Galactocerebrosidase from human urine: purification and partial characterization.

Galactocerebrosidase (GALC, EC 3.2.1.46) was purified from human urine by a series of hydrophobic affinity column chromatography steps. The activity was enriched 176,000-fold from concentrated urine by only four columns, including octyl Sepharose, hydroxylapatite, butyl Sepharose and ethyl-agarose. The overall recovery was about 20% but only low amounts were obtained due to its low abundance. The estimated final specific activities of several batches were between 1 and 2 mmol/h per mg protein. The final purified fractions were essentially free of other lysosomal enzyme activities. The most pure fractions showed a series of bands between 50 and 53 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis which were determined to have identical N-terminal amino acid sequence. In addition, gel filtration of partially purified GALC after disassociation showed one peak of activity estimated to have a molecular mass near 50 kDa. GALC was also purified from human brain and human placenta using the same methods demonstrating the usefulness of this procedure in obtaining GALC from solid human tissues. In addition to the bands migrating near 50 kDa from urine, there were also bands at 80 kDa and 30 kDa in some preparations. By N-terminal sequencing and the use of antipeptide antibodies, the 80 kDa band was demonstrated to have the same N-terminal amino acids as the 50-53 kDa bands. The 30 kDa band had a unique sequence. The relationship between the different molecular weight species remains to be determined. The purification of GALC and the securing of amino acid sequence information will aid in the cloning of the GALC gene. This enzyme is deficient in human patients with Krabbe disease and several animal species.

Purification and characterization of galactocerebrosidase from human lymphocytes.

Galactocerebrosidase was purified about 22,600-fold using several hydrophobic column and gel filtration steps with a 4.8% recovery, from human lymphocytes. Its specific activity was 1.54 x 10(5) nmol/h/mg with tritium-labeled galactocerebroside as the substrate in the taurocholate system. The optimal pH for galactocerebroside was 4.2 in the taurocholate system and 4.6 in the cholate system. The Km values for galactocerebroside were 5 microM in the taurocholate system and 25 microM in the cholate system. The molecular weight of the purified enzyme was estimated to be 90 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis and gel filtration. However, 70, 50, 40, and 30 kDa bands were also recognized on SDS-PAGE. The N-terminal amino acid sequences of the 70 kDa molecule and the three 50 kDa molecules were the same as that of the 90 kDa molecule. The N-terminal amino acid sequences of the 40 and 30 kDa molecules were unique. A monoclonal antibody raised against the purified enzyme effectively immunoprecipitated galactocerebrosidase activity, and an affinity column prepared with this monoclonal antibody bound the 90 and 50 kDa proteins. These results suggest that this enzyme is probably processed from the 90 kDa protein.

Serial MRI and neurophysiological studies in late-infantile Krabbe disease.

We report serial clinical, radiological, and neurophysiological findings of a patient with late-infantile Krabbe disease. At age 13 months, the patient was hospitalized for sudden stiffness and irritability and a diagnosis of spastic diplegia was made. At age 24 months, he was readmitted because of further psychomotor deterioration; neurologically, he manifested severe spastic tetraplegia with optic atrophy. MRI disclosed diffuse high intensity in the cerebral white matter on T2-weighted images. Nerve conduction velocity and evoked potential studies were markedly abnormal, as were the EEG and the EMG. Assay of galactocerebroside beta-galactosidase activity in leukocyte culture disclosed a marked deficiency of the enzyme, confirmatory of the diagnosis of late-infantile Krabbe disease. Serial MRI and neurophysiological studies performed every 6 months for 18 months demonstrated the progressive nature of the disorder, correlating with the clinical deterioration.

A taurodeoxycholate-activated galactosylceramidase in the murine intestine.

Highly active enzymatic hydrolysis of galactosylceramide was detected in the murine intestine in confirmation of an earlier report in the rat intestine (Brady, R. O., Gal, A. E., Kanfer, J. N., and Bradley, R. M. (1965) J. Biol. Chem. 240, 3766-3770). Unlike the classical galactosylceramidase (EC 3.2.1.46), which is present in other organs, as well as also in the intestine, this intestinal enzyme was not activated by sodium taurocholate and was inhibited by oleic acid. It was effectively activated by sodium taurodeoxycholate and had a pH optimum of 5.2. This taurodeoxycholate-activated galactosylceramidase did not appear to be present in the brain, liver, kidney, and spleen. Its activity was not deficient in affected twitcher mice, a newly discovered mutant caused by a genetic deficiency of the taurocholate-activated galactosylceramidase. Although it showed a relatively neutral pH optimum, the taurodeoxycholate-activated galactosylceramidase is not a nonlysosomal "neutral" beta-galactosidase, because unlike the latter, it was adsorbed to Concanavalin A-Sepharose after solubilization with 0.5% sodium taurodeoxycholate and was eluted by alpha-methylmannoside or alpha-methylglucoside. The taurodeoxycholate-activated galactosylceramidase could be completely separated from the taurocholate-activated galactosylceramidase and GM1-ganglioside beta-galactosidase (EC 3.2.1.23) by octyl-Sepharose hydrophobic chromatography. Thus, the taurodeoxycholate-activated galactosylceramidase localized in the intestine is distinct from the two known glycosphingolipid beta-galactosidases and the neutral beta-galactosidase.

Krabbe disease: isolation and characterization of a full-length cDNA for human galactocerebrosidase.

Human galactocerebrosidase, the enzyme deficient in Krabbe disease, was purified, through several hydrophobic column steps and gel filtration, 22,650-fold from human lymphocytes. Using information on its N-terminal and internal amino acid sequences, and the polymerase chain reaction method, we cloned a full-length cDNA for the enzyme. The deduced amino acid sequence matched all amino acid sequences determined. The 3780 nucleotide sequence included 2007 nucleotides which encoded a single chain peptide of 669 amino acid residues with a 26 amino acid N-terminal signal peptide and six potential asparagine-linked glycosylation sites. The galactocerebrosidase cDNA detected an about 4 kb mRNA band material in human cultured skin fibroblasts. A nonsense mutation was found at codon 369 (GAA-->TAA) in the coding sequence of cDNA amplified from cultured skin fibroblast mRNA from a patient with typical Krabbe disease.