Oscar P. Chilson

AMP deaminase: Stage-specific isozymes in differentiating chick muscle☆

The molecular basis of the developmental increase in AMP deaminase activity in chick muscle was investigated with a view toward determining whether isozymes of AMP deaminase exist in embryonic avian muscle and, if so, whether a stage-specific isozyme transition occurs during myogenesis in vivo and in vitro. Under specified conditions, AMP deaminase isozymes in adult chicken brain and muscle may be distinguished on the basis of differences in relative substrate specificities for 5′-dAMP and 5′-AMP (expressed as a ratio of the rates observed with these compounds; i.e., dAMPAMP ratios), as well as by differential immunoinactivation by antibody directed against breast muscle AMP deaminase. It was found that the AMP deaminase(s) that is (are) present in 6-day embryos is (are) catalytically and immunologically similar to the enzyme in adult brain. With mixtures of known amounts of adult muscle and brain enzymes, values for the dAMPAMP ratio (as well as the fraction of uninactivated AMP deaminase at antibody excess) were proportional to the fraction of muscle isozyme present. Standard curves constructed from these data were used to determine that the fraction of adult muscle-like AMP deaminase in developing muscle, as assessed by dAMPAMP ratios (and differential immunoinactivation), on days 6, 8, 10, and 15 were 23 (28), 55 (65), 83 (85), and 93% (96), respectively, Thus, parallel results were obtained for the two techniques, and the isozyme transition is virtually complete by the 15th day of incubation. Primary muscle cultures were used to investigate the isozyme transition of AMP deaminase during myogenesis in vitro. Comparison of the data obtained from primary muscle cultures treated with bromodeoxyuridine, cytosine arabinoside, and fluorodeoxyuridine with data from control cultures showed that biochemical differentiation of AMP deaminase in vitro could be attributed to the muscle cell. Also, the isozyme composition changed from a small percentage of adult muscle-like isozyme at the time of plating, to approximately 100% by the 6th day of culture.

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).

AMP-deaminases from avian brain and muscle: Catalytic and immunological differences

Abstract 1. 1. Purified adenylic acid deaminases from brain and breast muscle of adult chickens have been compared with respect to activation by ATP, substrate specificity and the effect of anti-breast muscle deaminase serum on enzyme activity. 2. 2. Both deaminases are activated by ATP; the effect is most pronounced on the brain enzyme. 3. 3. Although the brain and muscle deaminases will utilize deoxy-AMP, in addition to AMP, as a substrate, the ribonucleotide is deaminated at a faster rate. The brain enzyme is most specific. 4. 4. Rabbit anti-breast muscle deaminase serum strongly inhibits muscle adenylate deaminase but has no effect on the enzyme from brain.

An investigation of the subunit structure and AMP-deaminases from rabbit and chicken muscle

Abstract Native rabbit and chicken muscle AMP-deaminases have been compared by gel filtration chromatography. These studies, along with previously-reported ultracentrifugal analyses, suggest that these enzymes are virtually identical in size and shape. Chemical and physical evidence is presented which shows that: (a) each enzyme has four subunits; (b) the molecular weights of these subunits are indistinguishable (69,000 Daltons and 73,000 Daltons, as determined by gel filtration chromatography in guanidine hydrochloride and electrophoresis in SDS-containing polyacrylamide gels, respectively; and (c) the native enzymes are not stabilized by disulfiede bridges. Although previous studies suggest that these enzymes share certain structural features, the results presented here provide direct evidence, at the molecular level, that the adenylate deaminases from these sources have similar quaternary structures.

Antigenic structure of AMP-deaminase: isozyme specificity of antibodies directed against purified erythrocyte enzyme

AMP-deaminase is an allosteric enzyme that exists in several molecular forms [l-3]. Characterization of the antigenic structure of this enzyme is of interest because specific antibodies are useful probes for detecting developmental differences in isozyme distribution [4]. Interest in this protein as an antigen also arises from the observation that immunization of rabbits with skeletal muscle AMP-deaminase elicits production of circulating auto-antibody [S]. In an earlier report we compared the effect of rabbit antibody directed against homogeneous avian breast muscle AMP-deaminase on the enzymatic activity in relatively crude preparations from brain and erythrocytes [ 11. We have now purified erythrocyte AMP-deaminase -7000.fold and have carried out a reciprocal study of the interaction between rabbit antibody directed against highly-purified erythrocyte AMP-deaminase and purified isozymes from breast muscle and erythrocytes. The degree to which these isozymes share similar sequence and/or conformational antigenic determinants is not known. However, it is now clear that these isozymes are very similar with respect to the molecular weights of the native enzymes (i.e., 276 000), that each enzyme is a tetramer of subunits of identical size (i.e., 69 000; [6]), and that their amino acid compositions are similar, but not identical (Kruckeberg, S. L. and 0. P. C., unpublished observations). Antibodies obtained from rabbits following

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.

Red blood cell AMP-deaminase: Levels of activity in hemolysates from twenty different vertebrate species

Abstract 1. 1. The levels of adenylate deaminase activity have been measured in the erythrocyte hemolysates and hemolysate supernatants from twenty different vertebrate species. 2. 2. The chicken erythrocyte hemolysates proved to have the highest level of AMP-deaminase activity, with birds in general showing more enzyme activity than the other nucleated red cell types and the non-nucleated red blood cell hemolysates showed low levels of the enzyme activity. 3. 3. A developmental difference in red blood cell adenylate deaminase activity was discovered between 1-day old and adult ducks and was confirmed between 1-day old and adult chickens.

Chicken red blood cell adenylate deaminase: purification and comparison with the enzymes from chicken brain and muscle.

Abstract 1. 1. Lysates of erythrocytes from adult chickens were found to contain fifteen to twenty times as much adenylate deaminase acvitivy as lysates prepared from an equivalent volume of rabbit red blood cells, whereas erythrocytes from 1-day-hatched chicks had activities comparable to mammalian erythrocytes. 2. 2. The adult avian enzyme was purified 750-fold and some of its catalytic, physical and immunological properties were compared to the adenylate deaminases from chicken brain and muscle. 3. 3. The data suggest that at least three forms of adenylate deaminase are present in varying proportions in different chicken tissues.

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.