AMP-deaminases from chicken and rabbit muscle: partial primary sequences of homologous 17-kDa CNBr fragments: autorecognition by rabbit anti-[chicken AMPD].

The apparent size (87.5 kDa) of the major polypeptide in freshly isolated chicken muscle AMP deaminase (AMPD.M) was comparable with that predicted from the sequences of the genes for the major muscle isoforms from human and rat. The size of the subunit of AMP deaminase from chicken muscle is indistinguishable from that of the rabbit enzyme. The peptide profiles of cyanogen bromide digests of AMPD.M from chicken and rabbit share a 17-kDa fragment, representing approximately 20% of the intact subunits of these enzymes. The first 25 residues of these fragments are 88.5% identical; the rabbit and chicken segments are greater than 92% and 84% identical, respectively, to the sequences predicted for residues 310-335 for AMPD.M from human and rat. Polyclonal rabbit antisera directed against AMPD.M from chicken breast recognize the full-length AMPD.M polypeptides on immunoblots of extracts of both avian and rabbit muscle, including an antiserum from the rabbit in which the antibody was prepared. The 17-kDa fragments, derived by incomplete cleavage of highly conserved internal segments of the deaminase subunit, share epitopes involved in the autorecognition of rabbit AMPD.M by rabbit polyclonal antibodies directed against the avian AMPD.M.

Contrast in adenine uptake by chicken and rabbit erythrocytes in vitro.

1. Relative to rabbit erythrocytes, chicken red blood cells exhibit a much greater capacity to utilize [3H]adenine for nucleotide synthesis in vitro, even at 5 degrees C and in the absence of added inorganic phosphate. 2. This difference is largely due to a higher concentration of phosphoribosylpyrophosphate and greater activity of adenine phosphoribosyltransferase in the avian cells. 3. The capacity of avian erythrocytes for utilization of guanine and hypoxanthine is several fold less than that of adenine. 4. The data are consistent with lower activity for hypoxanthine/guanine phosphoribosyltransferase than for adenine phosphoribosyltransferase in intact chicken erythrocytes. 5. The results indicate that reutilization of adenine by chicken erythrocytes may be physiologically significant.

Pyrroline-5-Carboxylate Reductase in Soybean Nodules : Comparison of the Enzymes in Host Cytosol, Bradyrhizobium japonicum Bacteroids, and Cultures.

Characteristics of pyrroline-5-carboxylate reductase (P5CR) from Bradyrhizobium japonicum bacteroids and cultured rhizobia were compared with those of the enzyme in soybean nodule host cytosol. Reductase from host cytosol differed from that in bacteroids in: (a) the effect of pH on enzymic activity, (b) the capacity to catalyze both reduction of pyrroline-5-carboxylic acid and NAD(+)-dependent proline oxidation, (c) apparent affinities for pyrroline-5-carboxylic acid, and (d) sensitivities to inhibition by NADP(+) and proline. The K(1) for proline inhibition of P5CR in bacteroid cytosol was 1.8 millimolar. The properties of P5CR in B. japonicum and bacteroid cytosol were similar. The specific activities of P5CR in the cytosolic fractions of the nodule host and the bacteroid compartment were also comparable.

Pyrroline-5-carboxylate reductase in soybean nodules: isolation/partial primary structure/evidence for isozymes.

Electrophoretic evidence was obtained for two forms of pyrroline-5-carboxylate reductase (P5CR) in soybean nodules. One form was purified over 2300-fold. The apparent sizes of the polypeptides comprising the pyrroline-5-carboxylate reductases from soybean cytosol (29,700) and Escherichia coli (28,000) were consistent with those predicted from the sequences of the genes encoding them (Deutch et al., 1982 Nucleic Acid Res. 10, 7701-7714; Delauney and Verma, 1990 Mol. Gen. Genet. 221, 299-305). Primary structural analysis of the intact soybean P5CR subunit indicated that the amino-terminal residue is blocked. Analyses of a 12-mer and a 21-mer isolated from a cyanogen bromide digest were consistent with the proposition that the soybean P5CR isolated in these studies is very similar, although perhaps not identical, to the polypeptide predicted for the recently cloned soybean reductase (Delauney and Verma, 1990 Mol. Gen. Genet. 221, 299-305).

Mitogenic lectins bind to the antigen receptor on human lymphocytes.

