Immunogenicity of Plasmodium falciparum and Plasmodium vivax circumsporozoite protein repeat multiple antigen constructs (MAC).

In this study we characterized the immunogenic properties of three different multispecies multiple antigen constructs (MACs) carrying the circumsporozoite protein (CSP) repeats of human malaria parasites, Plasmodium falciparum and P. vivax. We synthesized tetrameric MACs containing the antigenic repeats from the CSP of P. vivax-like parasite in two arms and CSP repeat sequences of either P. vivax type-1 (vivax-like/vivax type-1 MAC), P. vivax type-2 (vivax-like/vivax type-2 MAC), or P. falciparum (vivax-like/falciparum MAC) in the other two arms. Mice of four different genetic backgrounds (H-2a, H-2b, H-2d, and H-2k) were immunized with these MACs in Freund's adjuvant. All three MAC preparations were found to elicit antibodies to P. vivax-like CSP repeats in B10.BR, B10.A, and C57BL/6 mice. On the other hand, in B10.D2 mice only vivax-like/vivax type-1 MAC, but not the other two MACs induced antibodies to the P. vivax-like CSP repeats. In mice immunized with vivax-like/vivax type-1 MAC, antibodies to P. vivax type-1 CS repeat peptides were induced in B10.BR, B10.A, and C57BL/6 mice, but not in B10.D2 mice. Antibody responses to P. vivax type-2 repeats were not induced in any of the four strains of mice that were immunized with vivax-like/vivax type-2 MAC. While B10.BR, B10.A, and C57BL/6 mice produced antibodies to NANP repeats of P. falciparum CSP following immunization with vivax-like/falciparum MAC, B10.D2 mice failed to elicit antibodies to this repeat. All the sera that showed positive reactivity to peptides in enzyme-linked immunosorbent assay were found to react with sporozoites by IFA. In conclusion, these results showed that naturally immunogenic epitopes from different species of malaria parasites can be incorporated in a single vaccine construct to induce immune responses against multiple epitopes.

Multiple antigen constructs (MACs): induction of sterile immunity against sporozoite stage of rodent malaria parasites, Plasmodium berghei and Plasmodium yoelii.

We prepared multiple antigen constructs (MACs) using circumsporozoite (CS) protein-based B-epitopes from Plasmodium berghei, (PPPPNPND)2 and Plasmodium yoelii, (QGPGAP)3QG, along with a P. berghei T-helper epitope KQIRDSITEEWS. Mice were immunized with individual MACs in oil-in-water or water-in-oil vehicles containing block copolymer (P1005) and detoxified RaLPS (RaLPS) as well as other adjuvants. Sporozoite challenge results demonstrated that MACs in adjuvant could induce antibodies capable of active and passive protection. Water-in-oil vaccines induced the highest level of protection in mice immunized with either P. berghei or P. yoelii MACs. In a study aimed at co-eliciting immunity against P. berghei and P. yoelii, three immunizations with MACs induced protective antibodies against P. berghei but not P. yoelii parasite challenge. Therefore, it can be concluded that individually MACs are capable of inducing strong and protective immune responses to either species of rodent malaria, and that protection can be passively transferred. When MAC formulations were used together as a combined vaccine, P. berghei MACs induced a strong protective antibody response while P. yoelii MACs induced a weaker nonprotective response.

Induction of protective antibodies in Saimiri monkeys by immunization with a multiple antigen construct (MAC) containing the Plasmodium vivax circumsporozoite protein repeat region and a universal T helper epitope of tetanus toxin.

