Isolation of galactosyltransferase from human milk and the determination of its N-terminal amino acid sequence

Galactosyltransferase (EC 2.4.1.22), purified to homogeneity from human milk by affinity chromatography, had an apparent molecular weight of 53,000 as determined by denaturing polyacrylamide gel electrophoresis. Subtration of the estimated contribution of the oligosaccharide portion of the molecule leaves a Mr of 47,000. An N-terminal amino acid sequence analysis of the isolated protein revealed a sequence similar to that found near the 5' end of a cDNA clone isolated by Shaper et al (11), which encodes a 35,500 molecular weight protein. Either the molecular weight of galactosyltransferase, has been overestimated, or a discrepancy exists between the actual molecular weight of galactosyltransferase and that predicted by the bovine cDNA clone isolated by Shaper et al (11).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26099/1/0000175.pd

Isolation of a cDNA coding for human galactosyltransferase

Human milk galactosyltransferase (EC 2.4.1.22) was purified to homogeneity using affinity chromatography. Edman degradation was used to determine the amino acid sequences of eight peptide fragments isolated from the purified enzyme. A 60-mer "optimal" oligonucleotide probe that corresponded to the amino acid sequence of one of the galactosyltransferase peptide fragments was constructed and used to screen a [lambda]gt10 cDNA library. Two hybridization-positive recombinant phages, each with a 1.7 Kbp insert, were detected among 3 x 106 recombinant [lambda]gt10 phages. Sequencing of one of the cDNA inserts revealed a 783 bp galactosyltransferase coding sequence. The remainder of the sequence corresponded to the 3'-region of the mRNA downstream from the termination codon.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26068/1/0000142.pd