The specificity of interactions between mitogenic and non-mitogenic lectins and disulfide-linked cell surface receptors on human lymphocytes was explored. Lysates (Nonidet-P40) of surface-radioiodinated tonsil lymphocytes and T lymphoblastoid cells (HPB-ALL) were absorbed with lectin-agarose derivatives (or bovine serum albumin, BSA-agarose) or immunoprecipitated with appropriate monoclonal antibodies (mAb). Lectin eluates and solubilized immunoprecipitates were analyzed by two-dimensional (nonreduced/reduced) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled polypeptides were visualized by autoradiography. Among the various lectin-binding polypeptides, two disulfide-linked heterodimers (II and III) and two apparent homodimers (I and IV) are bound by pea lectin, concanavalin A and lentil lectin on tonsil lymphocytes; II, III and IV are bound both leukoagglutinating (L)- and erythroagglutinating (E)-phytohemagglutinins from Phaseolus vulgaris (PHA). Pokeweed mitogen recognizes only II and III. These molecules are weakly bound by peanut agglutinin, soybean agglutinin, Ulex europaeus agglutinin-I, Dolichos biflorus agglutinin, Vicia villosa agglutinin and Sophora japonica agglutinin, but are not bound by Helix pomatia agglutinin or BSA-agarose. Heterodimer II (82-88 kDa), comprised of 50-55-kDa and 40-43-kDa subunits, probably represents the alpha/beta T cell antigen receptor (TcR alpha/beta). Heterodimer III (64-72 kDa), comprised of 41-kDa and 37-kDa subunits, may represent TcR gamma. The homodimers, I (120-130 kDa) and IV (55-61 kDa), comprised of 55-60-kDa and 30-kDa polypeptides, respectively, have apparently not been previously described. Evidence that H1-2D4, a mAb directed against the antigen receptor on HPB-ALL cells, and E-PHA interact with a common molecule includes: (a) immunoprecipitation of TcR with H1-2D4 from the glycopeptide fraction specifically eluted from insolubilized lectin with N-acetylgalactosamine; and (b) adsorption of TcR from a solubilized H1-2D4 immunoprecipitate by E-PHA-agarose. Recognition of CD3 by E-PHA is indicated by immunoprecipitation of CD3 protein by UCHT1 from the glycopeptide fraction specifically eluted from E-PHA. The results are consistent with the view that mitogenic lectins interact with certain disulfide-linked molecules on human lymphocytes, including the TcR alpha/beta and perhaps TcR gamma; while some nonmitogenic lectins also recognize these receptors, the interaction is of low affinity.

Disulfide-linked receptors for mitogenic lectins on piglet lymphocytes.

The interaction between leucoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), soybean agglutinin (SBA) and lentil lectin (LcH) with disulfide-linked cell surface receptors on lymphocytes from mesenteric lymph nodes of 3-day piglets (PMLN) was investigated. Surface radioiodinated PMLN lymphocytes were lysed with buffer containing Nonidet-P40. The lysates were adsorbed on lectin-agarose derivatives (or bovine serum albumin-agarose). Eluates from the lectin-agarose derivatives were analyzed by one-dimensional or two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis both under reducing and nonreducing conditions. Among the various-lectin-binding polypeptides, L-PHA recognizes a single 92-kDa disulfide-linked moiety in piglet lymphocyte lysate, comprised of polydisperse 52-kDa subunits. In addition to this apparent homodimer, SBA, Con A and LcH bind a much less prominent 82-kDa heterodimer comprised of 47-kDa and 37-kDa polypeptides; these molecules are not observed in eluates of L-PHA. Binding of the 92- and 82-kDa molecules by LcH is inhibited by methyl-alpha-D-mannoside. These results indicate that there are two lectin-binding disulfide-linked glycoproteins on lymphocytes from 3-day piglets which bind preferentially to potent mitogens. The electrophoretic properties of these molecules, under both reducing and nonreducing conditions, as well as their lectin-binding properties are very similar to those observed for antigen receptor molecules on lymphocytes from other species.

Phaseolus vulgaris phytohaemagglutinin (PHA) binds to the human T lymphocyte antigen receptor.

