Herbert G. Lebherz

Fructose-bisphosphate aldolases: an evolutionary history

Two mechanistically distinct forms of fructose-bisphosphate aldolase are known to exist. It has been assumed that the Class II (metallo) aldolases are evolutionary more primitive than their Class I (Schiff-base) analogs since the latter had only been found in eukaryotes. With the identification of prokaryotic Class I aldolases, we present here an alternative scheme of aldolase evolution. This scheme proposes that both aldolase classes are evolutionarily ancient and rationalizes the observed highly variable expression of both enzyme types in contemporary file forms.

Regulation of chicken apolipoprotein B: cloning, tissue distribution, and estrogen induction of mRNA.

Apolipoprotein (apo) B is a major protein component of plasma very low-density and low-density lipoproteins (VLDL and LDL, respectively) and serves as a recognition signal for the cellular binding and internalization of LDL by the apoB/E receptor. In contrast to the situation in mammals, avian apoB is also a component of specialized VLDL particles that are produced by the liver in response to estrogen. These particles transport cholesterol and triglyceride from the liver to the ovary for deposition in egg yolk. We report here the identification and characterization of cDNA clones for chicken apoB and their use in examining the tissue distribution and hormonal regulation of chicken apoB mRNA. The cDNA clones were identified by immunological screening of a phage lambda gt11 library constructed with hen liver mRNA and their identity was supported by sequence comparisons with mammalian apoB. The chicken apoB mRNA is approximately the same size as mammalian apoB mRNA (14 kb), and, as occurs in mammals, is present at high levels in liver and small intestine. Unlike mammals, the chicken apoB mRNA is also found at high levels in the kidney, consistent with previous protein biosynthetic studies. A DNA-excess solution-hybridization assay was used to quantitate apoB mRNA in these tissues and to examine its hormonal regulation. In control roosters the liver and kidney contained 65% and 10%, respectively, as much apoB mRNA as the small intestine. Within 24 h after estradiol administration, apoB mRNA was increased five- to seven-fold in liver but was unchanged in intestine and kidney. The increase in apoB mRNA content and the kinetics of induction parallel hepatic apoB synthesis, indicating that estrogen regulates apoB production through changes in the cellular abundance of apoB mRNA. The apoB mRNA increased rapidly following hormone treatment while the mRNA for another VLDL protein (apoII) showed a lag or slow phase of several hours before significant mRNA accumulation occurred. These data indicate that the liver can respond immediately to estrogen to increase apoB mRNA accumulation, while apoII mRNA accumulation appears to involve additional events or signals which occur slowly and are specific to this gene.

Characterization of transverse tubule membrane proteins: Tentative identification of the Mg-ATPase

Vesiculated fragments of chicken skeletal muscle transverse tubule (TT) membranes were analyzed for their content of loosely associated and integral membrane proteins. Of particular interest was the identification of the magnesium-stimulated ATPase (Mg-ATPase), which is characteristically located in native isolated TT vesicles of chicken skeletal muscle [R. A. Sabbadini and V. R. Okamoto (1983) Arch. Biochem. Biophys. 223, 107-119]. A number of the proteins found in vesicular TT preparations were found to be extractable by a mild Triton-X100 treatment and were identified as aldolase, enolase, creatine kinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and pyruvate kinase. Approximately 60% of TT-associated protein was extracted with Triton, resulting in a twofold enrichment of the Mg-ATPase. Concommitantly, one core integral membrane protein possessing a Mr of 102,000 was enriched, suggesting that it is responsible for the Mg-ATPase activity present in chicken skeletal muscle TT membranes.

Regulation of concentrations of glycolytic enzymes and creatine-phosphate kinase in “fast-twitch” and “slow-twitch” skeletal muscles of the chicken☆

