Kurloff cell lysosomal arylsulphatases: presence of both cationic and highly anionic isoforms of the sole B class.

Following the previous ultrastructural demonstration of the presence of arylsulphatase (Asase) activities in Kurloff cells (KC) and of their quasi-exclusive localization in the Kurloff body (KB), this work investigates their biochemical and zymographic properties after extraction from purified KC suspensions. Using the discriminative inhibitory conditions of both the Baum or Lee-Vaupel and Conzelmann methods, nitrocatechol sulphate hydrolyzing enzymes of the KC were assumed to belong to the B class of the type II Asase alone. After electrophoretic separation under non-denaturing conditions in a 4-23% polyacrylamide gel, they were characterized by 55 kDa and 62 kDa zymographic bands. After isoelectric focusing, 'classical' cationic isoforms (pI 8.5) and two anionic isoforms (pI 4.4 and 4.6) were observed on zymograms. As expected for class B Asase, the different zymographic forms of KC Asase were only recovered in the unadsorbed fraction after anion-exchange chromatography on DEAE-cellulose column equilibrated with high ionic strength buffer. Their Km (2.1 mM), their optimum pH (5.8) and their inhibitions by sulfite, phosphate, sulphate and ascorbic acid as well as their slight stimulation by AgNO3 were also characteristic of this class of Asase. Finally, chondroitin-4-sulphate was shown to potentially be a physiological substrate for these lysosomal enzymes.

Alpha naphthyl acetate esterase activities in guinea pig Kurloff cells: a cytochemical and electrophoretic study.

We established the presence of nonspecific esterases in the Kurloff cell (KC) by cytochemical methods at both light and electron microscope levels. Acid alpha-naphthyl acetate esterase (ANAE) activities were localized on the external face of the plasma membrane and on the external surface of the membrane surrounding the Kurloff body. Different cytosoluble KC extracts were obtained from purified splenic KC suspensions. About 18 isoenzymes were observed by isoelectric focusing, whereas after polyacrylamide gradient gel electrophoresis in native conditions almost all activity was observed on a few broad bands with very high apparent molecular weights, suggesting their oligomeric arrangement. After a first aqueous extraction step which released only a few isoenzymes, the remaining pellet was subjected to Triton X-100. This released almost all the isoenzymes observed after direct Triton X-100 extraction. These data suggest that almost all the KC esterases are membrane-bound enzymes, in agreement with the subcellular enzyme distribution. Different substrates were also used to characterize the different specificities of the KC isoesterases. Weak activity was detected with alpha-naphthyl butyrate by light cytochemistry, which essentially corresponded, on zymograms, to the membrane-bound esterase activity.

Cytochemical localization of arylsulfatase in guinea-pig Kurloff cells.

Kurloff cells are mononuclear cells characterized by a large metachromatic and PAS-positive inclusion called the Kurloff body. Bone-marrow and spleen Kurloff cells were incubated with p-nitrocatechol sulfate as substrate and barium chloride as capturing agent for the ultracytochemical detection of the lysosomal marker enzyme, arylsulfatase. Enzymatic reaction product was consistently found as a single spot-like deposit confined to the rim of the Kurloff body. These results, and the previously described presence of other acid hydrolases and sulfated glycosamino++glycans, emphasize the similarities between the Kurloff body and lysosomes. Reaction product could also be found occasionally in segments of the rough endoplasmic reticulum but it was absent from the Golgi apparatus. This arylsulfatase activity could be related to the natural killer activity of Kurloff cells.

Zymograms of Kurloff cell acid phosphatases. Thin layer isoelectric focusing and native polyacrylamide 4-15% gradient gel electrophoresis.

Following previous light and ultrastructural cytochemical reports, this paper presents the first zymograms of Kurloff cell acid phosphatases extracted from highly purified cell suspensions. Using isoelectric focusing, up to 20 isoenzymes were observed both with hexazotised pararosanilin or fast garnet GBC as coupler and naphthol ASB1 phosphate as substrate. Most were tartrate-labile. After Clostridium-derived neuraminidase digestion, the highly stained bands observed at pH 4-5 disappeared and, simultaneously, a few alkaline bands were enhanced. Two main bands of Mr 190,000 and 500,000 were characterized by their acid phosphatase activity after electrophoresis on a 4-15% gradient native polyacrylamide gel.

Cytochemical localization of acid phosphatase and trimetaphosphatase activities in Kurloff cells.

After stimulation of guinea pigs with estradiol to increase their Kurloff cell number, spleen imprints were prepared in order to detect non-specific acid phosphatase (AcPase) activity by light microscopic cytochemistry using naphthol AS-BI phosphate as substrate and pararosanilin or fast garnet GBC as coupler. For ultracytochemistry, Kurloff cells were prepared from spleens by filtration through a homogeneizer screen followed by repeated centrifugation. AcPase and trimetaphosphatase activities were tested using beta-glycerophosphate, cytidine-5'-monophosphate and inorganic trimetaphosphate as substrates. Significant enzymatic activities were demonstrated with all the substrates used in the cytoplasmic inclusion body of the Kurloff cells.