Abstract In contrast to all prokaryotic organisms previously studied, the fructose-P2 cleavage activity of crude Micrococcus aerogenes extracts was found to be insensitive to high levels of EDTA and o-phenanthroline. This observation suggested that M. aerogenes contained a Class I (Schiff base) fructose-P2 aldolase instead of the metallo (Class II)-aldolase typically found in other prokaryotes. The enzyme was purified about 1000-fold with 70% recovery. The purified aldolase, which appears essentially homogeneous by electrophoretic and gel filtration criteria, was completely active in the presence of 0.01 m EDTA and o-phenanthroline. In addition, direct Zn++ analysis on a metal-free preparation showed that a fully active enzyme sample contained only 0.01 g atom of Zn++ per mole. In contrast to Class II aldolases, the M. aerogenes enzyme lost activity on incubation with the substrate dihydroxyacetone-P in the presence of NaBH4. The degree of enzyme inactivation was directly correlated with the amount of [3H]dihydroxyacetone-P covalently bound to enzyme after treatment with NaBH4 and a stoichiometry of one dihydroxyacetone-P binding site per enzyme subunit was calculated. Identification of the substrate binding site as a lysyl residue by amino acid analysis establishes that this fructose-P2 aldolase is a Class I enzyme. The general catalytic features of the M. aerogenes enzyme resemble those of other Class I aldolases. (a) Its activity is not enhanced by the presence of K+. (b) It cleaves fructose-1-P at a significant rate (fructose-P2 to fructose-1-P activity ratio = 3.3). (c) It has a broad pH optima for fructose-P2 cleavage and a narrow optima for fructose-1-P cleavage; both pH profiles are similar to those of rabbit aldolases A and C. (d) The kinetic parameters Km and Vmax for the two substrates are very similar to those of rabbit aldolases. Km (fructose-P2) = 1 x 10-6 m; Km (fructose-1-P) = 1 x 10-2 m; Vmax (fructose-P2) = 8.2 units per mg; Vmax (fructose-1-P) = 2.4 units per mg. The enzyme, however, differs from other Class I aldolases in that its fructose-P2 cleavage activity is completely insensitive to digestion with carboxypeptidase A and its activity is unaffected by treatment with sulfhydryl reagents. M. aerogenes aldolase is distinct from all aldolases previously studied in that it is composed of a single polypeptide chain. The detection of only Class II aldolase activity in other micrococcus strains (Micrococcus lactilyticus, Micrococcus lysodeikticus, and Acidaminococcus fermentans) as well as all previous studies on the fructose-P2 aldolases of prokaryotic cells, suggest that the Class I aldolase described here has a very restricted distribution among the bacteria.
A full-length cDNA clone for the precursor form of chicken liver apolipoprotein A-I (apoA-I) was isolated by antibody screening of a chicken liver cDNA library in the expression vector lambda gt11. The complete nucleotide sequence and predicted amino acid sequence of this clone is presented. The identity of the clone was confirmed by comparison with partial amino acid sequences for chicken apolipoprotein A-I. Chicken preproapolipoprotein A-1 consists of an 18-amino acid prepeptide, a 6-amino acid propeptide, and 240 amino acids of mature protein. The sequence of the protein is homologous to mammalian apoA-I and is highly internally repetitive, consisting largely of 11-amino acid repeats predicted to have an amphipathic alpha-helical structure. The sequence of the propeptide (Arg-Ser-Phe-Trp-Gln-His) differs in two positions from that of mammalian apoA-I. The mRNA for chicken apoA-I is about 1 kilobase in length and is expressed in a variety of tissues including liver, intestine, brain, adrenals, kidneys, heart, and muscle. This quantitative tissue distribution has been determined and is similar to that observed for mammalian apoE and different from that of mammalian apoA-I mRNA. This reinforces the concept that avian apoA-I performs functions analogous to those of mammalian apoE. Moreover, comparisons revealed sequences of chicken apoA-I similar to the region of mammalian apoE responsible for interaction with cellular receptors. Previous studies have demonstrated striking changes in the rates of synthesis of apoA-I in breast muscle during development and in optic nerve after retinal ablation. We now demonstrate that these changes are paralleled by changes in mRNA levels. ApoA-I mRNA levels increase approximately 50-fold in breast muscle between 14 days postconception and hatching and then decrease about 15-fold to adult levels. The levels of apoA-I mRNA increase about 3-fold in optic nerve following retinal ablation. ApoA-I mRNA is also found in the brain in the absence of nerve injury. This may indicate that locally synthesized apoA-I has a routine or housekeeping function in lipid metabolism in the central nervous system.
