Effect of Antiserum on Maltase from Intestinal Microvillous Membranes of Ascaris suum

Abstract

This study is based on the premise that the nematode gut is the target of host-immune responses and is designed to test the hypothesis that antibodies reacting with antigenic sites on the surface of this organ interfere with its normal function. Ascaris suum was used as a model to investigate effects of antibodies on the digestion of maltose. Specific experiments involved (1) the isolation of ascaris intestinal brush border, (2) the development of agglutination and precipitin tests for antibodies to brush border antigens, and (3) the effect of antisera on the activity of membrane-bound and solubilized maltase. In the latter study 2 parameters, binding capacity and inhibition of enzyme, were measured. Reaction of antisera with solubilized brush border antigens resulted in the removal of up to 95% of maltase from solution. This was referred to as binding capacity, because the antigenantibody precipitate retained considerable maltase activity. Inhibition or inactivation of both solubilized and membrane-bound enzyme also occurred but was considerably lower than binding. These effects appeared to be associated with IgG immunoglobulins. Results suggest that antibodies induced by a complex array of brush border antigens from the gut of ascaris can react with specific catalytic components associated with this structure and alter activity. Evidence of immunity-induced changes in gut morphology of nematodes (Ogilvie and Hockley, 1968; Lee, 1969), contact of the gut with host tissue fluids, and the importance of the gut in nutrient assimilation (Castro and Fairbairn, 1969; Gentner, Savage, and Castro, 1972) and excretion (Harpur, 1969) suggest that this organ should be studied as a primary target for host-immune responses. Experiments were carried out to test the hypothesis that antibodies reacting with surface antigens on gut cells of nematodes can impair associated physiologic functions. Because of the relatively small size of most parasitic nematodes it is not technically feasible to study isolated gut tissue. Therefore, Ascaris suum was used as a model for this purpose. It was demonstrated previously that disaccharidases are localized in the brush border region of ascaris gut (Gentner, Savage, and Castro, 1972). A study was undertaken to determine the effect of antibody on the activity of membrane-bound maltase as well as solubilized enzyme obtained by treating brush borders with a detergent. It is presumed that effects of antibodies on enzyme components Received for publication 5 July 1973. * Work was supported by PHS Research Grant AI 11361 from the NIH. t Address reprint requests to: Dr. G. A. Castro, Program in Physiology, University of Texas Medical School, Houston, Texas 77025. of the gut surface reflect alterations in structural elements as well. Alterations in either catalytic or structural components would have a bearing on function. MATERIALS AND METHODS Pr paration of antiserum to brush border membranes Detailed procedures for collecting ascaris brush border have been published (Gentner, Savage, and Castro, 1972). Brush border membranes containing 7.1 mg protein (Lowry et al., 1951) were mixed with 1.5 ml of complete Freund's adjuvant (Difco Laboratories, Detroit, Mich.). Each of two New Zealand white rabbits weighing approximately 4 kg was injected subcutaneously (SC) in the nape with 1.0 ml and intramuscularly (IM) in each thigh with 0.25 ml of the mixture. Seventeen days later each rabbit was injected with a saline homogenate of brush border possessing a total protein content of 5 mg/ml; 5 mg were administered in a single SC injection and 2.5 mg were given IM in each thigh. Serum was collected from rabbits by heart puncture prior to antigen exposure and at weekly intervals following primary and challenge injections (i.e., days 7, 14, 24, 32, and 40 after primary exposure). Serum was separated from clots, centrifuged at 3,000 g for 1 hr to sediment cells, aspirated into vials, and stored at -20 C. Sera taken before and after primary antigen exposure will henceforth be referred to as normal sera and antisera, respectively. Solubilization of brush border Brush borders from approximately 200 worms were homogenized by hand in a Potter-Elvehjem