nitrate or adenosine triphosphate, the quantity of phosphate bound to and released from tissue was determined. Depending on the relative concentrations of lead and adenosine triphosphate, two situations may exist. With low lead or high adenosine triphosphate concentrations, phosphate release by tissue exceeds phosphate trapped by tissue and substantial quantities of phosphate are lost to the medium. With low adenosine triphosphate or high lead concentrations more phosphate is bound in tissue than can be attributed to tissue enzyme activity. Possible explanations for these phenomenon are discussed.

THE PARTICIPATION OF NUCLEOTIDE IN THE FORMATION OF PHOSPHATASE REACTION PRODUCT: A CHEMICAL AND ELECTRON MICROSCOPE AUTORADIOGRAPHIC STUDY

Data are presented which show that the reaction product formed in adenosine triphosphatase cytochemical procedures is not simple lead phosphate. Both by chemical and electron microscope autoradiographic techniques, reaction product was found to contain nucleotide as well as lead and phosphate. Nearly equimolar quantities of nucleotide and phosphate are present in tissue incubated in a standard Wachstein-Meisel medium. Electron microscope autoradiographs demonstrate that nucleotide is uniformly associated with adenosine triphosphatase reaction product, regardless of its cellular distribution. In vitro studies of the interaction of nucleotide with lead and phosphate are also presented. The relationship of these observations to the validity of lead-salt cytochemical procedures is discussed.