Isoform purification of gastric lipases. Towards crystallization.
Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.
Moreau
H
H
CNRS Centre de Biochimie et de Biologie Moléculaire, Marseille, France.
Abergel
C
C
Carrière
F
F
Ferrato
F
F
Fontecilla-Camps
J C
JC
Cambillau
C
C
Verger
R
R
D000818:Animals; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D006031:Glycosylation; D006801:Humans; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification*; D011817:Rabbits; D013270:Stomach / Q000201:enzymology*; D014961:X-Ray Diffraction
D007527:Isoenzymes; D008049:Lipase