Previous attempts in inducing protective immunity against Plasmodium vivax in human volunteers and nonhuman primates with recombinant circumsporozoite (CS) proteins have been unsuccessful, largely due to the failure of generating antibodies against the protective B epitope AGDR in the CS protein repeat region. We report here an immunization study in Saimiri monkeys with a multiple antigen construct (MAC) containing the P. vivax CS protein repeat region and a T helper epitope of tetanus toxin formulated in different adjuvants. Monkeys immunized three times with MAC in copolymer P1005, copolymer P1005 plus RaLPS, or MF-75 had titers of antibodies against CS repeat, sporozoites and the protective B epitope AGDR significantly higher than those immunized with MAC in alum or PBS (P < 0.05). Antibody levels in animals that received P1005 were maintained at high level for 7 months after the last immunization. Upon challenge with 10000 sporozoites 2 weeks after the last immunization, 75% (three of four) of monkeys from the alum group, 50% (three of six) of monkeys from the P1005 plus RaLPS group, 40% (two of five) of monkeys from the P1005 group, 33% (two of six) of monkeys from the MF-75 group, and 17% (one of six) of monkeys from the MAC alone group were fully protected. When immunized animals were challenged again with 30000 sporozoites 22 weeks after the last immunization. 40% (two of five) monkeys from the P1005 group were fully protected. The remaining (three) in this group developed low parasitemia (< 2000 parasites mm-3 of blood) after significantly longer prepatent period (P < 0.05). In addition, 17% (one of six) of monkeys each from the P1005 plus RaLPS and MF-75 groups were also fully protected. Protected animals had higher levels of prechallenge anti-AGDR antibody titers than unprotected (1933 vs 281 for the first challenge, P > 0.05; 21527 vs 196 for the rechallenge, P < 0.05). Anti-AGDR antibody titers were positively correlated with the prepatent period of infected animals (r = 0.42 for the first challenge, P > 0.05; r = 0.60 for the rechallenge, P < 0.05) and negatively correlated with the peak parasitemia (r = -0.39 for the first challenge, P < 0.05; r = 0.50 for the rechallenge, P < 0.05). The results suggested that when combined with the use of potent adjuvants and T helper epitopes, MAC subunit vaccines may potentially offer protection against malaria infection.

Rapid onset of malaria-induced mortality by immunizations with lipo-peptides: an experimental model to study deleterious immune responses and immunopathology in malaria.

We have recently shown that circumsporozoite (CS) protein-based cytotoxic T-cell epitope of Plasmodium berghei coupled to monoplamitic and tripalmitic acid was able to induce cytotoxic T-cell responses. In the present study, we investigated whether lipopeptide derivatized CS protein B and T helper epitopes in different combinations will be able to induce protective immune responses against sporozoite challenge. Several P. berghei CS peptides with monopalmitic fatty acid tails were prepared, suspended in an oil-in-water emulsion, and used to immunize and boost female A/J mice. The mice were challenged iv. with viable sporozoites of P. berghei (ANKA) two weeks after the last immunization. While immunization with some of these vaccine formulations induced protective immune responses, others shifted the typical bimodal pattern of P. berghei sporozoite induced death toward a rapid onset of death in a peptide specific manner. Therefore, demonstration that immunization with formulations of malarial peptides can cause enhanced malaria-related death provides an experimental model to delineate characteristics of deleterious immune responses.

Structural requirements of peptide and MHC for DR(alpha, beta 1*0401)-restricted T cell antigen recognition.

We identified functionally important regions of the DR(alpha, beta 1*0401) peptide binding site and present a model of bound peptide. DR(alpha, beta 1*0401)-restricted T cell recognition and peptide binding of Mycobacterium leprae (ML) peptide 38-50 and overlapping peptides from the 18-kDa heat-shock protein were analyzed. ML38-50 is unusual in its restricted binding pattern, binding to only one of five DR4 subtypes and no other DR molecules tested. Amino acid substitutions were introduced into ML38-50 and the DR(alpha, beta 1*0401) peptide binding site at positions likely to influence peptide-MHC or peptide- or MHC-TCR interactions. Peptide binding, T cell proliferation, and computer modeling studies suggest that residues 39F, 42E, and 44D of ML38-50 interact with pockets 1, 4, and 6, respectively, of the peptide binding site. Only DR(alpha, beta 1*0401) substitutions at residues in pockets 4 or 7 prevented binding of ML38-50, while multiple substitutions at other positions negatively affected its T cell recognition. In contrast, T cell recognition of some high affinity ML peptides that overlapped ML38-50, and contained N-terminal extensions, was only abolished with pocket 4 substitutions. An inverse correlation of peptide affinity for DR(alpha, beta 1*0401) with negative effects of MHC substitutions on T cell recognition of the overlapping ML peptides was observed. Thus, some regions, such as pocket 4, dominantly influence T cell recognition of multiple DR(alpha, beta 1*0401)-binding peptides. However, each DR(alpha, beta 1*0401)-binding peptide appears to have unique properties that determine the outcome of its MHC-peptide interactions and the relative importance of other polymorphic pockets.

