Absorption and analysis of clofazimine and its derivatives.

teinase inhibitors was included in the lysing solution. To preclude the precipitation of non-specific material, lysates were pre-cleared twice with goat anti-( mouse IgG)agarose, followed by incubation with NB-1, an irrelevant antibody [4]. Digestion of immunoprecipitated antigen for 4 h with trypsin at a final concentration of 1 mg/ml resulted in bands at 21 kDa, 16.5 kDa, 11.5 kDa, 10 kDa, 7.5 kDa and 5.8 kDa (Fig. la ; lane B). Non-digested NC-2 immunoprecipitates incubated at 37°C for 4 h exhibited the usual banding pattern, demonstrating that fragmentation was indeed due to the addition of trypsin and not due to proteolysis by enzymes present within the lysate. lmmunoprecipitated antigen was digested with 2.0 m-units of glycopeptidase F, an enzyme which hydrolyses all classes of asparagine-linked glycans, provided that both the aamino acid and carboxyl groups of the asparagine residues are present in a peptide linkage [S]. Digestion resulted in the usual bands and two additional bands, with molecular masses of 46 kDa and 38 kDa (Fig. 16). The persistence of the 50 kDa band in glycopeptidase F-digested material may indicate that the reaction did not proceed to completion. However, repetition of the experiment using 5.0 m-units of the glycosidase resulted in a banding pattern identical to that obtained in the experiment in which 2.0 m-units of glycopeptidase F was used. Treatment of immunoprecipitated antigen with neuraminidase for 18 h did not visibly alter the mobility of the 50 kDa band in the gel, suggesting that the antigen is a glycoprotein without a large amount of sialic acid residues, while the glycopeptidase F digests indicate the presence of N-linked oligosaccharides. We have examined the reactivity of the NC-2 antigen with cells from a variety of normal human tissues using the ‘APAAP’ immunocytochemical staining technique [6]. h e pared slides were obtained commercially, each containing tissue sections from ovary, testis, thyroid, pancreas, lung, heart, kidney, liver, spleen, adrenal glands and gastrointestinal tract. Some degree of positive staining was observed in all except thyroid, pancreas, lung and spleen. We have used immunofluorescence techniques to examine the internal location of the antigen and compare its distribution with that of a number of other well-characterized antigens expressed in HL-60 cells. Cells to be assayed for immunofluorescence were applied to microscope slides by cytocentrifugation, a process which flattens cells to some extent, enabling more internal detail to be distinguished than in unflattened cells. Antibodies directed against the X-hapten and a-tubulin respectively, produced staining patterns very distinct from that of NC-2. The pattern most similar to that of NC-2 was produced by the granulocytespecific antibody termed 82H 1, thought to be directed against carbohydrate sequences, some of which are present on the same 180 kDa protein as the X-hapten, although it has been shown not to be an anti-X antibody [7]. The pattern produced by NC-2 was granular, with distinct fluorescent spots visible internally. These fluorescent spots appear to be randomly distributed in the cytoplasm, suggesting a localization within granules. A similar pattern was observed by Baldari & Telford using a monoclonal antibody specific for a lysosomal antigen [8]. The biochemical analysis and the subcellular localization studies of the NC-2-reacting antigen presented here provide the basis for future studies to discern the functional role of this glycoprotein.