Abstract The distinctive contractile and metabolic characteristics of different skeletal muscle fiber types are associated with different protein populations in these cells. In the present work, we investigate the regulation of concentrations of three glycolytic enzymes (aldolase, enolase, glyceraldehyde-3-phosphate dehydrogenase) and creatine-phosphate kinase in “fast-twitch” (breast) and “slow-twitch” (lateral adductor) muscles of the chicken. Results of short-term amino acid incorporation experiments conducted both in vivo and with muscle explants in vitro showed that these enzymes turnover at different rates and that aldolase turns over 2 to 3 times faster than the other three enzymes. However, these differences in turnover rates were difficult to detect in long-term double-isotope incorporation experiments, presumably because extensive reutilization of labeled amino acids occurred during these long-term experiments. Mature muscle fibers synthesize these four cytosolic enzymes at very high rates. For example, 11 to 14% of the total labeled leucine incorporated into protein by breast muscle fibers was found in the enzyme aldolase. Results of short-term amino acid incorporation experiments also showed that the relative rates of synthesis of the three glycolytic enzymes were about fourfold higher in mature “fast-twitch” muscle fibers than in mature “slow-twitch” ones while the relative rates of synthesis of creatine-phosphate kinase were similar in the two fiber types. The relative rates of synthesis of these four enzymes and cytosolic proteins in general were found to be very similar in immature muscles of both types. More profound changes in the relative rates of synthesis of major cytosolic proteins, including the glycolytic enzymes, occurred during postembryonic maturation of fast-twitch fibers than occurred during maturation of slow-twitch fibers. Our work demonstrates that (1) the synthesis of creatine-phosphate is independently regulated with respect to the synthesis of the glycolytic enzymes in muscle fibers; and (2) the approximate fourfold higher steady-state concentrations of glycolytic enzymes in fast-twitch muscle fibers as compared with slow-twitch fibers are determined predominantly by regulatory mechanisms operating at the level of protein synthesis rather than protein degradation. Our demonstration that more profound changes in the relative rates of synthesis of major cytosolic proteins occur during maturation of fast-twitch fibers as compared with slow-twitch fibers is discussed in terms of the mode(s) of fiber-type differentiation proposed by others.

A Simple Procedure for the Isolation of Seven Abundant Muscle Enzymes

The present work describes procedures in which seven major muscle enzymes and serum albumin can be simultaneously isolated from chicken skeletal muscles. The seven enzymes isolated were: phosphorylase, enolase, creatine-P kinase, aldolase, glyceraldehyde-3-P dehydrogenase, phosphoglycerate mutase, and triose-P isomerase. The proteins isolated by these methods were judged to be greater than 97% pure on the basis of electrophoretic analysis in sodium dodecyl sulfate polyacrylamide gels. The procedure is applicable for isolation of the enzymes from large (greater than 100 g) or small (less than 0.5 g) amounts of muscle tissue and the entire procedure can be completed within two days. Particularly useful features of the procedures are: (1) preferential solubilization of the enzymes from myofibrils by extraction of muscle specimens in solutions of different ionic strength; (2) specific precipitation of phosphorylase, creatine-P kinase, and glyceraldehyde 3-Phosphate dehydrogenase from solutions of specified pH and degrees of ammonium sulfate saturation; and (3) an alternate method for isolation of glyceraldehyde-3-P dehydrogenase by specific elution of the enzyme from phosphocellulose columns with ATP. Because of the ease, rapidity, and reproducibility of the procedures, these methods may be useful for the routine isolation of the muscle enzymes in studies on biochemical regulation, as well as for obtaining large quantitites of the enzymes for structural analysis.

Content and synthesis of several abundant glycolytic enzymes in skeletal muscles of normal and dystrophic mice

In both 2- and 3-month-old 129 ReJ mice, the catalytic activity levels of three enzymes involved in glycogen breakdown (phosphorylase, enolase, and aldolase) were found to be 35-50% lower in hind limb muscles of dystrophic mice as compared with normal mice. The reduced activities of these enzymes in the diseased tissue was directly due to corresponding reductions in the number of enzyme molecules rather than being due to inactivation of the enzymes in the dystrophic muscle. Results of short term double isotope incorporation experiments conducted with muscle explants in vitro suggested that the rates of synthesis of these enzymes, and of most other abundant cytosolic proteins, relative to each other, were similar in hind limb muscles of normal and dystrophic mice. The present work on murine muscular dystrophy is discussed in terms of our previous studies into the influence of avian muscular dystrophy on the content and synthesis of abundant glycolytic enzymes in chicken skeletal muscles.

STUDIES ON THE REGULATION OF PROTEIN CONCENTRATIONS IN “RED” AND “WHITE” SKELETAL MUSCLES*

Publisher Summary This chapter presents the studies on the regulation of protein concentrations in red and white skeletal muscles. The structural and functional characteristics of animal cells are largely determined by the assortment of proteins they contain. As is the case with other types of animal cells, much of the fate of a muscle cell must be predetermined by events occurring during the process of cellular differentiation, and many of the biochemical changes that occur during embryogenesis in vivo have been observed when cultures of myogenic cells are allowed to differentiate in vitro. Myosin and actin occupy the same intracellular compartment, the myofibril. A final consideration on compartmentalization of the proteins concerns the possibility that certain muscle enzymes, especially those operating in the glycolytic pathway, may interact with each other to form a multienzyme complex at or near the myofibril.

