Conformational changes in the 20S proteasome upon macromolecular ligand binding analyzed with monoclonal antibodies.

Proteasomes interact with a variety of macromolecular ligands that modulate their ability to degrade peptide and protein substrates. The effector PA28 increases the peptidase activities of proteasomes whereas HSP90 and alpha-crystallin inhibit a peptide-hydrolyzing activity. Four monoclonal antibodies were used as probes to detect conformational changes of proteasome subunits. Conformational changes in alpha- or beta-subunits were found upon binding PA28, HSP90, alpha-crystallin, and the substrate casein but not with the peptide substrate analogs calpain inhibitor 1 (Ac-Leu-Leu-norleucinal), calpain inhibitor 2 (Ac-Leu-Leu-methioninal), or MG 132 (N-Cbz-Leu-Leu-leucinal).

Subunit arrangement in the human 20S proteasome.

In human 20S proteasomes two copies of each of seven different alpha-type and seven different beta-type subunits are assembled to form a stack of four seven-membered rings, giving the general structure alpha(1-7), beta(1-7), beta(1-7), alpha(1-7). By means of immunoelectron microscopy and chemical crosslinking of neighboring subunits, we have determined the positions of the individual subunits in the proteasome. The topography shows that for the trypsin-like, the chymotrypsin-like, and the postglutamyl cleaving activities, the pairs of beta type subunits, which are thought to form active sites, are nearest neighbors.

Human proteasomes analysed with monoclonal antibodies.

The proteasome or multicatalytic endopeptidase from eukaryotic cells consists of at least 14 subunits that fall into two families, alpha and beta. Subunit-specific monoclonal antibodies against ten different subunits of human proteasomes have been produced, together with an antibody that reacts with a motif (prosbox 1), common to alpha-type subunits. Four of the subunit-specific antibodies were able to precipitate proteasomes. The subunit composition of HeLa-cell proteasomes precipitated with these four different antibodies were identical, as judged from two-dimensional electrophoresis. One of the four antibodies was used to obtain proteasomes from cell lines (HeLa, Daudi, IMR90 and BSC-1) and human tissues (placenta, kidney, and liver). Electrophoretic analysis of these proteasomes, combined with peptide mapping of some subunits, suggests that they all contain 14 types of subunits as their major constituents. However, one subunit was present in two isoelectric isoforms in all cells examined. Two other subunits occurred in two or three isoelectric isoforms in placenta, liver and kidney, but not in the cell cultures. Extracts of human cells (HeLa, IMR90, Daudi and erythrocytes) were analysed by non-denaturing electrophoresis and immunoblotting. All of the 11 subunits detected by antibodies were present in a pair of ATP-stabilized protein complexes, presumed to be the 26 S proteinase, and in a doublet of complexes which migrated more slowly than purified proteasomes. Besides being present in proteasomes, one subunit was also found to occur in the free state in cell extracts.

Human proteasome subunits from 2-dimensional gels identified by partial sequencing.

Human proteasomes consist of 14 major subunits which can be resolved by two-dimensional polyacrylamide gel electrophoresis. Tryptic peptides from the subunits were sequenced. This allowed identification of all proteins in two-dimensional electrophoresis gels with proteasome subunits whose sequences are known from cDNA. The results also precisely define the specificity of a panel of subunit-specific monoclonal antibodies. A new proteasome subunit (Z) was discovered. In contrast, the putative subunit MECL-1 could not be detected in human placenta proteasomes.

Subunit stoichiometry of human proteasomes.

Subunits from human placental proteasomes were separated by two-dimensional polyacrylamide gel electrophoresis. The amino acid composition of proteins from individual spots were determined. Some of the spots had identical amino acid compositions, confirming that they contain isoforms of the same subunit. Proteasomes from HeLa cells, labelled with 3H-leucine, were precipitated with an antibody and similarly separated into subunits. The radioactivity in each subunit was measured. The subunit stoichiometry was then calculated from these data and the leucine contents in the subunits. Each of the 14 major subunits of human proteasomes are apparently present in equal amounts.

Monoclonal antibodies to the human multicatalytic proteinase (proteasome).

Multicatalytic proteinase is an intracellular enzyme composed of at least 12 different subunits. Seven murine hybridoma cell lines secreting antibodies to human multicatalytic proteinase (MCP) were established. The antibodies reacted with 4 different subunits of the oligomeric protein. Three of the antibodies bound to identical or closely spaced epitopes on the largest subunit, as shown by binding competition. Some of the antibodies cross-reacted with MCP from rat or rabbit, but none with lobster MCP. Glycoprotein components could not be detected in human MCP. The monoclonal antibodies and two polyclonal rabbit antibodies did not specifically inhibit the enzymatic activity of human MCP. Electrophoretic analysis of MCP immunoprecipitated from human placenta, liver, kidney, or HeLa cell extracts with antibodies to 3 different subunits suggested that the subunit compositions are very similar or identical.

