Giovanni Cassani

Autocrine saturation of pro-urokinase receptors on human A431 cells

Single-chain pro-urokinase (pro-uPA) is present both in the medium and lysate of the A431 epidermoid carcinoma cell line. Most of the cell-associated pro-uPA is on the cell surface, as shown by indirect immunofluorescence and by surface iodination. Pro-uPA is not an integral membrane protein but is bound to a specific surface receptor that is completely saturated. A mild acid treatment uncovers the surface receptors by dissociating pro-uPA. Resaturation of uncovered receptors has been studied by reincubating cells in normal medium; within 40 min, 50% of the free sites are reoccupied. Excess uPA-specific antibodies prevent rebinding of ligand to the receptors. Thus, A431 cells first secrete uPA, which then binds to the surface receptor. We propose that the synthesis of uPA and uPA receptor by the same cell may provide a pathway for the activation of the metastatic potential of malignant cells.

Synthesis and characterization of D-alanyl-D-alanine-agarose. A new bioselective adsorbent for affinity chromatography of glycopeptide antibiotics

A new affinity adsorbent, using D-alanyl-D-alanine as ligand, has been prepared. The dipeptide immobilized on Activated CH-Sepharose 4B (D-Ala-D-Ala-AGA) bioselectively binds the glycopeptide antibiotics teicoplanin, vancomycin, ristocetin A (vancomycinlike group of antibiotics) while it does not bind other antibiotics equally active on cell wall biosynthesis but with different target sites, such as penicillin G, cephalosporin C, gardimycin, and bacitracin. Teicoplanin, vancomycin, and ristocetin A have similar binding characteristics for the immobilized dipeptide, as indicated by equilibrium binding experiments. The affinity constants of the three antibiotics for D-Ala-D-Ala-AGA is of the same order of magnitude (105 L mol-1) and the number of effective binding sites is similar for each antibiotic (6–7 μEq/mL of gel). The adsorption is biospecific as no binding has been observed to immobilized L-alanyl-L -alanine.D-Ala-D-Ala-AGA has been successfully used to purify teicoplanin from mixtures of different complexity and for concomitant extraction and purification from fermentation liquors by both batch adsorption and column chromatography. The antibiotic can be recovered from the resin in high yields by elution at pH 11.

Comparison of the solid phase enzyme receptor assay (SPERA) and the microbiological assay for teicoplanin

A solid phase enzyme receptor assay (SPERA) for teicoplanin has been developed and recently presented in a kit form. The assay relies on the competition between antibiotic present in the biological fluid and peroxidase labelled teicoplanin for albumin-epsilon-amino-caproyl-D-alanyl-D-alanine (BSA-epsilon-ACA-D-Ala-D-Ala), a synthetic analog of the biological receptor. The kit contains BSA-epsilon-ACA-D-Ala-D-Ala coated microplates together with all the necessary reagents. The limit of detection of the SPERA is 0.5 mg 1(-1). The microbiological assay for teicoplanin uses Bacillus subtilis ATCC 6633 as test organism and a high salt medium to ensure a low limit of detection of 0.05 mg 1(-1). The previously established correlation between the two methods has been confirmed using the kits on 280 serum and 216 urine samples with concentrations up to 500 mg 1(-1).

Charge heterogeneity of recombinant pro-urokinase and urinary urokinase, as revealed by isoelectric focusing in immobilized pH gradients

When analysing homogeneous preparations of recombinant pro-urokinase and urinary urokinase by isoelectric focusing (IEF) in immobilized pH gradients, an extreme charge heterogeneity was detected (at least ten major and ten minor bands in the pH range 7-10). This extensive polydispersity was not caused by different degrees of glycosylation, or by IEF artefacts, such as binding to carrier ampholytes or carbamylation by urea. A great part of this heterogeneity could be traced back to the existence of a multitude of protein molecules containing Cys residues at different oxidation levels (-SH, -S-S-, even cysteic acid). Owing to the very large number of Cys residues in pro-urokinase (24 out of a total of 411 amino acids) and to the relatively high pI of its native forms (pI 9.5-9.8; the native form is believed to contain all Cys residues as -S-S- bridges), the presence of SH or cysteic acid residues would increase the negative surface charge, as even SH groups would be extensively ionized. In pro-urokinase, part of the heterogeneity was also due to spontaneous degradation to urokinase and possibly also to cleavage into lower-molecular-mass fragments. When all these causes of heterogeneity were removed, the pI spectrum was reduced to only four, about equally intense bands. The cause of this residual heterogeneity is unknown.

A rapid method for monitoring DNA labelling reactions with haptens

A rapid and simple method for evaluating the efficiency of DNA labelling reactions with haptens is described. The method, called the Flow-Through Hapten-DNA Assay (FT-HDA), relies on binding of anti-hapten antibodies/alkaline phosphatase conjugates to hapten-DNA, immobilized on disposable capillary absorbent filters, and visual detection of blue-grey coloured spots appearing on the filter after chromogenic reaction with enzyme substrates. FT-HDA of hapten-DNA is markedly faster and simpler than conventional diffusion assays on membranes.