ABSTRACT
The fungus Aspergillus flavus MTCC 873, a non-toxigenic isolate demonstrated its capability to synthesize mycoferritin (MF) upon induction with iron in yeast extract sucrose (YES) medium. The molecular mass, yield, iron and carbohydrate contents of the MF were 440 kDa, 0.015 mg/g of wet mycelia, 0.8 and 30.4%, respectively. Native gel-electrophoresis revealed a band corresponding to dimeric form of equine spleen ferritin (ESF). Subunit analysis by SDS-PAGE revealed a single protein band with an apparent molecular mass of 24 kDa, suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross-reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy (TEM) revealed an apparent particle size of 100 Å. N-terminal amino acid sequence of MF revealed a sequence of SLPLQDYA, which showed identities with other eukaryotic ferritin sequences. The spectral characteristics (UV/VIS, fluorescence and circular dichroic spectra) were similar to ESF. The fungus, unlike A. parasiticus 255 (non-toxigenic) was incapable of producing aflatoxins, when grown in YES media.
Subject(s)
Amino Acid Sequence , Animals , Aspergillus flavus/chemistry , Electrophoresis, Polyacrylamide Gel , Ferritins/chemistry , Ferritins/isolation & purification , Ferritins/metabolism , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Fungal Proteins/metabolism , Humans , Sequence Alignment , Spectrum AnalysisABSTRACT
Objective To purify a Rhizoma Pinelliae protein,determine its inhibiting activity and mechanism to trypsin,and analyze its N-terminal amino acid sequence. Methods Active Rhizoma Pinelliae protein was purified from the 40% (NH_4)_2SO_4 sediment of the crude extracted protein from Rhizoma Pinelliae by affinity chromatography of trypsin-Sepharose CL 4B and gel filtration of Sephadex G-50;12% of SDS-PAGE was used for determining the purity of the purified Rhizoma Pinelliae protein and estimating its molecular weight;N-terminal amino acid sequence of active Rhizoma Pinelliae protein was analyzed by Edman degradation. Results The purified active Rhizoma Pinelliae protein showed a sin-gle band on SDS-PAGE gel,which estimated molecular weight was about 1. 4?10～4,its specific activity was 1 059. 012 U/mg,and the yield was about 24.40%;The N-terminal sequence of the preceding six amino acid residues of active Rhizoma Pinelliae protein was DPVVDG;The quality inhibiting ratio of active Rhi- zoma Pinelliae protein to trypsin was 1:4. 72,and the Ki value ,the inhibition constant was about 7. 17 ?10～(-6) mol/L. Conclusion Active Rhizoma Pinelliae protein is a kind of serine protease inhibitor,a com-petive inhibitor to trypsin.