Peter Fischer

Comparative biochemical and immunological studies on gamma-glutamyltransferases from human kidney and renal cell carcinoma applying monoclonal antibodies

We have purified gamma-glutamyltransferases (GGT) from human kidneys and renal cell carcinomas, and fractionated them according to different lectin-binding properties of the isoenzymes. Native polyacrylamide gel electrophoresis and isoelectric focusing revealed different GGT-bands (even after desialylation) not only among kidney and renal carcinoma, but also among Con A-affine tumor fractions separated by ion-exchange chromatography. Mr of native GGTs were between 106 to 161 kDa, the pI ranged from pH 3 to 4 (pH 5 to 6 after desialylation). Monoclonal antibodies to GGT were produced. One of these, of IgG1 class and designed 138H11, recognizes human kidney GGT and, in addition, GGT from renal cell carcinomas and liver carcinomas. The specificity of mAb 138H11 for GGT was confirmed by Western blotting, by immunohistochemistry and by immunoprecipitation. The potential usefulness of mAb 138H11 in monitoring renal cancer patients and in identification of renal cancer metastases is currently being studied.

Differential diagnosis of histogenetically distinct human epithelial renal tumours with a monoclonal antibody against γ-glutamyltransferase

SummaryThe localization of membrane-bound γ-glutamyltransferase with monoclonal antibody (mAb) 138H11 proved to be of value for differential diagnosis of renal cancer, since it correlated with the histogenetic profile of human epithelial renal tumours. Immunoreactive γ-glutamyltransferase was located in the proximal tubule in all normal human kidneys (15/15) examined thus far by both ultrastructural and immunohistochemical techniques. From 68 epithelial renal cancers tested, 31/31 clear-cell carcinomas and 15/16 chromophilic carcinomas expressed the target epitope of mAb 138H11. In contrast, 0/11 oncocytomas, 0/9 chromophobic carcinomas, and 0/1 Duct-Bellini carcinoma were immunoreactive. These results support a model of histogenesis and classification of epithelial renal tumours, according to which clear-cell and chromophilic renal carcinomas originate from transformed proximal tubule cells, whereas oncocytomas, chromophilic and Duct-Bellini carcinomas originate from cells of the collecting duct.

Isolation and partial characterization of angiotensinase A and aminopeptidase M from urine and human kidney by lectin affinity chromatography and high-performance liquid chromatography.

Angiotensinase A (ATA) and aminopeptidase M (APM) were partially purified from human urine specimens and human kidney particles using wheat germ lectin affinity chromatography, anion-exchange Fast Protein Liquid Chromatography (FPLC) (Mono Q), chromatofocusing (Mono P, FPLC) and Superose 12 gel filtration. APM, a globular 5-nm glycoprotein, is localized in the brush border membrane of the proximal tubule; angiotensin II-degrading ATA is present on glomerular endothelia and podocytes and, to lesser extent, in the brush border. For the first time, both peaks of ATA and APM activity from urine samples were separated by the above-mentioned techniques with only slight overlap; ATP (146,000 dalton: pI4.8) was enriched more than 20-fold and APM (153,000 dalton, pI4.7) more than 50-fold compared with the activity of the starting material. Using similar separation steps, ATA and APM solubilized from kidney particles could not be resolved into two distinct peak fractions, however, except after hydrophobic interaction chromatography. Thus urine is a major source for the preparation of individual ATA and APM fractions, necessary to generate specific anti-enzyme antibodies for diagnostic purposes.

Isolation and characterization of dipeptidyl aminopeptidase IV from human kidney cortex

Intact dipeptidyl aminopeptidase IV (DAP IV) was solubilized by bromelain treatment from human kidney brush border plasma-membranes. Purification of DAP IV was performed by a 3-step method, applying lectin-affinity chromatography on WGA-Sepharose, gel filtration and anion-exchange chromatography. DAP IV from human kidney cortex showed a pH optimum of 8.7 and was totally inhibited by 1 mmol/l Zn2+. Isolated DAP IV revealed a relative molecular mass of 250 kDa as determined by the native-PAGE method and of 220 kDa by the gel filtration method. Analytical isoelectric focussing of DAP IV revealed an isoelectric point of pH 5.3. Ultrastructural analysis of isolated DAP IV fractions, using the negative staining technique, disclosed the presence of numerous globular particles with an average diameter of 5 nm which correspond to the structural substrate of the purified protein.

Hepatic expression of gamma-glutamyltranspeptidase in the human liver of patients with alcoholic liver disease

Background:u2002 Gamma‐glutamyltranspeptidase (GGT) has been recognized as an enzyme that converts glutathione into cysteine, and it is localized predominantly within the liver. Serum GGT is clinically recognized as the most useful marker for diagnosis of alcoholic liver disease (ALD).

Immunoscintigraphic localization of renal tumours in an extracorporeal perfusion model with a monoclonal antibody against γ-glutamyltransferase

SummaryMonoclonal antibody 138H11 against human γ-glutamyltransferase has been shown to react immunohistochemically with 98% of all tested clear-cell type and chromophilic renal cell carcinomas, but not with renal chromophobic carcinomas, Duct-Bellini carcinomas or oncocytomas. In normal kidney the target epitopes of mAb 138H11 are located in the luminal brush-border membrane of proximal tubule cells, whereas in renal carcinomas the epitopes are found surrounding the whole tumour cells. These results form the basis of the present immunoscintigraphic study designed to evaluate mAb 138H11 in an extracorporeal perfusion model. Immediately after nephrectomy, human tumour-bearing kidneys were perfused with99mTc-labelled mAb 138H11 in Euro-Collins solution. High specific uptake in 4/4 renal clear cell carcinomas could be demonstrated by planar immunoscintigraphy and single-photon-emission computed tomography, “regions of interest” investigation and immunohistochemistry. In contrast, a perfused oncocytoma showed up as an unlabelled lesion. The results indicate a possible use for mAb 138H11 in immunoscintigraphy or even therapy, provided high tumour uptake can be confirmed in patients.