Protein interactions within the N-end rule ubiquitin ligation pathway.

Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding.

N-end rule specificity within the ubiquitin/proteasome pathway is not an affinity effect.

The N-end rule relates the amino terminus to the rate of degradation through the ubiquitin/26 S proteasome pathway. Proteins bearing basic (type 1) or large hydrophobic (type 2) amino termini are assumed to be targeted through this pathway by their higher affinity for binding to the responsible E3 ligase compared with proteins bearing other residues (type 3). Paradoxically, a significant fraction of eukaryotic protein degradation occurs through the N-end rule pathway, although the majority of cellular proteins are type 3 substrates.

Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EPF and RAD6 are recognized by 26 S proteasome subunit 5.

Targeting of substrates for degradation by the ATP, ubiquitin-dependent pathway requires formation of multiubiquitin chains in which the 8.6-kDa polypeptide is linked by isopeptide bonds between carboxyl termini and Lys-48 residues of successive monomers. Binding of Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory complex commits the attached target protein to degradation with concomitant release of free ubiquitin monomers following disassembly of the chains.

Coordinated induction of the ubiquitin conjugation pathway accompanies the developmentally programmed death of insect skeletal muscle.

The developmentally programmed cell death of abdominal intersegmental muscles in the tobacco hawk-moth Manduca sexta is coincident with a 10-fold induction of the polyubiquitin gene as a hormonally regulated event (Schwartz, L. M., Myer, A.

[Interaction of rat hepatic glucose-6-phosphate dehydrogenase with some group-specific immobilized ligands].

Conditions of the interaction of rat hepatic glucose-6-phosphate dehydrogenase and some group-specific adsorbents (2'5'-ADP-sepharose Cl-6B, Red sepharose CL-6B and Blue sepharose CL-6B) have been examined. The extent of this interaction was estimated according to such parameters as the volume of enzyme elution (Ve) and the recovery of the enzyme activity (%). The effect of the NADP concentration, values of pH and ionic strength of buffer solution on the parameters of enzyme binding with immobilized ligand was studied.