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Isolation and Characterization of a Mutant Liver Aldolase in Adult Hereditary Fructose Intolerance

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Isolation and Characterization of a Mutant Liver Aldolase in Adult Hereditary Fructose Intolerance Isolation and characterization of a mutant liver aldolase in adult hereditary fructose intolerance. Identification of the enzyme variant by radioassay in tissue biopsy specimens Timothy M. Cox, … , Michael Camilleri, Arthur H. Burghes J Clin Invest. 1983;72(1):201-213. https://doi.org/10.1172/JCI110958. Hereditary fructose intolerance (HFI) is a metabolic disorder caused by enzymic deficiency of aldolase B, a genetically distinct cytosolic isoenzyme expressed exclusively in liver, kidney, and intestine. The molecular basis of this enzyme defect has been investigated in three affected individuals from a nonconsanguineous kindred, in whom fructose-l- phosphate aldolase activities in liver or intestinal biopsy samples were reduced to 2-6% of mean control values. To identify a putative enzyme mutant in tissue extracts, aldolase B was purified from human liver by affinity chromatography and monospecific antibodies were prepared from antiserum raised in sheep. Immunodiffusion gels showed a single precipitin line common to pure enzyme and extracts of normal liver and intestine, but no reaction with extracts of brain, muscle, or HFI liver. However, weak positive staining for aldolase in hepatocyte and enterocyte cytosol was demonstrated by indirect immunofluorescence of HFI tissues. This was abolished by pretreatment with pure enzyme protein. Accordingly, a specific radioimmunoassay (detection limit 7.5 ng) was established to quantify immunoreactive aldolase B in human biopsy specimens. Extracts of tissue from affected patients gave 10-25% immunoreactive enzyme in control samples; immunoreactive aldolase in intestinal extracts from four heterozygotes was reduced (to 55%) when compared with seven samples from normal control subjects (P < 0.05). In extracts of HFI tissues, there was a sevenfold reduction in apparent absolute specific […] Find the latest version: https://jci.me/110958/pdf Isolation and Characterization of a Mutant Liver Aldolase in Adult Hereditary Fructose Intolerance IDENTIFICATION OF THE ENZYME VARIANT BY RADIOASSAY IN TISSUE BIOPSY SPECIMENS TIMOTHY M. COX, MARTIN W. O'DONNELL, MICHAEL CAMILLERI, and ARTHUR H. BURGHES, Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 OHS, England A B S T R A C T Hereditary fructose intolerance (HFI) subjects (P < 0.05). In extracts of HFI tissues, there is a metabolic disorder caused by enzymic deficiency was a sevenfold reduction in apparent absolute specific of aldolase B, a genetically distinct cytosolic isoenzyme activity (1.02 vs. 8.82 U/mg) of immunoreactive fruc- expressed exclusively in liver, kidney, and intestine. tose-l-phosphate aldolase B, but the apparent specific The molecular basis of this enzyme defect has been activity in heterozygotes (7.71 U/mg) was only slightly investigated in three affected individuals from a non- impaired. Displacement radioimmunotitration of al- consanguineous kindred, in whom fructose-l-phos- dolase B in liver supernatants showed a significant (P phate aldolase activities in liver or intestinal biopsy < 0.005) decrease in antibody avidity for immuno- samples were reduced to 2-6% of mean control values. reactive protein in HFI tissue when compared with To identify a putative enzyme mutant in tissue ex- the pure enzyme or extract of normal control liver. tracts, aldolase B was purified from human liver by Immunoaffinity chromatography on antialdolase B- affinity chromatography and monospecific antibodies Sepharose facilitated isolation and purification of en- were prepared from antiserum raised in sheep. Im- zyme from liver biopsy specimens. Active aldolase in munodiffusion gels showed a single precipitin line normal liver, with substrate activity ratios and Mi- common to pure enzyme and extracts of normal liver chaelis constants identical to biochemically purified and intestine, but no reaction with extracts of brain, human enzyme, could be recovered from antibody muscle, or HFI liver. However, weak positive staining columns. Chromatography on monospecific Fab' an- for aldolase in hepatocyte and enterocyte cytosol was tialdolase B enabled pure enzyme protein to be re- demonstrated by indirect immunofluorescence of HFI trieved quantitatively from normal control and HFI tissues. This was abolished by pretreatment with pure liver: direct chemical assay showed 1.88 and 1.15 mg enzyme protein. Accordingly, a specific radioimmu- aldolase protein/g of tissue, respectively. This con- noassay (detection limit 7.5 ng) was established to firmed that the catalytic properties of the HFI aldolase quantify immunoreactive aldolase B in human biopsy were profoundly impaired with specific activities