Monopalmitic acid-peptide conjugates induce cytotoxic T cell responses against malarial epitopes: importance of spacer amino acids.

Cytolytic T cells (CTL) play a critical role in providing protection against the liver stage of malaria infection. Previous investigations have shown that induction of CTL against peptide or proteins can be achieved by attachment of lipids. In the present study, we used the Plasmodium berghei circumsporozoite protein CTL epitope (SYIPSAEKI (PL76)). This peptide with cysteine-serine (CS) as spacer amino acids was coupled to palmitic acid (PA). The same CTL epitope containing only an extra serine was linked to S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-cysteine (tripam-C). Inbred mice [(BALB/c x C57BL/6)F1] were immunized intravenously with the lipopeptides. Both types of lipopeptides induced significant CTL responses after one injection. Immunization of the monopalmitic acid-peptide conjugate intraperitoneally emulsified in Freund's complete adjuvant also induced a significant CTL response, but the magnitude was lower as compared to the intravenous route. The major advantages of the use of the simple monopalmitic acid-peptide conjugates are: (i) low costs of the fatty acid; (ii) coupling of lipid to peptide can be performed using the peptide synthesizer during standard peptide synthesis, and (iii) standard peptide methodology can be used for purification. To investigate whether a spacer amino acid sequence between the actual CTL epitope and PA is required for induction of an optimal CTL response, we prepared monopalmitic acid-peptide conjugates with different spacer amino acids. A lipopeptide without a spacer amino acid and another one containing the CS spacer sequence both induced a CTL response, whereas a lipopeptide with a serine as spacer failed to induce CTL. These results indicate that the amino acid spacer sequences influence the immunological properties of the palmitic acid-peptide conjugates.

Analysis of binding of monoclonal antibody to a malarial peptide by surface plasmon resonance biosensor and integrated rate equations.

Using biosensor technology and integrated rate equations, we have developed procedures to determine the kinetic parameters and equilibrium affinity constant of Ag-Ab interactions. The Ag used in these studies was a peptide that represents the major B cell epitope of the circumsporozoite protein of Plasmodium falciparum, a promising malaria vaccine candidate Ag. Measurements of association and dissociation rate constants of this peptide with the mAb 2A10 were determined by fitting integrated rate equations to binding data obtained with a BIAcore surface plasmon-resonance biosensor. We examined whether accurate estimates of initial velocity and final equilibrium levels of binding of Ab to peptides can be obtained using these methods, and whether kinetic rates and equilibrium constants obtained with systematic variation of the experimental parameters conform to a simple bimolecular model of binding. We found that initial velocity was approximately first order with respect to Ab concentration. When we used a series of four sensor cells with different peptides loads, however, we found that the initial velocity of binding appeared to be nearly independent of peptide concentration. Equilibrium analyses yielded dissociation constants of approximately 3 x 10(-7) M. Integrated rate treatment of biosensor data supports a critical examination of the assumptions on which the binding models are based and suggests a need to refine such models. Nevertheless, it provides a powerful quantitative tool for assessing the Ag-Ab binding reaction.

Antigenic diversity in the circumsporozoite protein of Plasmodium falciparum abrogates cytotoxic-T-cell recognition.