The human multicatalytic proteinase: affinity purification using a monoclonal antibody.

A monoclonal antibody, coupled to Sepharose CL-4B, was used for the rapid purification of the human multicatalytic proteinase in a single chromatographic step under mild conditions. The enzyme was homogeneous as judged by nondenaturing polyacrylamide gel electrophoresis. Electrophoresis under dissociating and reducing conditions revealed at least ten components with molecular masses in the range 22-34 kDa. Affinity-purified enzyme was identical to conventionally purified enzyme with respect to enzymatic properties, molecular mass and subunit composition.

Periplasmic nonspecific acid phosphatase II from Salmonella typhimurium LT2. Crystallization, detergent reactivation, and phosphotransferase activity.

The periplasmic nonspecific acid phosphatase II from Salmonella typhimurium was purified to homogeneity from a mutant strain that overproduces the enzyme (Uerkvitz, W., and Beck, C.F. (1981) J. Biol. Chem. 256, 382-389). It was shown that the enzyme transfers phosphate groups from organic phosphoric acid esters (donors) to water as well as to the 2'-, 3'-, or 5'-hydroxyls of nucleosides, nucleotides, and other compounds with free hydroxyl groups (acceptors). The enzyme was crystallized in two forms by precipitation with polyethylene glycol. Needles were formed in buffer containing Mg2+, whereas thin rectangular plates appeared in the presence of the non-ionic detergent n-octyl glucoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under partially or completely denaturating conditions revealed that the native enzyme is a tetramer consisting of identical 24-kDa monomers. Owing to surface inactivation, polyethylene glycol, non-ionic, or Zwitterionic detergents are indispensable for enzyme stability. The detergents are able to reactivate inactivated enzyme when present near or above their critical micelle concentration.

Identification of membrane proteins related to anion transport in Ehrlich ascites tumor cells.

DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) can interact covalently with membrane sites, resulting in an inhibition of anion exchange. This inhibition varies from reversible to irreversible, depending on the length of times DIDS interacts with the membranes. During the reversible phase, a kinetic analysis of the nature of its inhibitory effect on C1 self-exchange can be performed. The effect of variations in the chloride concentration on the inhibitory potency of DIDS is consistent with the concept that C1 and DIDS compete for the transport site of the anion exchange system in Ehrlich cells. The value of Ki for DIDS inhibition is approx. 0.5 microM. Since the reversible binding may be specific to the anion recognition site in the transport system, it is likely that the subsequent covalent reaction (which is very slow in the Ehrlich cell) involves a nucleophilic group in the membrane close to the transport site. In this case DIDS can be used as a covalent label for the transport protein. We have synthesized a 3-H labelled DIDS with a high specific activity (0.62 X 10(8) cpm/mumol). Using the labelled DIDS, it has been possible to demonstrate that in low C1 medium, only one membrane protein seems to be labelled, and that the number of binding sites per cell is 7 X 10(7). This value is very close to the known density of binding sites in the red blood cell.

Periplasmic phosphatases in Salmonella typhimurium LT2. A biochemical, physiological, and partial genetic analysis of three nucleoside monophosphate dephosphorylating enzymes.

Three periplasmic nucleoside monophosphate-splitting phosphatases from Salmonella typhimurium, 2':3'-cyclic nucleotide 2'-phosphodiesterase, nonspecific acid phosphatase I, and nonspecific acid phosphatase II, were separated by column chromatography. They are characterized with respect to their substrate specificities, Km values, pH optima, molecular weights, and sensitivity to inhibition by Pi and EDTA. Nonspecific acid phosphatase II has not been reported previously. The physiological roles of the various phosphatases are assessed by studies on mutant strains. Selection procedures were developed for the isolation of mutants defective in the synthesis of one or more of the phosphatase activities. Analysis of these mutant strains revealed that 2':3'-cyclic nucleotide 2'-phosphodiesterase is the major 3'-nucleotide dephosphorylating activity, and nonspecific acid phosphatase II is most active against 5'-nucleotides at concentrations below 1 mM. Triple mutants, lacking all periplasmic nucleotide-splitting activities, are viable. Regulatory mutants with elevated levels of nonspecific acid phosphatase II activity were isolated. The gene for 2':3'-cyclic nucleotide 2'-phosphodiesterase (pde) was located between purA and argI at 135 min on the S. typhimurium linkage map.