of specimens. Extracts of tissue from affected patients fructose-l-phosphate cleavage of 7.21 and 0.07 U/mg, gave 10-25% immunoreactive enzyme in control sam- respectively. Radioimmunoassay gave estimates of ples; immunoreactive aldolase in intestinal extracts 7.66 and 1.18 U/mg, respectively. Sodium dodecyl from four heterozygotes was reduced (to 55%) when sulfate-polyacrylamide electrophoresis indicated that compared with seven samples from normal control immunopurified aldolase from HFI liver possessed a single subunit size similar to material from control liver extracts: Mr 39,100 vs. 37,900±700 (SD) D, re- spectively. Electrofocusing under denaturing condi- Dr. Cox is a Wellcome Senior Clinical Resarch Fellow. His tions of aldolase isolated in parallel from control and current address is Massachusetts Institute of Technology, HFI liver revealed the same complement of subunits Division of Health Sciences and Technology, Cambridge, MA 02139. and, despite qualitative differences in distribution of Received for publication 22 July 1982 and in revised form bands during degradation, no additional charged spe- 17 March 1983. cies. J. Clin. Invest. The American Society for Clinical Investigation, Inc. - 0021-9738/83/07/0201/13 $1.00 201 Volume 72 July 1983 201-213 Fructose phosphate aldolase deficiency in hereditary Although the pathways of metabolic disturbance fructose intolerance is attended by the synthesis of an that occur in HFI are generally understood, the pri- immunoreactive, but functionally and structurally mary molecular basis of the aldolase defect is obscure. modified enzyme variant that results from a restricted The existence of a functionally modified enzyme pro- genetic mutation. tein in the liver of affected individuals has been sug- gested by the results of kinetic studies. FlP aldolase INTRODUCTION is usually reduced to below 5% of control activities and this residual enzyme possesses an increased apparent Hereditary fructose intolerance (HFI)' is an inborn Km (7). Clearly, however, the presence of the other error of carbohydrate metabolism characterized by aldolase isoenzymes may partly account for such re- vomiting, abdominal pain, and hypoglycemic symp- sidual activity, and more convincing evidence for the toms, which follow ingestion of fructose and related existence of a modified aldolase B stems from the use sugars (1, 2). These and other manifestations of the of type-specific antisera raised against the rabbit liver disorder are related to an enzymic deficiency of liver enzyme. Liver supernatants obtained from patients aldolase or aldolase B (3), which is transmitted by an with HFI contained material coprecipitating with autosomal recessive gene (4). wild-type aldolase B in immunodiffusion gels and Aldolase B (ketose-l-phosphate-aldehyde lyase, EC showing 20-30% apparent cross-reactivity in indirect 4.1.2.7) is chemically and genetically distinct from the antibody neutralization tests (7). A and C isoenzymes and, unlike them, has a powerful A body of evidence thus indicates that HFI is as- catalytic action against D-fructose-l-phosphate (F1P). sociated with the presence of an aberrant aldolase, but The enzyme is expressed almost exclusively in liver, the nature and extent of any intrinsic mutations of kidney, and intestinal mucosa; in HFI, it is these organs enzyme structure in this condition are unknown. A that suffer functional impairment and metabolic de- clear understanding of the molecular basis of such en- rangement during challenge with fructose or its co- zymic defects depends upon structural characteriza- geners. Under such circumstances, sequestration of tion of the mutant protein. In defining the magnitude phosphate-bond energy in the form of fructose ester of genetic defect involved, these investigations gen- generates an intracellular milieu that leads to profound erally indicate the level at which further detailed inhibition of both glycogenolysis and gluconeogenesis. molecular analysis is likely to prove tractable. Ac- Other important sequelae include hypophosphatemia, cordingly, we report studies in which immunochem- hyperuricemia and lactic acidosis (5). ical techniques were used to define the molecular pa- It appears that despite the profound deficiency of thology of aldolase deficiency in hereditary fructose aldolase B in viscera affected by HFI, sufficient activity intolerance. for preservation of glycolytic and some glucogenic fluxes in the absence of fructose challenge can be main- METHODS tained by the A and C isoenzymes. These isoenzymes Tissue specimens. Samples of tissue were obtained by have preferential activity against aldose diphosphates biopsy from control subjects; three members of a kindred and possess activity ratios of between 10 and 100:1 for affected by hereditary fructose intolerance; and four asymp- D-fructose-1,6-diphosphate (FDP) and FIP cleavage, tomatic
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