George L. Tritsch

A model for hydropathy-based peptide interactions.

Two peptides are specified when the noncoding DNA strand is read in the 5 to 3, or the 3 to 5 direction, and both peptides form strong complexes with the natural peptide, as found by J. E. Blalock and K. L. Bost with ACTH [1986) Biochem. J. 234, 679-683). We report here that strong hydropathic complementarity (pairing of hydrophobic with hydrophilic residues), the assumed basis of these interactions, is obtained only if the peptide resulting from reading in the 3 to 5 direction is aligned parallel to the natural peptide, or if the peptide derived by opposite reading of the DNA is aligned antiparallel to it. Complementary is abolished in other alignments, including all staggered ones. In the appropriate alignments of the constructs the amino acid residues opposite one another are specified by a pair of complementary codons in the DNA; Blalock and Bost have indeed shown that complementary pairs of codons specify amino acids of opposite hydropathy. A model is proposed to explain how hydropathic complementarity can lead to interaction between peptides. We propose that in the interacting peptides hydrophilic residues of both chains are oriented toward the aqueous solvent, while the hydrophobic ones form the interphase between the two chains. Tight packing is made possible by the stipulation that whenever a hydrophilic residue turns toward the aqueous phase, a space is liberated which can accommodate a hydrophobic residue from the opposing chain. This entropy-driven configuration can lead to strong interactions between portions of peptides consisting of hydropathically complementary residues.

Adenosine Deaminase Activity and Superoxide Formation During Phagocytosis and Membrane Perturbation of Macrophages

During phagocytosis and membrane perturbation, mouse macrophages generate superoxide in direct proportion to their intracellular adenosine deaminase activity. It is proposed that since adenosine deaminase controls the amount of substrate available to xanthine oxidase, and the latter produces superoxide during turnover of its substrates, the purine salvage pathway is an important contributor to the superoxide requirement of macrophages. It is further proposed that this may be the basis for the mechanism of the association of adenosine deaminase deficiency with immunodeficiency.

ECTO-enzyme activity of human erythrocyte adenosine deaminase

SummaryAdenosine deaminase is found primarily in the cytoplasm of many cell types. In the human erythrocyte, about 30 per cent of the total adenosine deaminase activity is membrane associated, and about two-thirds of this is inactivated by treatment of intact erythrocytes with the nonpenetrating reagent diazotized sulfanilic acid, without affecting lactate dehydrogenase, a soluble cytoplasmic enzyme. This indicates that within the cell membranes, the catalytic site of about two-thirds of the adenosine deaminase faces the external medium, i.e., ecto adenosine deaminase. Localization of adenosine deaminase activity at the cell membrane is demonstrated directly by electron microscopy by use of the substrate 6-Chloropurine ribonucleoside, which is dechlorinated by adenosine deaminase to produce Cl−, which is precipitated at its locus of formation by added Ag+, and the precipitated AgCl converted into the electron dense Ag0 upon exposure to light.From the Hydropathic Profile of the amino acid sequence of adenosine deaminase it is evident that there are two hydrophobic domains of sufficient length to span a biological membrane, and it is proposed that these domains could function to anchor the enzyme to the membrane.The importance of adenosine deaminase is indicated by the fatal immuno-deficiency which results from untreated genetic adenosine deaminase deficiency. It may be important to determine whether the amount of ecto adenosine deaminase activity is better suited to assess the clinical status of adenosine deaminase deficient patients that the currently used total cellular enzyme activity.

Modulation of macrophage superoxide release by purine metabolism

Metabolic flux through the purine salvage pathway appears to modulate superoxide secretion by elicited macrophages. Exogenous adenosine, the first substrate of this pathway, stimulates superoxide secretion, and Allopurinol, a specific inhibitor of xanthine oxidase, inhibits superoxide secretion. The effects of these agents are additive since it was possible for each to neutralize the effects of the other when given in combination. In these experiments, the purine salvage pathway was responsible for over ten times the superoxide production attributable to the NADPH oxidase system.

Positive correlation between superoxide release and intracellular adenosine deaminase activity during macrophage membrane perturbation regardless of nature or magnitude of stimulus

SummaryTuftsin stimulates macrophages to release superoxide in direct proportion to intracellular adenosine deaminase activity over a concentration range of 125 to 625 nM tuftsin. This relation is comparable to that previously observed for stimulation by single concentration of several agents. This finding led to the conclusion that the relation between superoxide and adenosine deaminase is independent of the nature or magnitude of the stimulus. In absolute terms, tuftsin increases superoxide secretion up to 375 nM tuftsin; further increases in tuftsin concentration cause a rapid decrease in superoxide secretion to near base line at 500 nM tuftsin. In contrast, the phagocytic response to tuftsin remains maximal up to 10 μM with no indication of inhibition at higher concentrations. Thus, tuftsin stimulation of phagocytosis and superoxide release may be at least partially independent phenomena.

STUDIES ON A FACTOR FROM BOVINE PLASMA TOXIC FOR MAMMALIAN CELLS CULTURED IN VITRO.

Abstract 1. 1. An extract prepared from Cohn Fraction IV-1 of bovine plasma has been shown to be toxic for two mammalian cell lines maintained in suspension culture in vitro. The active factor directs its effect against the viability of the cells, as evidenced by the permeability of trypan blue, and does not cause any appreciable lysis of cells. It appears to exhibit the properties of a protein, but is not identical with complement. 2. 2. The death curve obtained with this system is not that usually seen with single-cell suspensions of bacteria and may possibly be explained on the basis of a nonuniform sensitivity of the cell population. After 15–30 min contact with the cells, the toxicity of this agent cannot ne reserved by repeated washings. The toxicity is inhibited by whole serum or serum albumin.

