Biomedical subjects
Publications and source records attributed to E Shapira.
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The complex of trypsin with purified alpha2-macroglobulin from normals and patients with cystic fibrosis was studied. The formed complex failed to reveal any proteolytic activity toward a high molecular weight substrate whereas the esterolytic activity towards a low molecular weight substrate was retained. This esterolytic activity was resistant to inhibition by a high molecular weight inhibitor. During iincubation at 38 degrees C the complex with normal alpha2-macroglobulin was slowly inhibited by the high molecular weight inhibitor and regained activity with the high molecular weight substrate. This phenomenon was not obtained when the alpha2-macroglobulin from cystic fibrosis was examined. These data suggest that the gradual conversion of normal alpha2-macroglobulin-trypsin complex into an alpha2-macroglobulin fragment-trypsin complex is deficient in patients with cystic fibrosis.
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A child with recurrent pyogenic infections, eczema, markedly elevated serum concentrations IgA, and totally absent PMN chemotactic acticity is described. The chemotactic defect was characterized as a humoral inhibitor present in serumand plasma, directed toward autologous as well as homologous PMN's. Immunochemical studies revealed the purified inhibitor to be a cryoglobulin composed of an IgM-IgA complex. Incubation of the cryoglobulin complex with PMN's resulted in a quantitative dose-response curve.
Two acidic beta-galactosidase isozymes (designated A1 and A2) were separated by isoelectric focusing from beta-galactosidase A of human liver. Kinetic studies with 4-methylumbelliferyl-beta-D-galactopyranoside substrate revealed similar parameters for both. The Km value was 0.32 mmol/1 for A1 and 0.30 mmol/1 for A2 and Vmax values of 59.3 and mumol min-1 mg-1, respectively. The pH optimum was 4.2 for beta-galactosidase A1 and 4.5 for the A2 form. The A1 enzyme form was shown to be more heat labile than the A2. Significant differences were observed with antibody preparations against the two enzyme forms. Using the anti-A1 antibodies two precipitin arcs with residual enzymatic activity were obtained by immunoelectrophoresis of beta-galactosidase A whereas only one with anti-A2 antibodies. Anti-A1 precipated 85% of the original activity present in beta-galactosidase A and only 56% could be precipated by anti-A2. The possibility of common structural components is suggested.
The inhibition by specific antibody of carbonic anhydrase B activity towards bicarbonate was uncompetitive (Ki 9.5 X 10(-7) M) whereas that of activity towards p-nitrophenylacetate was mixed-type (Ki 9.2 X 10(-7) M). Differences in the immunological reactivity of the enzyme in the presence of the substrates was documented by quantitative precipitin tests and by the Farr technique. In the presence of bicarbonate, nearly half of the antigenic determinants of the enzyme were altered to such an extent that antibodies directed against these determinants in the native form did not bind. This antibody fraction was separated and shown to react with the native enzyme in different molar ratios than the total antibody population. It produced only a very limited inhibition of the activity towards CO2 and p-nitrophenyl acetate. The immunological reactivity of the enzyme with both the total antibody population and the fraction that is nonreactive in the presence of bicarbonate was identical in the absence of substrates and in the presence of CO2 and p-nitrophenyl acetate. It is suggested that the native enzyme form exhibits enzymatic activity towards CO2 and p-nitrophenyl acetate, whereas in the presence of bicarbonate a "conformational adaptation" is induced.
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Various fractions obtained from normal human liver homogenates by gel chromatography and selective adsorption and elution from insolubilized concanavalin A were compared as to their beta-galactosidase and beta-glucosidase activities. The high-molecular-weight acidic beta-galactosidase form was converted into the smaller major form by sodium dodecyl sulfate treatment. Electrophoresis and electrofocusing on acrylamide slabs revealed, in addition to the two major isoenzyme forms (acid and neutral), another five minor bands with enzymatic activity.
The present study using immunologic methodology confirms previous observations from this laboratory of an absence of a protease component with arginine esterase activity in plasma of patients with cystic fibrosis. In this study, the pooled plasma from control individuals was activated and partially purified after adsorption on columns of soybean trypsin inhibitor conjugated to Sepharose 4B followed by elution with benzamidine. The fraction was further purified by isoelectrofocusing on polyacrylamide gels. Proteins around the pI range of 5.5 were eluted and utilized to prepare an antiserum. Immunoelectrophoresis of activated plasma samples from control subjects and patients with cystic fibrosis was performed utilizing the antiserum. In controls, four precipitin arcs with residual esterase activity were observed, whereas only three were seen in plasma from patients with cystic fibrosis. Double gel diffusion experiments using specific antisera ruled out the presence of trypsin, chymotrypsin, plasminogen, prothrombin, C1 esterase, alpha one-trypsin inhibitor, and inter-alpha-trypsin inhibitor in the concentrated benzamidine eluate. The antisera to alpha two-macroglobulin gave an immunoprecipitate which was readily stained for proteolytic activity. On immunoelectrophoresis, the alpha two-macroglobulin precipitin band corresponded to the band absent in plasma of patients with cystic fibrosis. In contrast, the alpha two-macroglobulin levels were similar in plasma of control subjects and patients with cystic fibrosis. Using the antiserum to the protein fractith proteolytic activity could be demonstrated in control plasma. One specific enzyme-active "rocket" was absent in plasma of patients with cystic fibrosis. In a double blind study of 15 control samples and 15 samples from patients with cystic fibrosis, a specific "rocket" was shown to be present in 13 control samples and absent in 14 cystic fibrosis samples. alpha two-Macroglobulin was determined by both an immunologic procedure and by its trypsin binding (trypsin protein esterase concentration). The ratio of the immunologic assay to the biologic activity assay was 90 for the normal plasma samples and only 65 for cystic fibrosis samples.
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In certain cases of obscure chronic lung disease, the possibility of a genetic disorder, such as cystic fibrosis of the pancreas, should be considered. When the genetic defect fails to cause pancreatic involvement, the partial expression of cystic fibrosis is encountered. Elevation of sweat electrolyte levels is not sufficient to confirm the diagnosis of cystic fibrosis in adults since normal levels vary widely in both adolescents and adults. Other tests should, therefore, be utilized. The cilia dyskinesis test is positive in both homozygous and heterozygous patients with cystic fibrosis. Utilization of this technique in a family study confirmed the diagnosis of partial expression of cystic fibrosis in an adolescent female.
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An inactive mutant form of red cell carbonic anhydrase B is described in three members of a large kindred who manifest infantile renal tubular acidosis and nerve deafness. A combination of enzymatic and immunologic investigations permitted its detection, despite the fact that both antigenic and electrophoretic properties of the mutant were identical to those of the normal form.
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