Alan S. Rosenthal

Determinant Selection and Macrophage Function in Genetic Control of the Immune Response

The immune response to insulin, in both mouse and guinea pig, is under control of H-linked immune response genes. When immunized with either pork or beef insulin in CFA, both strain 2 and 13 guinea pigs respond by antigen-specific lymphocyte proliferation and synthesis of specific antibody. The specificities of the elicited antibodies and indistinguishable between these inbred strains. By constrast, strain 2 T cells recognized a distinct region of the A chain alpha loop consisting of amino acid residues 8, 9 and 10, while strain 13 T cells see an as yet undefined region of the B chain. H2b (A chain alpha loop responder) and H2d (B chain responder) mice similarly discriminate which areas of the molecule are recognized by their T lymphocytes. The function of the Ir gene in both the guinea pig and mouse appears to be an intramolecular selection of discrete regions within the antigen for recognition by the T cell. The data presented suggest that this function operates at the level of the macrophage.

Adrenal cholesterol: localization by electron-microscope autoradiography.

As determined by electron-microscope autoradiography of adrenal glands containing tritiated cholesterol and by modified differential centrifugation techniques, 70 to 80 percent of adrenal cholesterol is contained within lipid droplets of rat adrenal cortical cells.

Some characteristics of A Ca2+-dependent ATPase activity associated with a group of erythrocyte membrane proteins which form fibrils

A group of proteins with a fibrillar appearance and associated with ouabain-insensitive ATPase activity has been isolated from the human erythrocyte membrane. Some of the characteristics of a Ca2+-dependent, Mg2+-inhibited ATPase activity associated with this group of proteins are examined in this communication. The ATPase activity is not inhibited by ouabain. Mg2+ (0.5–6.0 mM) inhibited Ca2+ activation of ATP hydrolysis over the entire range of Ca2+ concentration tested (0–6 mM). GTP failed to serve as a substrate. A system of fibrils morphologically similar to the isolated fiber system can be seen on the erythrocyte membrane and has been identified in thin sections of ghosts. These fibrils were localized, at least in part, to the inner aspect of the unit membrane. A possible role for the Ca2+-inhibited ATPase and the associated fibrils in the maintainance of erythrocyte deformability is discussed.

PITFALLS IN THE USE OF LEAD ION FOR HISTOCHEMICAL LOCALIZATION OF NUCLEOSIDE PHOSPHATASES

The most commonly used method for the histochemical localization of nucleoside phosphatases is the procedure described by Wachstein and Meisel in 1957 (20). This procedure is based on a Gomori type metal precipitation reaction (7) and involves the incubation of tissue sections in a reaction mixture containing the nucleoside phosphate, magnesium sulfate (10.0 mM), lead nitrate (3.6 mM) and a buffer (pH 7.2). The customary scheme of reactions occurring with this procedure is shown in Figure 1. The inorganic phosphate released by the enzymatic hydrolysis of the nucleoside phosphate is precipitated by lead ions in the reaction mixture as insoluble lead phosphate, presumably at the site of enzyme activity. This deposit of reaction product is electron-dense because of its lead content and may be viewed directly in the electron microscope, or the lead phosphate deposit may be converted to black lead sulfide for viewing in the light microscope. The method has been widely used for the localization of apparent adenosine triphosphatase (ATPase) activity with both the light and electron microscopes (6, 13, 19). For the most part, the technique has been used with fixed tissues so that structural detail would be recognizable. The procedure has been employed with and without modification on a wide variety of tissues (5, 6), on cultured cells (3, 12) and in normal and pathologic tissue specimens (19). Problems in the use of fixed tissues or cells for enzyme studies have been partially circumvented by the use of unfixed or very briefly fixed material. However, disadvantages associated with the use of a heavy metal ion, lead, in enzyme reaction mixtures have not been obviated. It has been recently demonstrated that lead ion may catalyze the nonenzymatic hydrolysis of ATP and other nucleoside phosphates (17). In addition, the inhibitory effect of lead ion on the activity of specific enzymes and the physicalchemical characteristics of lead phosphate pre-

Lead ion and phosphatase histochemistry. I. Nonenzymatic hydrolysis of nucleoside phosphates by lead ion.

A previously undescribed catalytic action of lead ion on the nonenzymatic hydrolysis of nucleoside phosphates has been demonstrated. Lead ion (3.6 mM) hydrolyzed adenosine triphosphate (ATP) at pH 7.2 and 37°C. The presence of magnesium and imidazole was stimulatory. The rate appeared to increase with temperature from 25-60°C. The reaction was inhibited by ethylenediaminetetraacetate. Other nucleoside phosphates were hydrolyzed, some less rapidly than ATP. Adenosine triphosphate in the presence of a rate-limiting amount of lead acted as an inhibitor at high concentrations. With a rate-limiting concentration of ATP (0.72 mM), increasing concentrations of lead ion above 0.36 mM catalyzed a linear increase in ATP hydrolysis. It is suggested that this reaction may be a source of artifact in the lead salt method for the histochemical localization of nucleoside phosphatases.