Genetic analysis of field isolates of Plasmodium falciparum has shown selective accumulation of point mutations within the immunologically sensitive sites of the circumsporozoite (CS) protein, a vaccine candidate against malaria. This raised concern whether a vaccine containing the sequence of a selected strain of P. falciparum would be able to confer protection against other variant parasites. The answer to this question remained speculative, and in this study, we have formally tested the immunological impact of such natural variations within a known cytotoxic-T-cell (CTL) epitope, which is recognized by both human and murine CTLs. With a murine model, CTLs were generated against the 7G8 strain of P. falciparum. The ability of these CTLs to lyse histocompatible targets that were pulsed with synthetic peptides corresponding to polymorphic sequences of Brazilian, Papua New Guinean, and The Gambian isolates was determined. While these CTLs were able to recognize three of the four variant CS sequences found in Brazil and Papua New Guinea, they failed to recognize four of the five variant CS sequences found in The Gambia. Among the peptides that lost their reactivity to 7G8-specific CTL, all except one had amino acid variation in more than one residue. On the other hand, only one of the four peptides that showed a positive reaction had amino acid substitutions in more than a single residue. Thus, our findings demonstrate that natural amino acid variations in the CS protein abrogate CTL recognition. Therefore, it is important to consider the implications of these results in designing CS protein-based vaccines.

Laminin alterations after in vitro nonenzymatic glycosylation.

Laminin, a basement membrane protein derived from the matrix of the Engelbreth-Holm-Swarm murine tumor, was nonenzymatically glycosylated in vitro in the presence of increasing glucose concentrations. The amount of glucose incorporated per laminin molecule was shown to be proportional to the molarity of glucose used. Nonenzymatic glycosylation resulted in formation of cross-links and alterations of the cruciform shape of laminin molecules; these alterations were dramatic when high concentrations of glucose were used. One of the functions of laminin, the process of self-assembly, was shown to be impaired after in vitro nonenzymatic glycosylation. Glucose incorporation resulted in a dramatic decrease of long-to-long laminin dimers, which normally form during the initial steps of assembly. Furthermore, nonenzymatic glycosylation of laminin reduced its ability to self-associate into complexes larger than dimers, as judged by turbidimetry. The observed decrease of maximal turbidity was proportional to the degree of nonenzymatic glycosylation. Aminoguanidine, which has been suggested to inhibit cross-link formation, was shown to restore to a large extent the shape of laminin, the percentage of long-to-long arm dimers, and the maximal turbidity when included in the mixtures of laminin and glucose. These data suggest that structural and functional alterations of laminin may be primarily due to formation of cross-links. Such modifications of laminin (along with our basement membrane components) may contribute to the morphological and physiological changes observed in basement membranes under diabetic conditions.

Mobility of nucleoside transporter of human erythrocytes differs greatly when loaded with different nucleosides.

Time courses of transmembrane equilibration of 2-chloroadenosine, 2'-deoxyadenosine, 3'-deoxyadenosine, cytidine and 2'-deoxycytidine were measured by rapid kinetic techniques in human erythrocytes under equilibrium exchange and zero-trans conditions. The kinetic parameters for transport were computed by fitting appropriate integrated rate equations to the data pooled for seven concentrations and compared to the kinetic parameters for uridine, adenosine, thymidine and formycin B transport determined previously for human erythrocytes under comparable experimental conditions. The transport of all nucleosides conformed to the simple carrier model and was directionally symmetric. The Michaelis-Menten constants for equilibrium exchange (Kee) ranged from 22 microM for 2-chloroadenosine to about 4 mM for cytidine and the maximum velocities (Vee) differed in a similar manner, so that the first-order rate constants (Vee/Kee) were similar for all nucleosides. The kinetic parameters for 2'-deoxyadenosine transport were similar to those for adenosine transport, whereas the lack of the 3'-OH group greatly reduced the affinity of 3'-deoxyadenosine (cordycepin) for the carrier. 2', 3'-Dideoxynucleosides were transported less than 1% as efficiently as 2'- and 3'-deoxynucleosides. Thus, the 2'- and 3'-OH groups play an important role in nucleoside transport. The mobility of the carrier when loaded with pyrimidine nucleosides (reflected by Vee) was 5-10-times greater than that of the empty carrier, whereas the mobility of the adenosine-loaded or 2'-deoxyadenosine-loaded carrier was about equal to that of the empty carrier. Loading the carrier with 2-chloroadenosine or 3'-deoxyadenosine actually decreased its mobility. Thus, the differential mobility of the loaded and empty carrier differs greatly with the nucleoside substrate. The mobility of the loaded carrier as well as Kee increased with a decrease in lipid solubility of the nucleoside substrate, but the relationship was complex.

