J P Luzio

The Properties, Structure, Function, Intracellular Localisation and Movement of Hepatic 5′-Nucleotidase

5′-Nucleotidase (EC 3.1.3.5) was first described 50 years ago (Reis 1934) and was subsequently shown to be widely distributed in animal tissues (Reis 1951) as an intrinsic plasma membrane enzyme (Essner et al. 1958; Segal and Brenner 1960; Song and Bodansky 1967). Its ease of assay and stability made it a convenient and popular marker for the plasma membrane in subcellular fractionation (Evans 1978) and its measurement has also been found useful over many years as an indicator of cholestasis when raised levels of 5′-nucleotidase activity are present in patients’ sera (Ellis et al. 1978). The discovery that 5′-nucleotidase was an ectoenzyme in several cell types (De Pierre and Karnovsky 1974a, b; Trams and Lauter 1974; Newby et al. 1975) and the subsequent observation of its internalisation from the cell surface and re-cycling in the absence of protein synthesis (Stanley et al. 1980; Widnell et al. 1982) has led to more active investigation of its properties. This has been fuelled by a growing interest in its function which remains enigmatic (Stanley et al. 1982).

A monoclonal antibody capture assay for intestinal alkaline phosphatase and the measurement of this isoenzyme in pregnancy

Intestinal ALP was purified from meconium to a specific activity of 1,100 U/mg protein and used as antigen in the preparation of 7 monoclonal antibodies. Two of these antibodies were specific for intestinal ALP and reacted with different epitopes. Both bound adult intestinal ALP better than fetal intestinal ALP. One of these antibodies was used to establish a capture assay for intestinal ALP in human serum over the range 0.5-16 U/l. Reference ranges of serum intestinal ALP concentrations were established in relation to blood groups. Measurement of intestinal ALP in the serum of pregnant women showed no correlation with pre-term fetal passage of meconium.

Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance.

It has been proposed that killing of mammalian cells by ricin requires efficient endocytic delivery to the trans-Golgi network (TGN) prior to retrograde transport to the endoplasmic reticulum and entry to the cytosol. In polarized epithelial cells, an efficient membrane-traffic pathway to the TGN is present from the basolateral but not the apical plasma-membrane domain. Thus one can hypothesize that a ricin-resistant phenotype might be demonstrated by polarized cells that fail to differentiate and thus fail to develop an efficient membrane-traffic pathway from the basolateral plasma membrane to the TGN. We have isolated and studied a ricin-resistant Caco-2 cell clone (Caco-2-RCAr clone 2) which, when grown on plastic, was deficient in differentiation, measured by the development of polarized-cell-surface marker enzymes. The deficiency in differentiation was partially reversed, and ricin sensitivity was restored, when the cells were grown on filter supports. Our data provide the first evidence of a ricin-resistant cell line where resistance is due to the lack of development of polarized cell surfaces. The observed ricin resistance is consistent with the requirement that ricin is delivered to the TGN before its A chain enters the cytosol to mediate cell killing.

Approaches to Isolating Minor Membrane Components Using Monoclonal Antibodies, as in Purifying 5′-Nucleotidase

Isolation of a minor membrane protein requires optimal solubilization, then a protocol, with suitable affinity steps, that gives an active product in good purity and yield. Amphi-pathic detergents of diverse molecular structure are commonly used, aiming at “the smallest and least polydisperse active protein” [1] by criteria including non-sedimentability at high g forces and Polyacrylamide gel electrophoresis (PAGE) which also indicates the solubilized active protein’s molecular size. Immuno-affinity purification, very effective and of wide use because monoclonal antibodies can be prepared for a specific protein available only in partially purified form, entails immunization, production and purification of monoclonal antibody, preparation of immunoadsorbent, and adsorption and elution of antigen.