We recently observed that, around the time of hatching, chick skeletal muscles synthesize and secrete apolipoprotein A, (apo-A,) at high rates and that reinitiation of synthesis of this serum protein to high levels occurs in mature chicken breast muscle following sur-
It has been well documented that neural information, or the consequences of it, is required for the full phenotypic expression of different skeletal muscle fiber types. In the present work, we investigate the effect of removal of neural information, via surgical denervation, on the levels and rates of synthesis of several enzyme in mature breast (fast-twitch) white muscle fibers of the chicken. Denervation of these muscles resulted in reductions in the concentrations of several glycolytic enzymes to new steady state levels which were only about 50% of normal, and these decreases in enzyme levels were completed within 2 weeks after severing the nerves. In contrast, denervation for as long as 6 weeks did not have a significant effect on the levels of creatine-P kinase molecules in this muscle type. The decreased level of the skeletal muscle-specific aldolase A4 isoenzyme in denervated breast muscle fibers was associated with a 2- to 3-fold reduction in the relative rate of synthesis of this enzyme following denervation. As expected, denervation had no appreciable effect on the relative rate of synthesis of the muscle-specific MM isoenzyme of creatine-P kinase in this muscle. Our results show that neural information, or the consequences of it, is required to maintain the levels and rates of synthesis of glycolytic enzymes but not of creatine-P kinase in mature fast-twitch muscle fibers. We suggest that denervation results in a partial dedifferentiation of these fibers.
The quantitative and qualitative changes in fructose-P2 aldolase isoenzyme concentrations during development of red (leg) and white (breast) skeletal muscles of the chick were investigated. (a) The aldolase C to A subunit transition associated with muscle development is accompanied by large increases in aldolase activity (units/g, wet weight) and in specific catalytic activity (units/mg of protein). The accumulations in both muscle types follow pseudo-first order kinetics with doubling times of 2 to 3 days. The steady state level of aldolase activity in breast muscle (about 150 units/g) is approximately 4-fold higher than that in leg muscle (about 40 units/g). In contrast to leg muscle, the major increase in aldolase activity in breast muscle occurs during postembryonic development. (b) Immunotitration studies demonstrated a direct correlation between increases in enzyme activity and aldolase A subunits during postembryonic muscle development. It was calculated that under steady state conditions, aldolase A4 comprises about 1 percent and 0.26 percent, respectively, of the total wet weight of breast and leg muscle. (c) regulation at the level of protein synthesis in effecting the postembryonic accumulation of aldolase A4 in the muscle types was investigated in short term amino acid incorporation experiments. After a 1-hour pulse with [3H]leucine, aldolase from breast and leg muscle was isolated in a single step by affinity chromatography on phosphocellulose. Incorporation of tritum into aldolase A4 and into soluble or total protein was compared. Between 4 and 38 days after hatching, the rate of aldolase synthesis relative to the synthesis of soluble muscle protein increased about 7- and 3-fold, respectively, in breast and leg muscle. Relative to total protein, incorporation of [3H]leucine into A4 increased about 3-fold in breast muscle, and decreased slightly in leg muscle between 5 and 25 days after hatching. By 3 weeks after hatching, incorporation of [3H]leucine into aldolase A4 relative to incorporation into total protein was about 6-fold higher in breast muscle than it was in leg muscle. The present work, as well as other recent studies, are discussed in relation to the mechanism involved in controlling tissue-specific and stage-specific levels of aldolase isoenzymes in animal cells.