A spectrophotometric method for determination of fluorophore-to-protein ratios in conjugates of the blue fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)

7-Amino-4-methylcoumarin-3-acetic acid (AMCA) has been found to be a useful fluorophore for immunofluorescence. The present study describes a spectrophotometric method for determining the ratio of moles AMCA to moles protein (or the f/p ratio) in an AMCA-conjugated IgG. The concentration of a substance absorbing light can be determined spectrophotometrically using Beer's Law: Absorbance = Concentration x Extinction coefficient. From Beer's law, one can derive the following formula for determining the f/p ratio of AMCA-IgG conjugates: f/p = (epsilon 280IgG).A350 - (epsilon 350IgG).A280/(epsilon 350AMCA).A280 - (epsilon 280AMCA).A350 where A is the optical density of the conjugate at the given wavelength and epsilon is the extinction coefficient of a substance at the wavelength specified. Using conjugates of model proteins, it was found that the extinction coefficients of the AMCA moiety of AMCA-conjugated protein were 1.90 x 10(4) at 350 nm and 8.29 x 10(3) at 280 nm. Similarly, it was found that the extinction coefficients of swine IgG were 1.56 x 10(3) at 350 nm and 1.26 x 10(5) at 280 nm. Thus, for AMCA-conjugated swine IgG: f/p = (1.26 x 10(5)).A350 - (1.56 x 10(3)).A280/(1.47 x 10(4)).A280 - (6.42 x 10(3)).A350 [corrected]. Based on this formula, the f/p ratios of some AMCA-IgG conjugates useful for immunohistochemistry have been found to range between 6 and 24.

The effect of nonenzymatic glucosylation on the binding of the main noncollagenous NC1 domain to type IV collagen.

Type IV collagen has the ability to self-assemble by amino end, carboxyl end, and lateral associations to complex network-like structures which can be visualized by rotary shadowing. The main noncollagenous NC1 domain which is located at the carboxyl end of type IV collagen molecules binds to itself to form dimers and also binds along the length of type IV collagen. The latter binding initiates lateral assembly. Following in vitro nonenzymatic glucosylation of the isolated NC1 domain, binding to the helix-rich domain of type IV collagen was impaired. In turbidity experiments, the nonenzymatically glucosylated NC1 domain minimally suppressed the development of turbidity of collagen solutions when compared to control NC1 domain. In rotary shadowing experiments nonenzymatically glucosylated NC1 domain did not significantly inhibit lateral associations or networks formed by type IV collagen, whereas control NC1 domain caused a drastic decrease in laterally assembled structures. These data suggest that nonenzymatic glucosylation of the NC1 domain may interfere with normal assembly of type IV collagen in diabetes mellitus and may be related to abnormal functions of basement membranes in this pathological condition.

Purine and pyrimidine transport and permeation in human erythrocytes.

