Purification and properties of human urinary beta-D-mannosidase.
Two forms of the lysosomal enzyme beta-mannosidase were identified and purified from human urine. The purification strategy employed allowed sufficient quantities of both forms to be obtained for subunit analysis and for further characterizations. The two beta-mannosidases were identified as beta-mannosidase B and A, in order of their elution from an ion-exchange column. In all samples examined, the A form was predominant, and the B/A ratio was consistently 0.14. The two forms displayed the same optimum pH (i.e., 4.3) and both were retained by a Concanavalin-A Sepharose column, but showed different isoelectric points, molecular masses and subunit compositions. Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of pure beta-mannosidases B and A suggest that active protein B (160 kDa) consists of three subunits, one 75 kDa and two 49 kDa subunits. Protein A is smaller and appears to be composed of three subunits of 75 kDa, 49 kDa and 37 kDa. Two forms of beta-mannosidase, exhibiting a chromatographic behaviour comparable to the urinary forms, were also detected in human kidney. Nevertheless, in this tissue their relative distribution was different, the B/A ratio being 19.
Guadalupi
R
R
Dipartimento di Biologia Cellulare e Molecolare, Università degli Studi di Perugia, Perugia, Italy.
Bernard
M
M
Orlacchio
A
A
Foglietti
M J
MJ
Emiliani
C
C
D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000652:urine*; D007668:Kidney / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism / Q000652:urine*; D008970:Molecular Weight; D011487:Protein Conformation; D011489:Protein Denaturation; D013696:Temperature; D044902:beta-Mannosidase
D007527:Isoenzymes; D008361:Mannosidases; D044902:beta-Mannosidase