PubMed · 6856502
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E Portalska, J Sadowski. 1983-01-10. [Glomerular filtration: functional analysis. I].. https://pubmed.ncbi.nlm.nih.gov/6856502/
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Recent evidence indicates that efficient expression of major histocompatibility complex (MHC) complexes requires their interaction with the resident endoplasmic reticulum (ER) chaperone calnexin, which for certain proteins functions as a lectin specific for monoglucosylated glycans. In the current report, we studied the expression of MHC class I proteins in BW wild type thymoma cells (BW WT) and glucosidase II-deficient BW PHAR2.7 cells. Consistent with a requirement for glucose (Glc) trimming for interaction of class I proteins with calnexin, we found that nascent H-2Kk proteins associated with calnexin in untreated BW WT cells, but not in BW WT cells treated with the glucosidase inhibitor castanospermine (cas), or in untreated glucosidase II-deficient BW PHAR2.7 cells. Surprisingly, we found that H-2Kk expression occurred with similar efficiency in BW PHAR2.7 cells as in BW WT cells and that formation of nascent H-2Kk complexes was perturbed by cas treatment in BW WT cells but not in BW PHAR2.7 cells. Finally, it was noted that expression of the molecular chaperone Bip was markedly increased in BW PHAR2.7 cells relative to BW WT cells, which is suggested to play a role in regulating the expression of H-2Kk complexes in BW PHAR2.7 cells. The current study demonstrates that Glc trimming is required for efficient interaction of nascent H-2Kk proteins with calnexin; that expression of MHC class I proteins can, under certain conditions, proceed effectively in the absence of Glc trimming and calnexin association; and that Bip expression is markedly increased under conditions where diglucosylated glycans persist on nascent glycoproteins within the ER. These data are consistent with the hypothesis that alternative oligomerization pathways exist for class I proteins within the quality control system of the ER that have differential requirements for removal of Glc residues from nascent glycan chains.
3-oxo-delta 5-steroid isomerase (KSI) from Pseudomonas testosteroni catalyzes the isomerization of a variety of 3-oxo-delta 5-steroids to their conjugated delta 4-isomers through the intermediate formation of a dienolate ion. This dienolate is formed by proton transfer from C-4 of the substrate to Asp-38, which then protonates the dienolate at C-6. Catalysis is enhanced by electrophilic assistance (hydrogen bonding) to the 3-oxygen by Tyr-14. We have investigated the effect of modifying phenylalanine-101 (F101), a hydrophobic residue that is located in the binding pocket of KSI. Two mutant enzymes (F101L and F101A) of KSI were prepared, and their kinetic properties were examined with 5-androstene-3, 17-dione (1) as the substrate. Both of the mutants show reduced values of kcat compared to the wild type (WT), by about 30-fold (F101L) and by 270-fold (F101A), with only a small difference in Km values. There is little change in the Ki's ( < or = 4-fold) for the product 4-androstene-3,17-dione (3), although both enzymes bind the intermediate analog d-equilenin (4) about 25-fold less tightly than does the WT. Fluorescence spectra of 4 bound to each of these enzymes suggest that 4 is ionized at the active site of WT, un-ionized at the active site of F101A and a mixture of these ionization states at the active site of F101L. Free energy profiles are constructed for each of the mutant enzymes, and these are compared to the free energy profile for the WT.(ABSTRACT TRUNCATED AT 250 WORDS)
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