Time courses of the uptake of radiolabeled hypoxanthine, adenine and uracil were measured by rapid kinetic techniques over substrate ranges from 0.02 to 5000 microM in suspensions of human erythrocytes at 25 or 30 degrees C. At concentrations above 25 microM, the rate of intracellular phosphoribosylation of hypoxanthine and adenine was insignificant relative to their rates of entry into the cell and time courses of transmembrane equilibration of the substrates could be measured and analyzed by integrated rate analysis. Hypoxanthine and uracil are transported by simple facilitated carriers with directional symmetry, high capacity and Michaelis-Menten constants of about 0.2 and 5 mM, respectively. Adenine is probably transported by a carrier with similar properties but no saturability was detectable up to a concentration of 5 mM. Cytosine entered the cells much more slowly than the other three nucleobases, and its entry seems not to be mediated by a carrier. The hypoxanthine transporter resembles that of one group of mammalian cell lines, which does not exhibit any overlap with the nucleoside transporter and is resistant to inhibitors of nucleoside transport. Results from studies on the effects of the nucleobases on the influx and countertransport of each other were complex and did not allow unequivocal conclusions as to the number of independent carriers involved. At concentrations below 5 microM, radiolabel from adenine and hypoxanthine accumulated intracellularly to higher than equilibrium levels. Part of this accumulation reflected metabolic trapping, especially when the medium contained 50 mM phosphate. But part was due to an apparent concentrative accumulation of free adenine and hypoxanthine up to 3-fold at medium concentrations much less than 1 microM and when cells were incubated in phosphate-free medium. This concentrative accumulation could be due to the functioning of additional high-affinity, low-capacity, active transport systems for adenine and hypoxanthine, but other factors could be responsible, such as saturable binding to intracellular components.

Characterization of immunologically active cyanogen bromide peptide fragments of bovine and human retinal S-antigen.

Peptides which account for most, if not all, of the cyanogen bromide (CNBr)-generated peptide fragments of bovine retinal S-antigen have been identified and examined for their immunoreactivity with antisera raised to bovine and human S-antigen and with immune lymphocytes further selected twice in vitro with either bovine or human S-antigen. Amino-acid sequencing of a large fragment of S-antigen missing a small N-terminal peptide revealed the location of three overlapping CNBr peptides near the N-terminus. Amino-acid sequencing of several other CNBr peptides has allowed their position in a partial DNA-predicted sequence of the carboxy terminal half of the antigen to be determined. The total CNBr digest of human S-antigen was also prepared and compared with the fragments of the bovine antigen. Sera from rats immunized with bovine or human S-antigen were similar in their specificity in the enzyme-linked immunosorbent assay (ELISA) for purified bovine peptides except for the CB21 peptide which was not significantly bound by anti-human S-antigen sera. All of the other bovine peptides recognized by anti-bovine S-antigen sera were also bound by antibodies in the sera raised to the human antigen. The CNBr peptides of human and bovine S-antigen were extracted from gel slices and also assayed in the ELISA. Peptides of bovine S-antigen purified by HPLC were tested for their ability to stimulate an in vitro proliferative response in lymphocytes from Lewis rats immunized with either bovine or human S-antigen. Only quantitative differences in the proliferative response to human vs. bovine S-antigen and CNBr peptides were found. Methodology for the purification and analysis of the peptides is presented as well as the properties of the peptides.

S49 mouse lymphoma cells are deficient in hypoxanthine transport.

The rate of uptake of hypoxanthine in S49 cells was only about 2-5% of the rate of hypoxanthine transport observed in many other types of mammalian cells, and of the rate of uridine transport in this and other cell types. Part of the slow entry of hypoxanthine seems to be due to non-mediated permeation, but the remainder is saturable, strongly inhibited by uridine, nitrobenzylthioinosine and dipyridamole and not detectable in a nucleoside-transport-deficient mutant of S49 cells (AE1). The inhibition of hypoxanthine transport in S49 cells by nitrobenzylthioinosine resembles the inhibition of nucleoside transport in these and other mammalian cells, whereas it contrasts with the resistance of hypoxanthine transport to nitrobenzylthioinosine in all types of mammalian cells that have been investigated. We conclude that S49 cells lack the hypoxanthine transport system common to other types of cells and that hypoxanthine entry into these cells is mediated, although very inefficiently, by the nucleoside transporter. In contrast, adenine transport in S49 and AE1 cells was comparable to that in other types of cells.

Effects of nucleoside transport inhibitors on the salvage and toxicity of adenosine and deoxyadenosine in L1210 and P388 mouse leukemia cells.

