Protein chemical and kinetic characterization of recombinant porcine ribonuclease inhibitor expressed in Saccharomyces cerevisiae.
A cDNA encoding porcine ribonuclease inhibitor was used to express this protein in yeast under control of the PHO5 promoter. The recombinant protein was purified to homogeneity with a yield of 0.2 mg/g of yeast cells (wet weight) and was found to be indistinguishable from the inhibitor isolated from porcine liver on the basis of the following criteria: the amino acid composition, the number of free sulfhydryl groups, the molecular weight of the native and the denatured protein, peptide mapping, and amino acid sequence analysis of the N- and C-terminal regions of the protein. A simple method was developed for measuring accurately the slow, tight-biding kinetics of the inhibition of ribonuclease by ribonuclease inhibitor. From the dependence of the observed inhibition constant on the substrate concentration, it could be concluded that RI was competitive with the substrate UpA. The dependence of the observed association rate constant on the substrate concentration was consistent with a two-step mechanism in which the substrate only competed in the second (isomerization) step. The values for the inhibition constant for the inhibition of RNase by the recombinant inhibitor, 67 fM, the association rate constant, 1.5 x 10(8) M-1.s-1, and the dissociation rate constant, 8.3 x 10(-6) s-1, were in good agreement with those obtained for the porcine liver RNase inhibitor.
Vicentini
A M
AM
Friedrich Miescher-Institut, Basel, Switzerland.
Kieffer
B
B
Matthies
R
R
Meyhack
B
B
Hemmings
B A
BA
Stone
S R
SR
Hofsteenge
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors*; D012441:Saccharomyces cerevisiae; D013552:Swine
D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C066953:ribonuclease inhibitor, porcine; D004247:DNA; D012259:Ribonuclease, Pancreatic