[Interaction of rat hepatic glucose-6-phosphate dehydrogenase with some group-specific immobilized ligands]. / Vzaimodeistvie gliukozo-6-fosfatdegidrogenazy pecheni krys s nekotorymi immobilizovannymi ligandami obladaiushchimi gruppovoi spetsifichenost'iu.

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. The optimum conditions for the binding and specific elution of the enzyme were elucidated for each of the adsorbent studied. The scheme of glucose-6-phosphate dehydrogenase purification using the adsorbents studied is presented. It allows obtaining the preparation of the enzyme with specific activity about 100-240 U/mg and the extent of purification more than thousand fold.

[The effect of physical exertion on the activity of calpains and myeloperoxidase level in skeletal muscles of rats]. / Vliianie fizicheskikh nagruzok na aktivnost' kalpainov i soderzhanie mieloperoksidazy v skeletnykh myshtsakh krys.

It has been established that the activity of calpains and the amount of neutrophil myeloperoxidase in skeletal muscles of rats increases after the 24-48 h-rest, that followed an intensive single physical exercise. Adaptation which appears as a result of systematic physical exercises causes less pronounced changes in the activity of proteolytic enzymes. Free ubiquitin content in skeletal muscles remains unchanged during training and in posttraining periods.

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. Point mutants of ubiquitin (Lys-->Arg) were used to map the linkage specificity for ubiquitin-conjugating enzymes previously demonstrated to form novel multiubiquitin chains not attached through Lys-48. Recombinant human E2EPF catalyzed multiubiquitin chain formation exclusively through Lys-11 of ubiquitin while recombinant yeast RAD6 formed chains linked only through Lys-6. Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each bound to subunit 5 of partially purified human 26 S proteasome with comparable affinities. Since chains bearing different linkages are expected to pack into distinct structures, competition between Lys-11 and Lys-48 chains for binding to subunit 5 demonstrates that the latter possesses determinants for recognizing alternatively linked chains and precludes the existence of subunit 5 isoforms recognizing distinct structures. In addition, competition studies provided an estimate of Kd < or = 18 nM for the intrinsic binding of Lys-48-linked chains of linkage number n > 4. This result suggests that the principal mechanistic advantage of multiubiquitin chain formation is to enhance the affinity of the associated substrate for the 26 S complex relative to that of unconjugated target protein. Complementation studies with E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported isopeptide ligase-dependent degradation only through Lys-48-linked chains, while E2EPF retained the ability to target a model radiolabeled substrate through Lys-11-linked chains. Therefore, the linkage specificity exhibited by these E2 isozymes depends on their catalytic context with respect to isopeptide ligase.

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. We have exploited specific interactions between ubiquitin carrier proteins (E2/Ubc) and their cognate E3 ligases to purify for the first time the mammalian N-end rule ligase E3alpha/Ubr1 to near homogeneity. In vitro studies show that E3alpha forms lysine 48-linked polyubiquitin degradation signals on type 1-3 substrates and is absolutely dependent on Ubc2/Rad6 orthologs. Biochemically defined kinetic studies show that the basis of N-end rule specificity is a k(cat) rather than the K(m) effect originally proposed, since all three substrate classes show similar binding affinities (K(m) approximately 5 microm) but V(max) values that are 100- and 50-fold greater for type 1 and 2 versus type 3 model substrates, respectively. In addition, the N-end rule dipeptides lysylalanine and phenylalanylalanine are general noncompetitive inhibitors for E3alpha-catalyzed ubiquitination of type 1-3 substrates rather than type-specific competitive inhibitors as predicted. These observations are consistent with a model in which the N-end rule effect reflects substrate binding-induced transitions in E3alpha to a catalytically competent conformer, the equilibrium for which depends on the identity of the amino terminus or the presence of basic or hydrophobic surface features. The model reconciles conflicts between specific predictions and empirical observations relating N-end rule targeting in addition to explicating the efficacy of selected dipeptides as potent in vivo inhibitors of this pathway.