Incubation of deoxycoformycin-treated L1210 leukemia cells with dipyridamole or nitrobenzylthioinosine, inhibitors of nucleoside transport, enhanced the long-term incorporation of 2'-deoxyadenosine and adenosine into the nucleotide pool and the toxicity of 2'-deoxyadenosine for the cells. In contrast, 2'-deoxyadenosine uptake in deoxycoformycin-treated P388 leukemia cells, which was about 10 times greater than that in L1210 cells, was inhibited by dipyridamole and nitrobenzylthioinosine, and 2'-deoxyadenosine toxicity was not significantly affected by the transport inhibitors. P388 cells also were about 6 times more resistant to 2'-deoxyadenosine than were L1210 cells, in spite of the greater uptake of the nucleoside. We found that purine nucleoside transport in L1210 and P388 cells exhibited similar kinetic properties and sensitivity to dipyridamole and nitrobenzylthioinosine (both influx and efflux) and that the stimulation of 2'-deoxyadenosine uptake by the inhibitors in L1210 cells is not mediated at the level of its transport into the cells but rather reflects an enhanced intracellular net accumulation of deoxyadenosine nucleotides.

Adenosine uptake, transport, and metabolism in human erythrocytes.

Using rapid kinetic techniques, we have determined the kinetics of zero-trans influx and equilibrium exchange of adenosine, and its uptake and in situ phosphorylation at 25 degrees C in human erythrocytes which were pretreated with 2'-deoxycoformycin to inhibit deamination of adenosine. Both the Km and Vmax for adenosine transport were about 300 times higher than those for the in situ phosphorylation of adenosine (Km about 0.2 microM), so that the first order rate constants for both processes were about the same. In contrast, the first order rate constant for adenosine deamination by untreated, intact cells was about 20% of that of adenosine transport or phosphorylation. These kinetic properties of the various steps, in combination with substrate inhibition of adenosine phosphorylation above 1 microM adenosine, assure that, at extracellular concentrations of physiological relevance (less than 1 microM), adenosine is very rapidly and efficiently salvaged by the erythrocytes and converted to ATP, whereas at extracellular concentrations of 10 microM or higher, practically all adenosine transported into the cells is deaminated. When the concentration of adenosine was 0.1 microM, a 10% (v/v) suspension of erythrocytes depleted the extracellular fluid of adenosine within 1 min of incubation at 25 degrees C.

Adenine nucleotide metabolism and nucleoside transport in human erythrocytes under ATP depletion conditions.

The adenine nucleotides of human red cells were labeled by incubation of the cells with [3H]adenosine. Then, the cells were incubated in Tris-saline with various supplements that cause the loss of cellular ATP, and the degradation products were quantitated as a function of time of incubation at 37 degrees C. Incubation of the cells with 2.5 or 5 mM iodoacetate, iodoacetamide or 1 mM HCHO in combination with 5 mM KF and 50 mM deoxyglucose, 50 mM D-glucose or 10 mM inosine was most efficient in depleting the cells of ATP (100% in 0.5-1 h) without causing cell lysis. In iodoacetate- and iodoacetamide-treated cells practically all catabolism of ATP occurred via ADP----AMP----IMP----inosine----hypoxanthine with hypoxanthine accumulating in the medium. In HCHO-treated cells and in cells incubated in Tris-saline or in Tris-saline with deoxyglucose with and without KF, a substantial proportion of ATP (up to 50%) was catabolized via ADP----AMP----adenosine----inosine----hypoxanthine. Under all conditions, AMP deamination and IMP and AMP hydrolysis were rate-limiting reactions. IMP degradation was more rapid in iodoacetamide- and HCHO-treated than in iodoacetate-treated red cells. It was also more rapid in fresh than in outdated red cells, and it was inhibited by Pi. Treatment with iodoacetamide and HCHO under ATP-depletion conditions resulted in a 60-80% inhibition of uridine transport by the cells. Treatment with iodoacetate or deoxyglucose plus KF had only minor effects on nucleoside transport; thus, cells treated in this manner might be useful for studying the transport of adenosine and deoxyadenosine under conditions were their phosphorylation is prevented.