Monoclonal antibodies to renal brush border membrane maltase: age-associated antigenic alterations.

Abstract

Monoclonal antibodies to maltase (a-D-glucoside glucohydrolase, EC 3.2.1.20) from young adult and aged rats were prepared by the hybridoma technique. Four cell lines producing antibodies of the IgGI subclass to maltase were established. Two, designated IF12E1 and 8B1G6, produced monoclonal antibodies specific for the catalytically active form of the enzyme found predominantly in enzyme preparations from young animals. The other two clones designated 7G1OH3 and 2E1C1O produced monoclonal antibodies that reacted exclusively with an enzymatically inactive form of maltase found mostly in enzyme preparations from aged rats. The increased prevalence of an inactive form of the enzyme in the old rat accounts for the decreased maltase-specific activity previously reported in the senescent rat. The active and inactive maltase species were separated by immunoaffinity chromatography by using the monoclonal antibodies as ligands. The separated forms of the enzyme were not distinguished by NaDodSO4/ polyacrylamide gel electrophoresis, peptide mapping of the CNBrcleaved proteins, and the NH2-terminal residues of these peptides. This study demonstrates the presence of an altered, antigenically distinct enzyme in senescent animals. Critical issues on the mechanism of the aging process may be addressed by application of these findings.Maltase (a-D-glucoside glucohydrolase, EC 3.2.1.20) is the major disaccharidase in rat kidney brush border membranes (1, 2). Maltase-specific activities in renal cortex homogenates and brush border membranes prepared from aged rats decrease 30-40% relative to the activities of the enzyme from young adult animals (3,4). When the enzyme from senescent and mature rats is solubilized and purified to homogeneity, the same decrement with age is found (5). This finding indicates that the decrease in maltase activity with age results from an alteration in the enzyme per se, rather than from a change in the enzyme's membrane environment, which is reflected secondarily in a loss in activity. The pure "young" and "old" enzymes do not differ measurably in molecular weight, electrophoretic mobility, and amino acid composition. However, significant differences are found in their circular dichroism spectra, perhaps suggesting a conformational alteration with age (5). These results prompted us to examine whether this age-dependent molecular change is associated with modification in the antigenicity of the enzyme. The present communication shows that monoclonal antibodies (MAbs) specific to young and old forms of maltase can be obtained by hybridoma technology and that the two species of enzyme can be separated by using the MAbs as immunoaffinity ligands. Thus, we demonstrate the presence of an altered, antigenically distinct enzyme in senescent animals. MATERIALS AND METHODSEnzyme Purification. Kidney brush border maltase was purified to homogeneity from mature (6 mo) and aged (24 mo) female Wistar-derived rats as described (5, 6). The animals were obtained from the Animal Resources Facility, Gerontology...

Microdomains of distinctive glycoprotein composition in the kidney proximal tubule brush border.

“…Recognizes both gp330 and maltase (by immunoprecipitation) . FIGURE 14 Immunogold localization of gp330 to several coated pits (cp) on glomerular epithelial cells . Clusters of protein A-gold are found in three coated pits located on the sides of the foot processes .…”

“…Monoclonal antibodies directed against gp330 (the Heymann nephritis antigen) were obtained from clone D 1551`21 (IgG2 subclass) (13) and those for active maltase from clone 1F12G1 (IgG, subclass) . IgG was purified from ascites by ammonium sulfate precipitation followed by ion-exchange chromatography on DEAE-Biocel A (15) or by affinity chromatography on a Protein A-Sepharose 4B column (14).…”

Structural Modifications of Proteins During Aging

“…Thus, Sharma and Rothstein 96 demonstrated that the young and old forms of the glycolytic enzyme enolase (isolated from young and old nematodes, respectively), which display different activities, share the same amino acids sequence; however, when these two enzyme species were unfolded by guanidine:HCl (a strong denaturant of proteins) and then allowed to refold, they formed an identical structure similar to that of old enolase. Strong evidence was also found for conformational modifications as the origin of the observed aging effects in rat kidney maltase 33 and in rat muscle GAPDH 27 . None of these studies, however, provided direct, conclusive, evidence that there were no other changes in the old forms of these proteins and, hence, that purely conformational alterations were indeed responsible for the effects seen.…”

“…These modifications have been found to involve the functional properties, the stability, as well as the structural integrity of these proteins. Old proteins (i.e., those found in tissues of old animals) often, though not always, display a reduction in their biological activity, 26–28 a modified heat inactivation pattern reflecting alterations in their stability, 29–31 and frequently possess a modified affinity to antibodies raised against the young form of the protein, a reflection of changes in structural integrity 32,33 . The fact that, as a rule, those proteins whose activity is affected by aging, display reduced (rather than enhanced) biological competence is not surprising as evolution has strongly selected for each protein a structure that possesses a high biological activity, and a change in the former is likely to reduce the latter.…”

Sexual dimorphism in rat cerebrum and cerebellum: different patterns of catalytically active creatine kinase isoenzymes during postnatal development and aging

“…Glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase, also have catalytically inactive isoforms during aging (Dovrat et al, 1984;Hopkirk and Bloxham, 1979). Ornithine decarboxylase in the mouse brain (Laitinen, 1985), as well as maltase (Reiss and Sacktor, 1983), and glucose 6-phosphate dehydrogenase (Dovrat et al, 1986) are other examples of inactive isoenzymes that could be part of cell structures.…”