Glycosidases in Leishmania and their importance for Leishmania in phlebotomine sandflies with special reference to purification and characterization of a sucrase.
Culture forms of Leishmania (Leishmania) amazonensis (IFLA/BR/67/PH8) produce an extracellular enzyme that hydrolyzes sucrose molecules into their component monosaccharides. This is important because phlebotomine sand flies, the invertebrate hosts of Leishmania, ingest plant sap or aphid and coccid honeydew rich in sucrose between blood meals and Leishmania promastigotes cannot uptake sucrose. The sucrase was purified and characterized; its molecular weight, estimated by gel filtration chromatography and SDS-PAGE electrophoresis, was about 73 kDa. K(m) and V(max) measured with sucrose as substrate were respectively 4.4 mM and 6.9 mumole glucose.min-1 (mg sucrase)-1, with maximum pH activity at pH 5.5. A series of natural and p-nitrophenyl-derived substrates were assayed, characterizing the enzyme as a highly specific beta-D-fructofuranoside fructohydrolase. When 11 species of Leishmania and 7 genera of trypanosomatids were screened, only the species of the genus Trypanosoma did not produce an enzyme with saccharolytic activity. These data are in agreement with the fact that the latter vectors do not acquire sucrose or raffinose in their meals. Searching for glycolytic enzymes other than sucrase, we found an N-acetyl-beta-D-galactosaminolytic activity. This N-acetyl-galactosaminidase, here described for the first time, might have a role in peritrophic membrane disruption. The importance of sucrase and N-acetyl-beta-D-galactosaminidase in the Leishmania life cycle is discussed.
Gontijo
N F
NF
Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Brazil.
Melo
M N
MN
Riani
E B
EB
Almeida-Silva
S
S
Mares-Guia
M L
ML
D000818:Animals; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D006026:Glycoside Hydrolases / Q000378:metabolism*; D007303:Insect Vectors / Q000469:parasitology*; D007894:Leishmania mexicana / Q000201:enzymology* / Q000254:growth & development; D008969:Molecular Sequence Data; D011576:Psychodidae / Q000469:parasitology*; D013393:Sucrase / Q000302:isolation & purification* / Q000378:metabolism; D013395:Sucrose / Q000737:chemistry / Q000378:metabolism
D002241:Carbohydrates; D003470:Culture Media; D013395:Sucrose; D006026:Glycoside Hydrolases; D013393:Sucrase