Characterization of the inactive form of fructose-1,6-bisphosphate aldolase isolated from livers of fasted rabbits.

Pontremoli, S.; Melloni, Edon; Michetti, M.; Salamino, Franca; Sparatore, Bianca; Horecker, B.L.

doi:10.1073/pnas.79.17.5194

Cited by 10 publications

(2 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Treatment of liver aldolase with carboxypeptidase decreases the activity towards both hexose substrates by approx. 50 % [40,42] ; starvation-induced proteolysis of liver aldolase in mice, which also cleaves C-terminal residues, shows no selective effects on F-1-P activity [43]. Although the partly denatured Val$$( protein might be degraded more rapidly in i o compared with the wildtype enzyme, this would not explain the loss of function specific to aldolase B.…”

Section: Discussionmentioning

confidence: 99%

Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337→Val) in fructose intolerance

RELLOS*¹,

ALI*²,

VIDAILHET³

et al. 1999

Biochem. J.

View full text Add to dashboard Cite

A molecular analysis of human aldolase B genes in two newborn infants and a 4-year-old child with hereditary fructose intolerance, the offspring of a consanguineous union, has identified the novel mutation Ala337-->Val in homozygous form. This mutation was also detected independently in two other affected individuals who were compound heterozygotes for the prevalent aldolase B allele, Ala149-->Pro, indicating that the mutation causes aldolase B deficiency. To test for the effect of the mutation, catalytically active wild-type human aldolase B and the Val337 variant enzyme were expressed in Escherichia coli. The specific activities of the wild-type recombinant enzyme were 4.8 units/mg and 4.5 units/mg towards fructose 1,6-bisphosphate (FBP) and fructose 1-phosphate (F-1-P) as substrates with Michaelis constants of 4 microM and 2.4 mM respectively. The specific activities of purified tetrameric Val337 aldolase B, which affects an invariant residue in the C-terminal region, were 4.2 units/mg and 2.6 units/mg towards FBP and F-1-P as substrates respectively; the corresponding Michaelis constants were 22 microM and 24 mM. The FBP-to-F-1-P substrate activity ratios were 0.98 and 1.63 for wild-type and Val337 variant enzymes respectively. The Val337 mutant aldolase had an increased susceptibility to proteolytic cleavage in E. coli and rapidly lost activity on storage. Comparative CD determinations showed that the Val337 protein had a distinct thermal denaturation profile with markedly decreased enthalpy, indicating that the mutant protein is partly unfolded. The undegraded mutant had preferentially decreased affinity and activity towards its specific F-1-P substrate and maintained appreciable activity towards FBP. In contrast, fluorescence studies of the mutant showed an increased binding affinity for products of the aldolase reaction, indicating a role for the C-terminus in mediating product release. These findings in a rare but widespread naturally occurring mutant implicate the C-terminus in the activity of human aldolase B towards its specific substrates and demonstrate its role in maintaining the overall stability of the enzyme tetramer.

show abstract

Section: Discussionmentioning

confidence: 99%

Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337→Val) in fructose intolerance

RELLOS*¹,

ALI*²,

VIDAILHET³

et al. 1999

Biochem. J.

View full text Add to dashboard Cite

show abstract

“…In the case of the starved rabbit, it was suggested that the inactive enzyme is a slightly truncated form derived from cathepsin M activity (20,21). A similar truncated form was found in the rat irradiated with x-rays and was attributed to cathepsin B activity (22,23).…”

Section: Discussionmentioning

confidence: 91%

Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.

Reznick¹,

Rosenfelder²,

Shpund³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A method has been developed that enables us to identify intracellular degradation intermediates of fructosebisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This method is based on the use of antibody against thoroughly denatured purified aldolase. This antibody has been shown to recognize only denatured molecules, and it did not interact with "native" enzyme. Supernatants (24,000 x g for 30 min) of liver and kidney homogenates were incubated with antiserum to denatured enzyme. The antigen-antibody precipitates thus formed were subjected to NaDodSO4/PAGE, followed by electrotransfer to nitrocellulose paper and immunodecoration with antiserum to denatured enzyme and 1251-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of the intact subunit) to 18,000, which cross-reacted antigenically with denatured fructose-bisphosphate aldolase, could be identified in liver. The longest three peptides were also present in kidney. The possibilit that these peptides were artifacts of homogenization was ruled out as follows: 1251-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were then subjected to NaDodSO4/PAGE followed by autoradiography, and the labeled enzyme was shown to remain intact. This method is suggested for general use in the search for degradation products of other cellular proteins.The various stages that comprise the process of intracellular protein degradation and the proteases involved have remained obscure despite extensive studies in many laboratories (1, 2). So far no high molecular weight intermediates of intracellular degradation of specific native proteins have been identified in eukaryotes. The identification of such intermediates is obviously necessary in order to elucidate the mechanism of intracellular degradation of proteins. Several previous experimental results from our laboratory have indicated that a direct search for degradation intermediates of specific proteins might be feasible for the following reasons:

show abstract

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

1983

View full text Add to dashboard Cite

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, 1982), 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-terminal 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 proteins per 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 the relative 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.

show abstract

Characterization of the inactive form of fructose-1,6-bisphosphate aldolase isolated from livers of fasted rabbits.

Cited by 10 publications

References 18 publications

Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337→Val) in fructose intolerance

Alteration of substrate specificity by a naturally-occurring aldolase B mutation (Ala337→Val) in fructose intolerance

Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.

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

Contact Info

Product

Resources

About