Similarities in properties, content, and relative rates of synthesis of fructose-P2 aldolase in livers of fed and starved rats

The present work gives evidence that, in contrast to the situation reported by Pontremoli et al. for the rabbit (Proc. Natl. Acad. Sci. U.S.A. 76, 6323–6325, 1979; Arch. Biochem. Biophys. 203, 390–394, 1980; Proc. Natl. Acad. Sci. U.S.A., 79, 5194–5196, 1992), starvation for as long as 3 days does not cause intracellular covalent modification and inactivation of fructose-P2 aldolase molecules in rat liver cells. This conclusion is based on our observations that liver aldolase molecules isolated from fed and starved rats in the presence of proteolytic inhibitors were not distinguished on the basis of specific catalytic activity, electrophoretic mobility, subunit molecular weight, NH2-terminat structure, or COOH-terminal structure. Further, the approximate 40% loss in rat liver mass which occurred during the 3-day fast was not associated with appreciable changes in the content of aldolase and most other abundant cytosolic proteinsper gram of rat liver, as judged by electrophoretic analysis of 100 000-g soluble fractions of liver extracts . Finally, a 3-day fast had no appreciable effect on therelative rates of synthesis of aldolase and most other abundant cytosolic proteins in rat liver. Our findings suggest that nutrient deprivation has no preferential effect on the concentration or metabolism of aldolase in rat liver cells.

A selective reaction of fructose bisphosphate aldolase with fluorescein isothiocyanate in chicken muscle extracts

The present work describes the selective covalent modification of fructose bisphosphate aldolase in crude extracts of chicken breast muscle by fluorescein 5′‐isothiocyanate (5′‐FITC) at pH 7.0 and 35°C. The modification was observed after 1 min while no other major soluble protein was labeled even after 30 min. We calculated that ca. one 5′‐FITC molecule was incorporated into each aldolase tetramer after a 30 min reaction which resulted in a minimal loss of enzyme activity. The “native” structure of aldolase was required for the selective modification by 5′‐FITC since high pH, high temperature, and ionic detergents either inhibited or prevented the reaction of 5′‐FITC with aldolase. Certain metabolites (ATP, ADP, CTP, GTP, FBP) and erythrosin B also inhibited the 5′‐FITC modification of aldolase. In contrast, F‐6‐P, AMP, NADH, and NAD+ as well as free lysine and most importantly, the 6′‐isomer of FITC exhibited no competition with 5′‐FITC for the labeling of aldolase. Alone, the 6′‐isomer of FITC did not exhibit preferential reaction when combined with aldolase. 5′‐FITC‐labeled and ‐unlabeled aldolases were not distinguished by their ability to bind to muscle myofibrils (MFs) or by their abilities to refold following reversible denaturation in urea. Structural analysis revealed that 5′‐FITC‐labeled a tryptic peptide corresponding to residues 112–134 in the primary structure of aldolase, a peptide that does not contain lysine, the amino acid believed to be the primary target of this reagent. Unlike chicken and rabbit muscle aldolases, chicken brain and liver aldolase isoforms along with several other aldolases derived from diverse biological sources did not exhibit this highly selective modification by 5′‐FITC. Copyright

Specific, limited tryptic modification of wheat-germ fructose-bisphosphate aldolase subunits: destruction of catalytic activity but not of ability to establish precise subunit-subunit recognition

We have been using the glycolytic enzyme fructose-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) as a model system to investigate the assembly of oligomeric enzymes. In the present work, we investigate the effect of specific, limited tryptic modification on the properties of aldolase isolated from wheat germ. The wheat-germ enzyme was selected, since several aldolases isolated from animal sources were not readily susceptible to the specific tryptic modification seen with this plant enzyme. We will show that: Low levels of trypsin cause a first-order inactivation of wheat-germ aldolase activity which is associated with a fairly specific cleavage of the enzyme which reduces its subunit molecular weight from 41000 to 39000. The proteolytic modification is greatly inhibited in the presence of the aldolase substrate, fructose bisphosphate. The intact and modified enzymes appear to have similar surface changes, as judged by their behavior during electrophoresis in polyacrylamide gels under non-denaturing conditions. The modified aldolase is not specifically eluted from phosphocellulose columns by fructose bisphosphate under the conditions used in the affinity chromatographic isolation of the intact enzyme, suggesting that the modified enzyme may no longer be able to bind substrate. Although enzymatically inactive, the modified aldolase subunits are able to refold and reassociate into tetrameric combinations following unfolding of the subunits by treatment at low pH; thus, this specific proteolytic modification does not interfere with the ability of wheat-germ aldolase subunits to refold and to establish precise subunit-subunit recognition in vitro.