RELATIONSHIP BETWEEN THE ACID-PHOSPHATASES OF THE KURLOFF BODY AND THE MAJOR 30-35 KDA GLYCOPROTEINS OF THE KURLOFF CELL

This study deals with the acid phosphatase (AcPase) of the Kurloff bod y (KB), a large (10 mu m diameter) periodic acid-Schiff-positive lysos omal inclusion body present in Kurloff cells (KC). KC AcPase were extr acted, with similar yields, either with non-ionic detergent solution o r after Dounce homogenization in low ionic strength buffer suggesting that they mainly correspond to hydrosoluble AcPase. After DEAE-cellulo se chromatography of such crude Dounce-extracts, 97% of KC AcPase acti vity was recovered in the unbound glycoprotein fraction (peak I). The main protein content consisted of, as testified by SDS-PAGE analysis, major KC glycoproteins of 30-35 kDa. Thus, KC AcPase, and particularly sialoAcPase, may be assumed to correspond to these N-glycosylproteins among which the presence of alpha(2,6) sialoglycoproteins was previou sly established. Following electrophoresis on a 4-15% gradient native polyacrylamide gel or isoelectric focusing, the two size populations ( 200 kDa and 500 kDa) and up to 20 isoforms of KC AcPase were respectiv ely detected by zymography in peak I. After Clostridium-derived sialid ase digestion of peak I, the highly active bands observed at pH 3.5-5. 2 disappeared. These zymographic patterns were similar to those obtain ed with crude extracts. After Concanavalin A (ConA)-Sepharose chromato graphy of peak I, the single ConA-bound glucosamine-labelled peak, elu ted at 200 methyl-alpha-D-mannopyranose, contained the AcPase activity while the ConA-unbound peak was devoid of any acid phosphatase activi ty. After SDS-PAGE analysis, the ConA-bound fraction appeared to corre spond only to a single broad protein band in the 30-35 kDa zone. The S ambucus nigra agglutinin-reactivity of the latter band confirmed the a lpha(2,6) sialylation pattern of KC AcPase. Moreover, their strong bin ding to ConA-Sepharose suggests that they could only correspond to the hybrid type of the N-glycosylproteins, since they could nt correspond to the oligomannosidic type considering the absence of Galanthus niva lis agglutinin-positive structures.