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H S Lu

Publications and source records attributed to H S Lu.

81 records · Page 5Linked to original sources
PubMed1981

Chemical modification of critical catalytic residues of lysine, arginine, and tryptophan in human glucose phosphate isomerase.

Human glucose phosphate isomerase was subjected to a series of chemical modifications aimed at identifying residues essential for catalytic activity. A specific lysine was found to stoichiometrically react with pyridoxal 5'-phosphate forming a reversible Schiff base which could be reduced with NaBH4. The covalently modified enzyme was specifically cleaved with hydroxylamine at three labile Asn-Gly sequences yielding a series of peptides which were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis. The modified lysine was located in the COOH-terminal peptide. A critical arginine residue/subunit was found to be stoichiometrically modified with either 2,3-butadione or cyclohexadione. At high concentrations of butadione, an irreversible nonspecific modification of essentially all arginines occurred. An essential tryptophan residue was found to be stoichiometrically modified with N-bromosuccinimide in a similar fashion. Each of the chemical modifications of these three residues followed pseudo-first order and rate saturation kinetics and the modifications were prevented by the presence of substrates or competitive inhibitors. Circular dichroic spectral studies and analytical gel filtration indicated that these modifications have no effect on the quarternary structure and little effect on the secondary and tertiary structures of the enzyme. However, the extensive modification of arginine with butadione caused a dissociation of the enzyme into monomers and significant changes in tertiary structure. These studies provide new insights into functional aspects of isomerization and also provide an effective method for evaluating structural consequences of chemical or genetic modification of the enzyme.

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Comparison of N-linked oligosaccharides of recombinant human tissue kallikrein produced by Chinese hamster ovary cells on microcarrier beads and in serum-free suspension culture.

Glycosylation heterogeneity in recombinant human tissue kallikrein (r-HuTK) produced by Chinese hamster ovary (CHO) cells from microcarrier culture and from a serum-free suspension cell recycle process has been compared. Significant differences in the degree of sialylation were observed in glycoform distribution and oligosaccharide heterogeneity. High-performance liquid chromatography with a pellicular anion-exchange column under low pH eluant conditions was used to characterize the number and types of N-linked complex type oligosaccharides present. The oligosaccharides were released by N-glycanase and, after reduction, were resolved into a number of peaks containing one, two, three, and four sialic acids with an additional subfractionation based on the nature of the antennary structure. The microcarrier process resulted in a reduced amount of sialylated oligosaccharide species as compared to the suspension cell process. Removal of sialic acid followed by chromatography of the asialooligosaccharides under high pH anion-exchange conditions indicated that the same antennary structures were present but in slightly different relative amounts. The oligosaccharide profiles are indicative of a highly complex array of microheterogeneity present, encompassing mono-, di-, tri-, and tetrasialylated complex type oligosaccharides.

Animals↗
PubMedDate not supplied

Neuregulin-beta induces expression of an NMDA-receptor subunit.

Neuregulins (also known as ARIA, NDF, heregulin, GGF) are a family of widely expressed growth and differentiation factors. Neuregulins secreted from motor neurons accumulate at maturing neuromuscular junctions, where they stimulate transcription of genes encoding specific acetylcholine receptors. How these factors function at central synapses, however, is unknown. In the maturing cerebellum, neuregulins are concentrated in glutamatergic mossy fibres that innervate granule cells in the internal granule-cell layer. We have analysed the effects of neuregulins on the expression of genes encoding NMDA (N-methyl-D-aspartate) receptors in the cerebellum, because receptor composition changes dramatically as expression of the receptor NR2C subunit is specifically induced in neurons in the internal granule-cell layer during synaptogenesis. Here we report that addition of a neuregulin-beta isoform to cultured cerebellar slices specifically increases the expression of NR2C messenger RNAs by at least 100-fold; effects are only minor with a neuregulin-alpha isoform. This stimulation of NR2C expression requires synaptic activity by NMDA receptors, as well as neuregulin-beta. Addition of the NMDA-receptor-channel blocker AP-5 prevents upregulation of the NR2C subunit by neuregulin, whereas an AMPA/kainate-receptor antagonist does not. Consistent with these effects of neuregulin, we find that granule cells express its receptors ErbB2 and ErbB4 before the NR2C subunit of the NMDA receptor. Our results indicate that neuregulins regulate the composition of neurotransmitter receptors in maturing synapses in the brain, in a manner analogous to the neuromuscular junction.

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