The interaction of phytohaemagglutinin (PHA) with the human T lymphocyte antigen receptor (Ti) was explored. Nonidet-P40 lysates of surface-labelled HPB-ALL cells were immunoprecipitated with PHA, using a rabbit anti-(PHA)-serum, as well as clonotypic monoclonal antibodies (H1-2D4 and T40/25) and a rabbit antiserum (R-43) against Ti. One- and two-dimensional SDS-polyacrylamide electrophoresis under reducing and non-reducing conditions showed that both the clonotypic antibodies and PHA precipitated a disulphide cross-linked heterodimer having a mol. wt. of approximately 79 000 (unreduced) and a comprising subunits of mol. wts. approximately 50 000 and 39 000 (reduced). Further evidence that PHA binds Ti was obtained by (i) cross-immunodepletion with H1-2D4 and PHA; (ii) immunoprecipitation with H1-2D4 of a glycoprotein fraction specifically eluted from a PHA immunoprecipitate; (iii) immunoprecipitation with PHA of a solubilised H1-2D4 immunoprecipitate; (iv) 2-D (non-equilibrium pH gradient electrophoresis/SDS) analyses of H1-2D4 and PHA immunoprecipitates, indicated that H1-2D4 and PHA recognise coincident beta polypeptides. PHA also binds a Ti-like disulphide cross-linked heterodimer on tonsil lymphocytes and two other T-cell leukaemias (HUT-78 and J6). The data further suggest that PHA and R-43 recognise a subpopulation of Ti molecules on HPB-ALL cells that are not bound by H1-2D4, suggesting that there may be at least two forms of Ti. Similar experiments indicate that Concanavalin A (Con A) and wheat germ agglutinin (WGA) also probably bind Ti, whereas Helix pomatia agglutinin (HPA) does not.

Regulation of avian erythrocyte AMP-deaminase.

1. Kinetic data for avian erythrocyte AMP-deaminase in lysate supernatants and 2000-fold purified enzyme were consistent with an allosteric model having four binding sites for substrate. 2. Relative to the purified enzyme, AMP-deaminase in lysate supernatants exhibited a greater S0.5 and enhanced sensitivity toward phytic acid, but was far less sensitive toward potassium ion. 3. In the absence of potassium chloride, the enzymatic activity in lysates exhibited hysteresis at subsaturating 5'-AMP. This response was modified reversibly by allosteric ligands. 4. It is concluded that the characteristics of avian RBC AMP-deaminase, as expressed in lysates, may reflect important intermolecular interactions and better represent the regulatory properties of this enzyme in erythrocytes.

Erythrocyte AMP-deaminase: an investigation of the increase in activity during chick maturation.

1. AMP-deaminase activity in erythrocytes increases gradually during chick (Gallus domesticus) maturation, reaching the adult level of enzymatic activity at about 16 weeks after hatching. 2. Adenosine deaminase activity increases approximately two-fold during this period. 3. Substrate specificity and immunoinhibition studies indicate that erythrocytes from adult chickens and newly-hatched chicks contain the same AMP-deaminase isozyme. 4. Comparison of temporal changes in RBC AMP-deaminase with those previously described for this enzyme in muscle and brain suggests that the level of this enzyme is regulated differently in these tissues.

Subunit structure of AMP-deaminase from chicken and rabbit skeletal muscle.

The AMP-deaminases from chicken and rabbit muscle have been investigated by techniques which include sedimentation equilibrium, sodium dodecyl sulfate gel electrophoresis, amino acid analysis, NH2- and COOH-terminal analyses, and tryptic peptide mapping. The molecular weights of the native chicken (276,000) and rabbit (271,000) enzymes obtained by sedimentation equilibrium studies are in good agreement with values of 276,000 (chicken) and 275,000 (rabbit) calculated from amino acid analyses. The enzymes were reduced, carboxymethylated, and treated with either maleic or succinic anhydride in the presence of 6 M guanidine hydrochloride. Sodium dodecyl sulfate gel electrophoresis of the chemically modified enzymes resulted in a single electrophoretic species having an apparent molecular weight of 85,000. This observation is consistent with previous studies on the nonacylated enzymes and suggests that the muscle AMP-deaminases from chicken and rabbit do not contain noncovalent linkages which are readily disrupted by a large increase in negative charge. NH2-terminal analyses by the method of Stark and Amyth as well as the dansyl technique, indicate that the NH2-terminal positions of these enzymes are blocked. The enzymes are also resistant to digestion with carboxypeptidases A or B (or both) in the presence of sodium dodecyl sulfate. The most distinctive feature of the amino acid compositions of both the chicken and rabbit AMP-deaminases is the presende of eight half-cystine residues per 69,000 g of protein. Tryptic digests of the S-14C-carboxymethylated proteins were fractionated by ion exchange chromatography and high voltage electrophoresis. Six and five radioactiviely labeled peptides were detected in the electrophoretograms of the chicken and rabbit enzymes, respectively. This observation and the number of ninhydrinposition spots, together with the physical data on the molecular weights of the native enzymes and their subunits, suggest that the AMP-deaminases from chidken and rabbit muscle consist of four identical or very similar polypeptide chains.