Abstract
The molecular weight of Streptomyces subtilisin inhibitor (SSI), a protein proteinase inhibitor, and that of the complex of SSI and subtilisin BPN' [EC 3.4.21.14] were determined by a sedimentation equilibrium method in 25 mM phosphate buffer, at pH 7.0, ionic strength 0.1 M (NaCl), 25.0 degrees C. The molecular weight of SSI was found to be 23,000 over a wide concentration range, 0.01-10 mg/ml, the range used for inhibitory, spectrophotometric, and kinetic measurements. Based on the amino acid sequence, the molecular weight of SSI has been calculated to be 11,500 (Ikenaka, T., et al. (1974) J. Biochem. 76, 1191-1209); therefore, the molecular weight of 23,000 obtained above suggests that SSI is in a dimeric form under usual conditions in the concentration range of 5 X 10(-7)-5 X 10(-4) M. The molecular weight of the subtilisin BPN'-SSI complex was determined to be 78,000 in the concentration range of 0.03-5.0 mg/ml by sedimentation equilibrium of the crystallized preparation and by that of a mixture of subtilisin BPN' and SSI treated as a multicomponent-polydisperse system. The molecular weight obtained here, combined with the results of binding stoichiometry (Inouye, K., et al. (1977) J. Biochem. 82, 961-967) that showed that one mol of SSI (molecular weight, 11,500) and one mol of the enzyme (molecular weight, 27,500) are tightly bound (Kd less than 1 nM), demonstrate that one mol of dimeric SSI binds two mol of the enzyme to form a stable complex, E2I2.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.