PubMed · 4268481
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G Seidel. 1973. Two functionally different kininogens in human plasma.. https://doi.org/10.1007/bf02023845
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The purification of glycosyltransferases involved in wall matrix polysaccharide synthesis has been attempted. A number of activities readily demonstrated in isolated Golgi membranes are lost following detergent solubilization. However, solubilization releases pyrophosphorylases and phosphatases that hydrolyse the substrate in enzyme assays, whether UDP-glucose, -arabinose or -xylose is used. This hydrolysis, which cannot be completely inhibited, appears to be the major factor in the apparent loss of activity. Separation of this hydrolytic activity during further purification by ion-exchange and gel exclusion leads to recovery of glycosyltransferase activity. Thus two xylosyltransferases and one arabinosyltransferase could be partially purified. These appeared to be differentially expressed. The arabinosyltransferase of apparent M(r) 70,000 on size-exclusion chromatography was isolated from cells undergoing rapid growth and division. A xylosyltransferase of apparent M(r) 38,000 on size-exclusion chromatography was associated with cell expansion and primary wall synthesis. A second xylosyltransferase, which was purified to near homogeneity with M(r) 40,000, showed a peak of activity during the period of maximum secondary wall synthesis.
Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.
The kinetics of the hydrodynamic volume change accompanying the reversible unfolding of staphylococcal nuclease have been observed by size-exclusion chromatography at 4 degrees C and pH 7.0 using the denaturant guanidine hydrochloride. The observed chromatographic profiles have been simulated by a six-component unfolding/refolding mechanism using a consistent set of equilibrium and kinetic parameters. The native protein is an equilibrium mixture of the cis and trans isomers of the peptide bond preceding proline-117. The native conformation containing the cis isomer dominates the equilibrium mixture, is more stable, and unfolds more slowly at its transition midpoint. The denatured protein is an equilibrium mixture of at least four components, the cis/trans isomers of proline-117 and one of the five remaining prolines. The dominant refolding pathway is initiated from the denatured component containing the trans isomer of proline-117. The six-component mechanism is consistent with tryptophan fluorescence kinetic measurements of the wild-type protein and with chromatographic measurements of a mutant P117G protein.