LEAD ION AND PHOSPHATASE HISTOCHEMISTRY II. EFFECT OF ADENOSINE TRIPHOSPHATE HYDROLYSIS BY LEAD ION ON THE HISTOCHEMICAL LOCALIZATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY

The lead method of Wachstein and Meisel for the histochemical localization of adenosine triphosphatase (ATPase) involves the incubation of sections of fixed tissue in reaction mixtures containing ATP, lead nitrate, magnesium sulfate and a Tris-maleate buffer, pH 7.2. Both fixation and the presence of lead ion were shown to inhibit tissue ATPase activity markedly and to inactivate the sodium- plus potassium-dependent membrane ATPase. In addition, recent studies have demonstrated that lead ion, in the concentration used in the Wachstein-Meisel system, will catalyze the hydrolysis of ATP. Studies on the effect of this nonenzymatic reaction on the histochemical localization of ATPases demonstrated that plasma membrane localization occurred only with lead and ATP concentrations which gave significant nonenzymatic hydrolysis of ATP by lead. In addition, nuclear and mitochondrial localization without accompanying plasma membrane localization could be obtained in formalin-fixed tissue with decreased concentrations of lead or with increased concentrations of ATP in the reaction mixture. The amount of lead-catalyzed hydrolysis was in the same order of magnitude as fixed tissue ATPase activity and could quantitatively account for the amount of phosphate needed to give recognizable localization of lead salt deposits in sections of fixed tissue.

ON THE SIGNIFICANCE OF LEAD-CATALYZED HYDROLYSIS OF NUCLEOSIDE PHOSPHATES IN HISTOCHEMICAL SYSTEMS

lead (Novikoff et at., J. Hislochem. Cytochem. 9: 434. 1961). 7. Finally, it should be noted that the Wachstein-Meisel medium remains free of lead phosphate precipitate through the incubation periods generally used for tissue sections, even at 37#{176}C. Thus the staining patterns of cytomembranes in tissue sections are more likely to result from enzyme activities than from specific lead phosphate adsorptions. Sections fail to show adsorption of lead phosphate: Sections were floated in the media, and lead phosphate was produced slowly by dropwise addition of 0.005 M phosphate, pH 7.4, as the medium was stirred. We tested: (1) heat-inactivated frozen sections of formaldehyde-calciumfixed tissues (90#{176}C,5 mm for rat sciatic nerve; 10 mm for rat kidney), at 4#{176} and 37#{176}C, with phosphate addition to Wachstein-Meisel medium over 10 mm; (2) nerve and kidney sections that had not been inactivated, in the Wachstein-Meisel medium to which 0.1 M sodium fluoride was added, with phosphate addition over 10 mm at 4#{176}C; and (3) noninactivated sections in the Wachstein-Meisel medium to which 0.01 M PCMB was added, with phosphate addition over 5, 10 and 30 mm at 4#{176}C. We also tested the effect upon staining of noninactivated sections in the usual Wachstein-Meisel medium to which phosphate was slowly added over 5 and 10 mm at 4#{176}C. In no instance did the presence of nonenzymatically produced lead phosphate in the medium produce staining of plasma membranes or other structures. To favor adsorption, the rate of lead phosphate production was kept low, in a manner reported in 1951 to produce nuclear adsorption in the alkaline phosphatase medium (Novikoff, Science 113: 320. 1951). It could also be argued, as Moses et at. do, that “difficulties in interpretation arise because most methods of inactivating ATPases also alter tissue proteins, phospholipids and other constituents.” We used not only heat-inactivated sections but also untreated sections. We exposed them to forming lead phosphate as they were incubated in the Wachstein-Meisel medium in the presence of fluoride or PCMB and in the absence of enzyme inhibitors. No sign of adsorption of nomenzymatically produced lead phosphate was observed. These considerations make it reasonable to conclude that the staining reactions obtained in the Wachstein-Meisel and similar media reflect real enzymatic activities. We would subscribe to our 1962 statement, “One may be tempted to suggest on the basis of such differences as are found in rat placenta and toad bladder, or from the wide substrate specificity seen in capillary endothelium or the narrow specificity of the -cytomembranes of kidney and parotid, that different enzymes are present in different membranes and they take part in the transport of special molecules or in different membrane activities” (Novikoff et at., Symp. mt. Soc. Cell Biol. 1: 149. 1962).

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.

INTERPRETATION OF PHOSPHATASE CYTOCHEMICAL DATA

In his recent Letter to the Editor (J. Histochem. Cytochem. 18: 366. 1970), Dr. Novikoff has objected to the conclusions but not the substantive data recently presented as a series of articles published in this journal (J. Histochem. Cytochem. 17: 608, 641, 839. 1969). Such an objection would be reasonable and proper if our conclusions were based upon unsound reasoning or drawn from incorrect data. Since neither of these objections was raised, nor does Dr. Novikoff present conflicting new data, we must assume that our disagreement results from a difference in interpretation of the literature. Such a difference is likely to be the result of our lack of knowledge concerning the chemical mechanisms involved in cytochemical reactions and the relationship of enzymatic activity to reaction product localization. Our published conclusions include the following. (a) Lead ion at concentrations greater than 1 mM results in ATP hydrolysis (if ATP is 1 mM or less) and as such is a source of free phosphate in tissue which in turn may result in reaction product artifacts (J. Histochem. Cytochem. 17: 608. 1969). The rate of this nonenzymatic lead-catalyzed ATP hydrolysis is dependent on the relative concentration of lead and ATP and the presence of other divalent cations (J. Histochem. Cytocheni. 17: 85. 1969). (b) Lead-catalyzed hydrolysis of ATP cannot of itself account for the localization of all reaction product seen in tissue exposed to phosphatase cytochemical procedures (J. Histochem. Cytochem. 17:641. 1969). (c) Reaction product is not lead phosphate but is a complex containing lead, nucleotide and phosphate (J. Histochem. Cytochem. 17: 839. 1969). We are aware that reaction product may be found at membrane sites at concentrations of lead at which there is no detectable nonenzymatic ATP hydrolysis. Such relative ATP-lead concentrations are, however, those at which the phosphate-trapping efficiency is low and diffusion artifacts are possible (J. Histochem. Cytochem. 17: 608. 1969). Furthermore, the localization of

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.