Photolysis of the lysosomal neuraminidase in cultured human skin fibroblasts in the presence of a photoreactive competitive inhibitor.

Photolysis of the lysosomal neuraminidase in crude homogenates of cultured human skin fibroblasts was carried out using the potent competitive enzyme inhibitor, 9-S-(4-azido-2-nitrophenyl)-5-acetamido-2,6-anhydro-2,3,5,9-tetradeoxy-9 -thio-D - glycero-D-galacto-non-2-enonic acid (9-PANP-2,3-D-NANA). Irradiation of the homogenate and the inhibitor (2 min, pH 4.3, 10 degrees C) with a medium pressure mercury lamp resulted in about a 24% reduction of enzyme activity compared to irradiated controls that did not contain additives. No significant loss of activity was observed with homogenate that contained a photoreactive thioglycoside of sialic acid that was not an inhibitor of the enzyme. Similarly, the enzyme activity was not affected when 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid was photolyzed with the homogenate. The latter is a potent competitive inhibitor but it is not photoreactive. Also, the products obtained by prephotolyzing 9-PANP-2,3-D-NANA gave similar enzyme levels under standard assay conditions when compared with the nonirradiated material. Together, these results demonstrate that the photoinactivation is highly specific and both the aryl azide and the unsaturated pyran portion of the molecule are required for inactivation. The title compound may be useful as a potential photolabeling reagent which may facilitate purification of the enzyme and permit further characterization of the mutation in sialidosis patients.

Target size analysis by radiation inactivation: a large capacity tube rack for irradiation in a Gammacell 220.

Target size analysis by radiation inactivation is now a well-established method to study structure-function relationships in biologically active macromolecules without prior purification or even solubilization. Recently, it was reported that a relatively low-dose-rate but commonly available gamma source such as the Gammacell 220 (Atomic Energy of Canada, Ltd.) can be used to carry out radiation inactivation experiments providing it is appropriately calibrated with enzymes of known radiation sensitivities (G. Beauregard and M. Potier (1982) Anal. Biochem. 122, 379-384). In this report, a tube rack designed to fit into the irradiation chamber of the Gammacell 220 which allows five experiments (at 30 tubes per experiment) to be carried out simultaneously with both standard and unknown samples is described. The dose rates delivered at different positions in the rack were determined by irradiating rat liver cytosolic neuraminidase, an enzyme of known radiation sensitivity. A better than 2.7% agreement was obtained between experimental dose rate and computed values from isodose curves previously published by other authors (O. A. Curzio and H. O. Quaranta (1982) Int. J. Appl. Radiat. Isot. 33, 1-3).

Adjuvant immunotherapy of malignant melanoma.

A vaccine made of irradiated Vibrio cholerae neuroaminidase (VCN) treated autochthonous tumor cells plus BCG was utilized in combination with surgery or with chemotherapy for Stage II and Stage III malignant melanoma, respectively. A few patients with Stage I melanoma were treated with surgery and BCG. Most of the studies were carried out on a prospective, randomized protocol. When the results with conventional therapy were compared with the results of conventional therapy plus immunotherapy, no beneficial effects of the immunotherapy were seen. Stratification insured comparability in both immunotherapy and nonimmunotherapy groups. We conclude that VCN treated tumor cells plus BCG, when administered according to the protocol utilized here, offer patients with malignant melanoma no substantial benefit when compared with conventional therapy.