Linked Life Data

6437271

Source:http://linkedlifedata.com/resource/pubmed/id/6437271

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1984-12-18
pubmed:abstractText
Two Sepharose-bound substrate analogs, 6'-aminohexanoyl-(2-N-sphingosyl-O-beta-D-glucoside) and 6'-aminohexyl-dodecanedioyl-1-(2-N-sphingosyl-1-O-beta-D-glu coside), were synthesized and used sequentially for the affinity purification of lysosomal beta-glucosidase (N-acyl-sphingosyl-1-O-beta-D-glucoside:glucohydrolase, EC 3.2.1.45). The capacities of these nondegradable affinity supports were 0.1 and 0.15 mg enzyme/ml settled gel, respectively. The purified enzyme had a specific activity of 75 mumol min-1 mg-1. The preparation had a single protein band with a molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, evidencing its apparent homogeneity. Isoelectric focusing on granular gels revealed four molecular forms of the enzyme with pI values of 4.0, 4.5, 4.7, and 5.8 to 6.2. The purified enzyme hydrolyzed glucosyl ceramide and 4-methylumbelliferyl-beta-D-glucoside with Km and Vmax values of 0.6 and 2.5 mM, and 101 and 26.1 mumol min-1 mg-1, respectively. The enzyme also hydrolyzed octyl beta-glucoside, a linear mixed-type inhibitor of the enzyme. Binding constants (Ki) were determined for the inhibitors, sphingosyl-1-O-beta-D-glucoside (Ki = 20 microM) and its N-hexyl derivative (Ki = 0.3 microM). The enzyme had a half-life of 65 and 30 min at 50 degrees C and pH 5.0 or 6.0, respectively. In addition, two other classes of ligands were used for the purification of lysosomal beta-glucosidase, and their capacities and specificities were compared to those of the substrate analog affinity supports. These included (i) the alkyl amine inhibitors octylamine, decylamine, and tetradecylamine; and (ii) the inhibitors, 6-aminohexanoyl-beta-glucosylamine and aminododecanoyl-1-(2-N-sphingosyl-1-O-beta-D-glucoside). Compared to these other ligand columns, the substrate analog affinity supports had about 100- to 1000-fold greater capacities or afforded 8- to 40-fold greater purification of human lysosomal beta-glucosidase.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
141
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
267-79
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1984
pubmed:articleTitle
Human lysosomal beta-glucosidase: purification by affinity chromatography.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't