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Biomedical subjects

R B Freedman

Publications and source records attributed to R B Freedman.

At least 55 records · Page 3Linked to original sources
PubMed1995

Characterization of the subunits in an apparently homogeneous subpopulation of Clostridium thermocellum cellulosomes.

Clostridium thermocellum cellulosomes isolated by cellulose affinity chromatography were fractionated by anion exchange chromatography into apparently homogeneous subpopulation that differed with respect to enzyme activity and subunit composition. One such subpopulation contained predominantly six subunits and was closely similar to the "subcellulosome" described by Kobayashi et al. (Kobayashi, T., Romaniec, M. P. M., Fauth, U., and Demain, A. L., Appl. Environ. Microbiol., 1990, 56, 3040-3046). Avicelase specific activity of this homogeneous subpopulation was slightly higher than that of unfractionated cellulosomes, but the two preparations were similarly affected by Ca2+, dithiothreitol, and cellobiose. Determination of their N-terminal sequences and enzyme activities has enabled three of the six major subunits of the subpopulation of cellulosomes to be positively identified as known components of the C. thermocellum cellulase complex; the other three subunits did not match up with previously characterized cellulosomal proteins.

Amino Acid Sequence↗
PubMed1995

The formation of protein disulphide bonds.

The past year has provided more detail on the formation of native disulphide bonds during protein folding at biosynthesis and has identified important cellular factors in the oxidative folding compartments, namely the eukaryotic endoplasmic reticulum and the bacterial periplasm. This information has enabled traditional in vitro refolding studies to be re-evaluated and their relevance as models for folding in the cell to be established.

Animals↗
PubMed1995

N-glycosylation of recombinant human interferon-gamma produced in different animal expression systems.

Recombinant human interferon-gamma (IFN-gamma) was expressed in Chinese hamster ovary cells, baculovirus-infected Sf9 insect cells and the mammary gland of transgenic mice. The N-linked carbohydrate populations associated with both Asn25 and Asn97 glycosylation sites were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. A site-specific analysis of dual (2N) and single (1N) site-occupancy variants of IFN-gamma derived from Chinese hamster ovary cells showed that N-glycans were predominantly of the complex bi- and triantennary type. Although Asn25-linked glycans were substituted with a core fucose residue, Asn97 N-glycans were predominantly non-fucosylated, and truncated complex and high-mannose oligosaccharide chains were also evident. Transgenic mouse derived IFN-gamma exhibited considerable site-specific variation in N-glycan structures. Asn25-linked carbohydrates were of the complex, core fucosylated type, Asn97-linked carbohydrates were mainly of the oligomannose type, with smaller proportions of hybrid and complex N-glycans. Carbohydrates associated with both glycosylation sites of IFN-gamma from Sf9 insect cells were mainly tri-mannosyl core structures, with fucosylation confined to the Asn25 site. These data demonstrate the profound influence of host cell type and protein structure on the N-glycosylation of recombinant proteins.

Animals↗
PubMed1995

Recombinant pro-regions from papain and papaya proteinase IV-are selective high affinity inhibitors of the mature papaya enzymes.

Proteolytic enzymes require the presence of their pro-regions for correct folding. Of the four proteolytic enzymes from Carica papaya, papain and papaya proteinase IV (PPIV) have 68% sequence identity. We find that their pro-regions are even more similar, exhibiting 73.6% identity. cDNAs encoding the pro-regions of these two proteinases have been expressed in Escherichia coli independently from their mature enzymes. The recombinant pro-regions of papain and PPIV have been shown to be high affinity inhibitors of all four of the mature native papaya cysteine proteinases. Their inhibition constants are in the range 10(-6) - 10(-9) M. PPIV was inhibited two to three orders of magnitude less effectively than papain, chymopapain and caricain. The pro-region of PPIV, however, inhibited its own mature enzyme more effectively than did the pro-region of papain. Alignment of the sequences of the four papaya enzymes shows that there is a highly variable section towards the C-terminal of the pro-region. This region may therefore confer selectivity to the pro-regions for the individual proteolytic enzymes.

Amino Acid Sequence↗