The S-peptide and S-protein fragments of ribonuclease S (RNase S, no EC no. assigned) have been immobilized onto separate Sepharose gels via a "leash" of polycytidylic acid substrate. Each of these gels releases its RNase fragment when treated with the complementary enzyme fragment or with RNase A (EC 3.1.27.5), and the released fragments recombine to give RNase S activity. Thus this system pro...

The amino-terminal 20 amino acids are required for microinjected ribonuclease A (RNase A) to be taken up by lysosomes and degraded at an enhanced rate during serum withdrawal. We used water-soluble carbodiimides to covalently attach the RNase S-peptide (residues 1-20) to [3H]RNase S-protein (residues 21-124) at unspecified locations. We then measured catabolism of the [3H]S-protein-S-peptide co...

As a core factor in S-RNase-based gametophytic self-incompatibility (GSI), the SCF (SKP1-Cullin1-F-box-Rbx1) complex (including pollen determinant SLF, S-locus-F-box) functions as an E3 ubiquitin ligase on non-self S-RNase. The SCF complex is formed by SKP1 bridging between SLF, CUL1, and Rbx1; however, it is not known whether an SCF complex lacking SKP1 can mediate the ubiquitination of S-RNas...

Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas-phase environment. Solution binding affinities may or may not be accurate predictors o...

Self-incompatibility (SI) in flowering plants entails the inhibition of fertilization by pollen that express specificities in common with the pistil. In species of the Solanaceae, Rosaceae, and Scrophulariaceae, the inhibiting factor is an extracellular ribonuclease (S-RNase) secreted by stylar tissue. A distinct but as yet unknown gene (provisionally called pollen-S) appears to determine the s...

The differences between stylar protein of the Japanese pear (Pyrus pyrifolia (Burm.f.)) cultivars ‘Kosui’ (SS) and ‘Kikusui’ (SS) were compared by two-dimensional difference gel electrophoresis (2-D DIGE), and were labelled and visualized with different fluorescent dyes (IC3-OSu, IC5-OSu) on a single 2-D gel. The individual different expressed proteins spots were subjected to identification. Th...

RNase A introduced into the cytoplasm of IMR-90 human diploid fibroblasts by erythrocyte-mediated microinjection is degraded with a half-life of approximately equal to 75 hr in the presence of fetal bovine serum. In response to serum deprivation the degradative rate of microinjected RNase A is enhanced 2-fold. RNase S protein (amino acids 21-124) is degraded with a half-life similar to that of ...

RNase E of Escherichia coli is an essential endoribonuclease that is involved in many aspects of RNA metabolism. Point mutations in the S1 RNA-binding domain of RNase E (rne-1 and rne-3071) lead to temperature-sensitive growth along with defects in 5S rRNA processing, mRNA decay and tRNA maturation. However, it is not clear whether RNase E acts similarly on all kinds of RNA substrates. Here we ...

In attempting to relate the covalent structure of ribonuclease to its enzymic activity, controlled proteolytic degradation is a useful approach. Although native ribonuclease is markedly resistant to a variety of proteolytic enzymes, the separated components of ribonuclease S (1, 2), S-peptide, and S-protein, are extensively degraded by carboxypeptidase. Previous studies (3) on the sequence of a...

S-RNase-based self-incompatibility in Petunia is a self/non-self recognition system that allows the pistil to reject self-pollen to prevent inbreeding and to accept non-self pollen for outcrossing. Cloning of S-RNase in 1986 marked the beginning of nearly three decades of intensive research into the mechanism of this complex system. S-RNase was shown to be the sole female determinant in 1994, a...