Glucocerebrosidase: reconstitution from macromolecular components depends on acidic phospholipids (Short Communication)

Abstract

The reconstitution of glucocerebrosidase activity from its inactive macromolecular components depended on acidic membrane phospholipids.

Specificity of low molecular weight glycoprotein effector of lipid glycosidase

“…Phosphatidic acid or phosphatidylinositol replaced phosphatidylserine completely to give a selective binding of glucocerebrosidase catalytic protein, in agreement with the previous finding [8] that all three phospholipids were effective in enabling association of the proteins in free solution. No binding of any of the three enzymes were obtained between pH 5.5-7.0, all activities co-eluted in the early fractions.…”

“…These results demonstrate the specific binding of glucocerebrosidase catalytic protein to insolubilized effector protein under the same conditions that gave optimum association between the proteins in free solution [8]. The most likely explanation is that a specific effector exists for glucocerebrosidase.…”

“…A crude solubilized preparation of the catalytic protein from the total membrane fraction of normal human spleen was made as previously described [8]. This was then precipitated with 35 g/ 100 ml solid ammonium sulphate at 4°C.…”

“…The column was eluted sequentially with 4 or more column vols of the following buffers all containing 0.05% (v/v) Triton X-100: buffer A, buffer B (0.05 M sodium acetate, pH 5.3) and buffer C (0.02 M sodium phosphate, pH 7.0). Fractions of 3 ml were monitored for /3-N-acetyl-glucosaminidase, /I-galactosidase and glucocerebrosidase activities as described in detail elsewhere [3,8,12]. …”

“…Association between effector and catalytic proteins occurs at acid pH: incorporation of the effector into a membrane preparation of the catalytic protein is heat and time dependent, and parallels the appearance of enzyme activity in the membrane [2]. When the catalytic protein is solubilized [3,8], association with effector to produce active enzyme requires the addition of acidic membrane phospholipids, such as phosphatidic acid, phosphatidylserine and phosphatidylinositol [8]. In the presence of optimum concentrations of acidic phospholipid, the association reaches equiliNorth-Holland Publishing Company -Amsterdam brium instantaneously [ 1,9] .…”