Functional lysosomal hydrolase size as determined by radiation inactivation analysis

Abstract

Electron inactivation analysis with 16 MeV electrons was used to determine the functional target size of a number of commonly studied lysosomal hydrolases. Observed values ranged from a low of 62 000 +/- 4000 Da for beta-galactosidase to a high of 200 000 +/- 17 500 Da (mouse beta-glucuronidase). One group of lysosomal hydrolases (N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, alpha-galactosidase, beta-mannosidase, beta-glucosidase, arylsulphatase A and sphingomyelinase) had target sizes in the range 100 000-120 000 Da, whereas alpha-glucosidase and alpha-fucosidase exist as complex multimers in the 150 000-160 000 Da range. Analysis of freeze-dried cell material showed little evidence of species (mouse versus human) variation in the functional size of most lysosomal hydrolases with the exception of beta-glucuronidase. Our findings suggest the potential usefulness of lysosomal hydrolases as endogenous marker enzymes in studies where the target size of proteins of unknown molecular mass is to be determined.

Molecular-size standards for use in radiation-inactivation studies on proteins

“…The present results show that the use of enzyme standards provides an accurate alternative to empirical formulae for Mr determination in radiation-inactivation experiments. A greater range of standards should be developed, and where possible endogenous enzymes present in the preparation under investigation should be used, as in [16]. Careful selection of standards and interpretation of results must be made.…”

“…These included ,igalactosidase, yeast alcohol dehydrogenase and pyruvate kinase, but in a subsequent study McIntyre & Churchill [11] found these three enzymes produced variable results in irradiation studies on frozen samples and recommended only glucose-6-phosphate dehydrogenase. An alternative is to use endogenous enzymes of known Mr [16]. However, suitable endogenous enzymes will not always be present.…”

A Guided Tour of the Structural Biology of Gaucher Disease: Acid-β-Glucosidase and Saposin C

“…Specifically, in apo structures, half of the molecules in the asymmetric unit are in the extended and other in the helical conformation. It is not currently known whether GCase, which appears to be a functional dimer [ 70 , 71 ], can only take on one helical conformation at a time, or if this observation is trapped by crystal packing [ 38 ]. Comparison of the thermal B-factors reveals that the IFG-bound loop 1 conformation, with its additional secondary structure, is better locked in position than when present as an extended loop [ 35 , 38 ].…”

Gaucher Disease

“…Molecular weights, estimated by sedimentation and molecular exclusion chromatography, have varied between about 60,000 and 450,000 (Pentchev et aI., 1973;Prence et al, 1985;Garrett et al, 1985). In comparison, estimates of the native molecular weight in tissues by in situ radioinactivation of the enzymatic activity were consistent with either a monomeric or dimeric structure (Dawson and Ellory, 1985;Maret et al, 1984Maret et al, , 1983. About 13% of the residues are basic (lysine, arginine, or histidine) and the calculated pI value is 7.2, which is consistent with experimental values (PI = 7.3-7.8) for placental enzyme preparations containing only the neutral mannosyl oligosaccharide core (Furbish et al, 1981).…”