Studies on purine transport and on purine content in vacuoles isolated from Saccharomyces cerevisiae

The transport of purine derivatives into vacuoles isolated from Saccharomyces cerevisiae was studied. Vacuoles which conserved their ability to take up purine compounds were prepared by a modification of the method of polybase-induced lysis of spheroplasts.Guanosine > inosine = hypoxanthine > adenosine were taken up with decreasing initial velocities, respectively; adenine was not transported.Guanosine and adenosine transporting systems were saturable, with apparent K_m values 0.63 mM and 0.15 mM respectively, while uptake rates of inosine and of hypoxanthine were linear functions of their concentrations.Adenosine transport in vacuoles appeared strongly dependent on the growth phase of the cell culture.The system transporting adenosine was further characterized by its pH dependency optimum of 7.1 and its sensitivity to inhibition by $\text{S-}\text{adenosyl-}\text{l}\text{-methionine}$.In the absence of adenosine in the external medium, [¹⁴C]adenosine did not flow out from preloaded vacuoles. However, in the presence of external adenosine, a very rapid efflux of radioactivity was observed, indicating an exchange mechanism for the observed adenosine transport in the vacuoles.In isolated vacuoles the only purine derivative accumulated was found to be $\text{S-}\text{adenosyl-}\text{l}\text{-homocysteine}$.     

In vitro dissociation-recombination of malate dehydrogenase subunits in Corydalis solida

Summary Two allelic forms of NAD specific malate dehydrogenase were found in samples of a wild population of Corydalis solida. The dimeric nature and the origin of the heterodimeric form has been demonstrated by in vitro dissociation and recombination of the subunits detected by subsequent electrophoresis. The method is applicable for polyacrylamide gel electrophoresis of crude leaf extracts of individual MDH isozyme forms.     

Antigen-binding receptors on T cells from long-term MLR. Evidence of binding sites for allogeneic and self-MHC products

Antibody inhibition of radiolabelled stimulator membrane vesicle binding by T blasts activated in the mixed lymphocyte reaction (MLR) was used to identify responder-cell determinants involved in the binding phenomenon. Antisera or monoclonal antibodies against Thy-1, Lyt-1, Lyt-2 and Ly-6 antigens were not inhibitory. However, antibodies against heavy-chain V region (V_H) determinants strongly inhibited vesicle binding by both primary and longterm MLR blasts. Anti-Ia (both alloantisera and monoclonal reagents) caused inhibition of antigen binding by primary MLR blasts only. T blasts from long-term MLR lines were neither Ia-positive, nor susceptible to blocking of antigen binding with anti-Ia. However, these cells were capable of specifically absorbing soluble syngeneic Ia material, with the concomitant appearance of vesiclebinding inhibition with anti-Ia sera. Acquisition of syngeneic Ia by T blasts was effectively blocked with the anti-V_H reagent. Passively bound self-Ia did not interfere with vesicle binding in the absence of anti-Ia. These results strongly suggest the existance of specific self-Ia acceptor sites closely linked to the receptors for stimulator alloantigens on T cells proliferating in MLR. A receptor model based on these findings is briefly discussed.Abbreviations used in this paper B10 C57BL/10 - Con A concanavalin A - FcR Fc receptor - FCS fetal calf serum - H heavy chain - Ia I-region associated antigen - Ig immunoglobulin - LPS lipopolysaccharide - Lyt T-lymphocyte differentiation antigen - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - PM plasma membrane - T thymus derived - Tcr T-cell receptor - V variable region of Ig     

Genetic control of T-cell proliferative responses to poly(glu40ala60) and poly(glu51lys34tyr15): subregion-specific inhibition of the responses with monoclonal Ia antibodies

The relationship between Ir genes and Ia antigens was studied in the T-cell proliferative responses to two synthetic polypeptides poly(glu40ala60) (GA) and poly(glu51lys34tyr15) (GLT15). The response to GA was found to be controlled by an Ir gene in the I-A subregion, whereas the anti-GLT15 response was shown to be under dual control, one Ir gene mapping probably in the I-A subregion, and the other in the I-E subregion. We obtained two different lines of evidence suggesting identity of Ir and Ia genes. First, the presence of certain serologically identified allelic forms of the I-A-encoded A molecule correlated with the responder status to GA both in inbred strains and in B10.W lines, the latter carrying wild-derived H-2 haplotypes. Thus the Ir and Ia phenotypes were not separable in strains of independent origin. Second, the anti-GA response was completely inhibited by monoclonal antibodies against determinants on the A molecule (Ia.8, 15, and 19), but not by a monoclonal antibody against a determinant on the E molecule (Ia.7). In contrast, the anti-GLT15 response was only inhibited by a monoclonal antibody against the E molecule, but not by antibodies against the A molecule. Our data support the hypothesis that Ia antigens, as restriction elements for T-cell recognition, may in fact be the phenotypic manifestation of Ir genes.     

Antigen-binding receptors on T cells from long-term MLR. evidence of binding sites for allogeneic and self-MHC products

Antibody inhibition of radiolabelled stimulator membrane vesicle binding by T blasts activated in the mixed lymphocyte reaction (MLR) was used to identify responder-cell determinants involved in the binding phenomenon. Antisera or monoclonal antibodies against Thy-1, Lyt-1, Lyt-2 and Ly-6 antigens were not inhibitory. However, antibodies against heavy-chain V region (VH) determinants strongly inhibited vesicle binding by both primary and long-term MLR blasts. Anti-Ia (both alloantisera and monoclonal reagents) caused inhibition of antigen binding by primary MLR blasts only. T blasts from long-term MLR lines were neither Ia-positive, nor susceptible to blocking of antigen binding with anti-Ia. However, these cells were capable of specifically absorbing soluble syngeneic Ia material, with the concomitant appearance of vesicle-binding inhibition with anti-Ia sera. Acquisition of syngeneic Ia by T blasts was effectivelly blocked with the anti-VH reagent. Passively bound self-Ia did not interfere with vesicle binding in the absence of anti-Ia. These results strongly suggest the existance of specific self-Ia acceptor sites closely linked to the receptors for stimulator alloantigens on T cells proliferating in MLR. A receptor model based on these findings is briefly discussed.     

An H-2 haplotype possibly derived by crossing-over between the (A α A β ) duplex and the E βlocus

The B10.STA62 strain carries the H-2 ^w27 haplotype derived from a wild mouse captured in the vicinity of Ann Arbor, Michigan. Products of two class II loci composing this haplotype, A and A , are serologically, biochemically (by tryptic peptide mapping), and functionally indistinguishable from products controlled by the A ^b and A ^/b genes of the B10.A(5R) strain. In contrast, the polypeptide chain controlled by the third class II locus, E , is different from that controlled by the E ^/b gene. This E ^/w27 chain lacks an antigenic determinant present on the E^b molecule and carries determinants lacking on the E^b molecule, the E ^/b and E ^/w27 peptide maps differ in at least six peptides, and cytotoxic T cells specific for the E ^b chains do not react with B10.STA62 target cells. This great difference between the E ^/b and E ^/w27 chains suggests that the corresponding genes have not been derived from one another by a direct mutational conversion; instead, H-2 ^w27 appears to be a recombinant haplotype derived by crossing-over between the A A duplex and the E locus. This is the first recombinant discovered separating these class II loci.