Multiple forms of fructose diphosphate aldolase in mammalian tissues.

Abstract

\'Jany of the molecular and catalytic properties of aldolase A, the classical muscle enzyme, and aldolase B, liver aldolase, have been defined.' Although these enzymes have generally similar molecular properties, they have different primary structures and can also be readily distinguished by their catalytic properties.2 In this paper, we report the existence of a third fructose diphosphate aldolase termed "aldolase C" which is distinct from aldolases A and B. These three parent forms of aldolase appear to be structurally homologous since hybrids of these enzymes can be formed by dissociation and reassociation of mixtures of the parent aldolases. Five forms, including three hybrids, are detectable by zone electrophoresis and by chromatography for each two-membered set of parent aldolases. Hybrid forms of the aldolase A-B and aldolase A-C sets are found in extracts of tissues which are known to contain aldolases A and B or A and C, respectively. The fructose diphosphate aldolase activity of all tissues examined can be related to the presence of the parent FDP a-dolases A, B, C, or their hybrids. The number of hybrids found (three) in each two-membered aldolase set is difficult to reconcile with a three-chain model of the enzyme, but it is easily harmonized with a molecule composed of four subunits. In spite of considerable chemical and physical evidence supporting the three-chain model, we have been led to consider the latter possibility seriously.Materials and Methods.-Enzymes and reagents: Rabbit muscle aldolase A (Boehringer) had a specific activity ranging from 10 to 12 units/mg protein. Aldolase B, prepared by the method of Rajkumar, Woodfin, and Rutter,3 had a specific activity of 1.5 units/mg protein. Glyceraldehyde-3-phosphate dehydrogenase was purchased from Boehringer. NaFDP, NAD, and NADH were procured from Sigma Chemical Co. Nitroblue tetrazolium and phenazine methosulfate were obtained from Aldrich Chemical Co.Assays: Aldolase activity was assayed as described elsewhere.4 A unit of enzymatic activity is defined as the cleavage of 1 Mmole of substrate (FDP or F1P) per min.Immunochemical analysis: Preparation of antibodies against aldolases A and B and the methods of immunochemical analyses have been described elsewhere.4 5 Preparation of tissue extracts: The tissues were excised, minced, and homogenized in the cold for 15 sec in presence of 2 vol (w/v) of 0.01 M Tris Cl and 0.001 M EDTA, pH 7.5. The homogenate was centrifuged at 100,000 g at 20 for 60 min.Zone electrophoresis: Zone electrophoresis was performed in 0.06 M barbital, 0.01 M jB-mercaptoethanol, pH 8.6, on 63/4' cellulose acetate strips (Gelman) at 250 v for 90 min with the origin equidistant from the electrodes. The strips were stained for aldolase activity by a modification of a procedure developed by Crawford.6 A 0.5% Noble agar solution in 0.01 M NaAsO4, pH 7.5, containing 0.01 M NaFDP (Calbiochem), 0.001 M NAD, 0.12 mg/ml glyceraldehyde-3-phosphate dehydrogenase, 0.024 mg/ml phenazine methosulfate, and 0.4 mg/ml nitroblue tetrazolium ch...

Purification of Aldolase C from Rat Brain and Hepatoma

“…This specific activity in the rat is lower than in the chicken [lo] but higher than in the rabbit [6] ox [7] or man [ll]. The fmctose-l,6-P2/fructosel-P activity ratios (7.3, 7.4) are characteristic of aldolase C [19]. The K, value for both substrates were identical for the two enzymes studied, suggesting that the same protein is present in brain and in hepatoma.…”

“…The purity of the protein was checked by polyacrylamide gel, using the method of Davis [18] followed by Coomassie brilliant blue staining, or by the aldolase-specific staining procedure according to Penh d t and Rutter [19].…”

Expression of aldolase isozyme mRNAs in fetal rat liver

“…However, the expression of each subunit is controlled independently. The accumulations of these subunits are tissuespecific or organ-specific in normal animals; aldolase A subunit is predominant in muscle cells, whereas the B subunit is produced in liver cells, and the C subunit is synthesized in brain cells [4]. Moreover, change in the isozyme: pattern in fetal liver during development has been observed.…”

A Comparative Study of the Structure of Muscle Fructose 1,6‐Diphosphate Aldolases

“…From its mobility on cellulose acetate electrophoresis, the rabbit enzyme has a slightly greater net negative charge at pH 8.0 than the pig and ox enzymes. However, since it is known that aldolases from different tissues within a given animal have different mobilities on cellulose acetate and are still capable of forming active hybrids [6], it would appear that the importance of net charge to both catalytic function and formation of quaternary structure is small.…”