Enzymatic estimation of galactose in the presence of galactolipids

TY - JOUR

T1 - Enzymatic estimation of galactose in the presence of galactolipids

AU - Gatt, Shimon

N1 - Funding Information: Fig. 2 shows the results of two experiments in which a glycolipid was hydrolyzed by enzyme preparations from rat brain, and the galactose released was estimated by the above procedure. The substrate was a tetrahexosyl ceramide (galactosyl N-acetyl-galactosaminylgalactosylglucosylceramide, also termed “asialo Gmr ganglioside”) and the enzymes were (a) a partially purified rat brain p-galactosidase” and (b) a suspension of “particle E” of KOENIG et aLla. Straight lines were obtained in either case when the absorption at 340 nm was plotted against enzyme concentration. In similar experiments where the enzyme sources were rat brain homogenates or crude mitochondrial particles, an increased absorption at 340 nm was not obtained, most probably because of reoxidation of the NADH by NADH oxidase. To overcome this, the reaction mixtures were placed for several minutes in a boiling-water bath prior to the addition of the alkaline Tris buffer. Most enzymatic hydrolyses of galactolipid are performed by lysosomal enzymes operating at pH 5 or less (reviewed in ref. I) ; these enzymes are inoperative at pH values above 8. Advantage was taken of this finding to assay the galactose split off galactolipids, by adjusting the pH to 8.6 and performing the NAD+ reduction coupled to galactose dehydrogenation in the original reaction tube. Since lipid dispersions are too opaque to enable a direct reading of the increased absorption at 340 nm, the NADH is partitioned into an aqueous-methanolic upper phaselo. This leaves the unreacted lipid and detergents in the lower chloroform phase and enables a direct reading of the absorption of the clear upper phase in a spectrophotometer. When crude tissue preparations were used as source for j3_galactosidase, increased absorption at 340 nm due to NADH was not obtained. This is most probably due to reoxidation of the NADH by NADH oxidase at the alkaline pH. This may be avoided by heat-denaturing the proteins in a boiling-water bath prior to the addition of the alkaline Tris buffer. Similar heat denaturation is also recommended for purified galactosidases which have hydrolytic activity at pH 8.6 and which will therefore release galactose during the hour-long incubation with galactose dehydrogenase. This work was supported in part by a U.S. Public Health Service Grant (NS 02967). The expert technical assistance of Mr. A. Herzl is acknowledged.

PY - 1970/10/6

Y1 - 1970/10/6

UR - http://www.scopus.com/inward/record.url?scp=0014935324&partnerID=8YFLogxK

U2 - 10.1016/0005-2760(70)90106-2

DO - 10.1016/0005-2760(70)90106-2

M3 - Article

C2 - 4319688

AN - SCOPUS:0014935324

SN - 0005-2760

VL - 218

SP - 173

EP - 175

JO - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids

JF - Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids

IS - 1

ER -