PubMed · 10857082
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C L Spence, P Bailon. 2000. Fluidized-bed receptor-affinity chromatography.. https://doi.org/10.1007/978-1-60327-261-2_3
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The small ubiquitin-like modifier (SUMO) family in vertebrates includes three different family members that are conjugated as post-translational modifications to target proteins. SUMO-2 and -3 are nearly identical but differ substantially from SUMO-1. We used quantitative proteomics to investigate the target protein preferences of SUMO-1 and SUMO-2. HeLa cells were established that stably express His6-SUMO-1 or His6-SUMO-2. These cell lines and control HeLa cells were labeled with stable arginine isotopes, and His6-SUMOs were enriched from lysates using immobilized metal affinity chromatography. 53 SUMO-conjugated proteins were identified, including 44 novel SUMO targets. 25 proteins were preferentially conjugated to SUMO-1, 19 were preferentially conjugated to SUMO-2, and nine proteins were conjugated to both SUMO-1 and SUMO-2. SART1 was confirmed by immunoblotting to have both SUMO-1- and SUMO-2-linked forms at similar levels. SUMO-1 and SUMO-2 are thus shown to have distinct and overlapping sets of target proteins, indicating that SUMO-1 and SUMO-2 may have both redundant and non-redundant cellular functions. Interestingly, 14 of the 25 SUMO-1-conjugated proteins contain zinc fingers. Although both SUMO family members play roles in many cellular processes, our data show that sumoylation is strongly associated with transcription because nearly one-third of the identified target proteins are putative transcriptional regulators.
Lipoxygenase was purified homogeneously from cups of Pleurotus ostreatus by Sephacryl S-400 HR gel filtration, Dyematrex Green A affinity, and DEAE-Toyopearl 650M ion-exchange chromatographies. The molecular weight of the enzyme was estimated to be 67,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 66,000 by gel filtration; the isoelectric point was pH 5.1. The optimum pH and temperature of the enzymatic activity were 8.0 and 25 degrees C, respectively. The enzyme contained non-heme iron, and a thiol group seemed to be involved in its activity. The K(m), V(max), and k(cat) values of the enzyme for linoleic acid were 0.13 mM, 23.4 micromol.min(-1).mg(-1), and 25.7 s(-1), respectively. The enzyme showed high specificity toward linoleic acid. When linoleic acid was incubated with the enzyme, 13-hydroperoxy-9Z,11E-octadecadienoic acid was found to be the main oxidative product.
The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase (PP1(C)) is encoded by the essential gene GLC7 and is involved in regulating diverse cellular processes. To identify potential regulatory or targeting subunits of yeast PP1(C), we tagged Glc7p at its amino terminus with protein A and affinity-purified Glc7p protein complexes from yeast. The purified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by peptide mass fingerprint analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. To confirm the accuracy of our identifications, peptides from some of the proteins were also sequenced using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. Only four of the Glc7p-associated proteins that we identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previously been shown to interact with Glc7p, and multiple components of the CPF (cleavage and polyadenylation factor) complex involved in messenger RNA 3'-end processing were present as major components in the Glc7p-associated protein fraction. To confirm the interaction of Glc7p with this complex, we used the same approach to purify and characterize the components of the yeast CPF complex using protein A-tagged Pta1p. Six known components of the yeast (CPF) complex, together with Glc7p, were identified among the Pta1p-associated polypeptides using peptide mass fingerprint analysis. Thus Glc7p is a novel component of the CPF complex and may therefore be involved regulating mRNA 3'-end processing.