Protein sequencer maintenance and troubleshooting.
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Maintenance↗
Biomedical subjects
Bryan Dunbar
Publications and source records attributed to Bryan Dunbar.
A 60-66 kDa protein with gonadotrophin surge attenuating factor bioactivity is produced by human ovarian granulosa cells.
We aimed to confirm the ovarian site of gonadotrophin surge-attenuating factor (GnSAF) production and produce granulosa/luteal cell-conditioned medium (G/LCM) containing GnSAF for purification studies. Blue dye affinity chromatography followed by pseudochromatofocusing of G/LCM yielded bioactive fractions at pH 5.74 and 5.77. The former had a major 60-66 kDa band with an internal amino acid sequence of EPQVYVHAP following tryptic digestion. A rat polyclonal antiserum (rPAb) raised against this band completely blocked in-vitro GnSAF bioactivity in human follicular fluid, serum and G/LCM. GnSAF bioactivity was localized to a 64 kDa band of serum-free G/LCM and following 2D gel electrophoresis, one of the spots recognized by Western blotting with the GnSAF rPAb had an N-terminal amino acid sequence of NH-XVPQGNAXXN. Neither amino acid sequence had significant homology with proteins in the human genome database. When ovarian tissues from spontaneously cycling women were cultured under serum-free conditions, neither theca- nor stroma-conditioned media contained GnSAF bioactivity. However, granulosa cell-conditioned medium significantly reduced GnRH-induced LH secretion, an effect that was reversed by incubation with the GnSAF rPAb. In conclusion, we have confirmed that human granulosa cells produce GnSAF within the ovary and have two candidate amino acid sequences for GnSAF. We have also demonstrated that serum-free granulosa cell culture constitutes the method of choice for the characterization of GnSAF since recovery of bioactivity is superior in the presence of fewer serum proteins.
Biological Factors↗
A catalase from Streptomyces coelicolor A3(2).
Catalase was purified from the Gram-positive bacterium Streptomyces coelicolor A3(2) in a three-step purification procedure comprising (NH4)2SO4 fractionation, Phenyl-Sepharose chromatography and Mono Q chromatography. The purification of catalase, as judged by the final specific activity of 110,000 U mg-1, was 250-fold with a 35% yield. The native protein was a homotetramer with a subunit M(r) 55,000. N-terminal and internal peptide sequence analyses showed that there was a high degree of sequence similarity between the S. coelicolor catalase and other microbial and mammalian catalases. Southern blot analysis indicated that there was a single catalase gene in S. coelicolor. The specific activity of catalase throughout the growth of batch cultures was investigated and elevated catalase activity was found in stationary-phase cells.
Amino Acid Sequence↗