Purine Salvage Pathway Enzyme Activity in Tuftsin‐stimulated Macrophages

The regulation of metabolic flux to produce superoxide ions (01) in phagqcytes in response to chemotactic and phagocytic stimuli has not been fully elucidated. Oxidation of NADPH has been discussed as a source of Oj , but we have been the only ones so far to evaluate the significance of Ojproduction during purine metabolism by xanthine oxidase (FIG. 1). Several investigators have made observations that relate purine metabolism to immunological activity. In addition to the considerable literature that relates adenosine deaminase (ADA) and purine nucleoside phosphorylase activities to the magnitude of an immune response, phagocytosis has been shown to be accompanied by increased uric acid excretion by macrophages,2 infection has been observed to be associated with increased xanthine oxidase activity, elicited macrophages have been found to contain about twice as much adenosine deaminase activity (per mg protein) as resident cells,4 and ADA has been associated with maturation of monocytes into macrophages and with normal chemotaxis.6 We have shown previously that intracellular ADA activity in elicited macrophages increases in direct proportion to Oj secretion during phagocytosis and membrane perturbation by several soluble agent^.^-^ In addition, we have been able to demonstrate the localization of ADA activity in the macrophage membrane near phagocytic vacuoles by electron micros-

Purine catabolism as a source of superoxide in macrophages.

The underlying thesis of this paper is that purine catabolism has a role in the production of superoxide by macrophages. These cells are the first line of defense against infection primarily in the lung; elsewhere they probably function as the backup for plymorphonuclear leukocytes (PMN). These two types of phagocytes differ with regard to their stem cell of origin and in their ability to divide. Recent evidence indicates PMN leukocytes do not make superoxide via purine catabolism. Macrophages are not only phagocytes, but also function as secretagogues to modulate both humoral and cellular immunity. Evidence is beginning to accumulate that control of these secretagogue functions may be related to control of phagocytic efficacy. Thus the regulation of metabolic flux through purine catabolism by adenosine deaminase (ADA) may have broader influence on the immune system than implied by the data presented herein. It is therefore suggested that the macrophage may be able, at least in part, to participate in the mediation of the effects of ADA deficiency on immunodeficiency. The metabolic source of the cytocidal superoxide free radical secreted by phagocytic leukocytes remains to be definitively established. The respiratory burst that accompanies phagocytosis includes increased oxygen uptake and activation of the hexose monophosphate shunt pathway, and the production of peroxide and superoxide.’ Oxidation of NADPH has been favored as the source of the superoxide. The finding that the K,,, of the oxidase is lowered by one order of magnitude in phagocytizing cells as compared to resting cells is part of the considerable evidence for this view. Although it has been questioned whether NADPH oxidase levels are sufficient to cover the whole cell phenomenon,’ there do not appear to be extensive evaluations of alternate sources of the superoxide. Our interest in the enzymology of purine metabolism focused in 1980 on purine catabolism as a source of the superoxide in the phagocytic process. We selected the macrophage as the phagocyte for study because of the ease with which these cells can be elicited and obtained in relatively pure form in murine model systems. Furthermore, the isolation of these cells does not require centrifugation through density gradients, usually Ficoll-Hypaque, which has been reported to elevate the cyclic AMP content of the cell.’ As it now turns out, this choice was fortuitous because had we selected the PMN leukocyte, our results would have been very different.”‘ There are several observations recorded in the literature that suggest involvement of purine metabolism with phagocytosis: macrophages secrete large amounts of uric acid into the medium during phagocytosis’; infection is accompanied by increased xanthine oxidase activity*; elicited macrophages have higher ADA activity than the resting cells’; monocyte maturation into macrophageso and normal chemotaxis’ both require ADA activity. The rationale for the experimental design was to examine ADA activity as related to chemotactic and phagocytic stimulation of macrophages. It was decided to look at

Extracts of plasma and serum toxic for mammalian cells cultured in vitro

1. n1. The participation of two components in the cytotoxic activity of extracts of certain preparations of bovine plasma alpha-globulins (Cohn Fraction IV-1) is described. One is a heat-labile, non-dialyzable material assumed to be a protein. The other, lipid in nature, can be extracted from whole and lyophilized calf serum as well as from bovine alpha-globulins. n n2. n2. The role of the protein has not been established; it apparently binds the lipid. When it is present, it must be in the native state for the expression of the cytotoxic property. n n3. n3. Lipid extracts are clearly toxic per se. Even after denaturation of the protein and apparent loss of cytotoxic activity, active lipids can be released with organic solvents. They have been separated and identified by column and thin layer chromatography. Active compounds taken from thin layer chromatograms appear to be fatty acids.

Adenosine deaminase activity and superoxide formation during phagocytosis at different cell densities

Abstract Direct proportionality between intracellular adenosine deaminase activity and superoxide generation during phagocytosis at cell densities between 0.1 and 10 million cells/ml has been observed. Since adenosine deaminase controls the amount of substrate available to xanthine oxidase, and the latter produces superoxide during turnover of its substrates, it is suggested that the purine salvage pathway is an important contributor to the superoxide requirement of macrophages. Because macrophages participate in many immune processes, it is proposed that this may be the basis for the association of adenosine deaminase deficiency with immunodeficiency.