We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The ...proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as 14Cmethylated casein optimally at pH 8.5 and 90°C. The enzyme activity is enhanced severalford by Mg2+ and Ca2+ at 25–500 mM. This increase in activity results primarily from a change in Km. The serine‐proteinase inhibitors diisopropylfluorophosphate and 3,4‐dichlorosiocoumarin irreversibly inhibit the enzyme, obviously by modification of both the α and β subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz‐Leu‐Leu‐CH2Cl and Cbz‐Ala‐Ala‐Phe‐Ch2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.
The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The molecule has a molecular mass of approximately 2000 kD and has a highly conserved structure ...in eukaryotes. The 26S proteasome is formed by a barrel-shaped 20S core complex and two polar 19S complexes. The 20S complex has C2 symmetry and is formed by four seven-membered rings of which the outer rings (alpha-type subunits) are rotated by 25.7 degrees relative to the inner rings while the inner rings (beta-type subunits) are in register. From a comparison of the activity and regulation of the 26S and 20S particles it can be deduced that the 20S particle contains the protease activity while the 19S complex contains isopeptidase, ATPase and protein unfolding activities. In this article we describe the structures of various proteasome complexes as determined by electron microscopy and discuss structural implications of their subunit sequences.
In an attempt to settle the question of whether the multicatalytic proteinase or proteasome exist in all three kingdoms of life--eukaryotes, archaebacteria, and eubacteria--we have undertaken a ...search for them in the eubacterium Comamonas acidovorans. We have, in fact, isolated and purified a cylinder-shaped particle. However, according to various structural and biochemical criteria this turned out to be more reminiscent of the groEL protein from Escherichia coli and its homologs than to proteasomes of eukaryotic or archaebacterial origin. N-terminal sequencing provided definite proof for its belonging to this family of molecular chaperonins. Image analysis of electron micrographs revealed that the C. acidovorans groEL-like protein and proteasomes in spite of their significantly different dimensions have certain principles of organization in common.
The structure of several eubacterial and archaebacterial surface (glyco)proteins as determined by three-dimensional electron microscopy is described. Particular emphasis is placed on surface proteins ...which interact with membranes. Some structure-function relationships deduced from the structural information, such as shape maintenance and molecular recognition phenomena, are discussed.
The phosphoenolpyruvate synthase of the hyperthermophilic archaeonStaphylothermus marinusforms an unusually large homomultimeric complex of 93 kDa subunits. Electron image analysis of negatively ...stained and low-dose unstained preparations showed that the complex has a single, stable characteristic view and a well-preserved core with threefold rotational symmetry. The periphery of the assembly is composed of a nebulous, possibly flexible, component. Mass measurements by scanning transmission electron microscopy yielded a molecular weight of 2250 ± 230 kDa, confirming the well-defined nature of the structure and indicating that it is composed of 24 ± 2.5 subunits. The stability and symmetry of the characteristic projection views suggest a polyhedral three-dimensional architecture. The novel quaternary arrangement of this enzyme might be a consequence of its adaptation to an extreme environment.
Three archaeological sites at Katanda on the Upper Semliki River in the Western Rift Valley of Zaire have provided evidence for a well-developed bone industry in a Middle Stone Age context. Artifacts ...include both barbed and unbarbed points as well as a daggerlike object. Dating by both direct and indirect means indicate an age of ∼90,000 years or older. Together with abundant fish (primarily catfish) remains, the bone technology indicates that a complex subsistence specialization had developed in Africa by this time. The level of behavioral competence required is consistent with that of upper Paleolithic
Homo sapiens sapiens
. These data support an African origin of behaviorally as well as biologically modern humans.
The thermosome, a chaperonin from the archaebacterium Thermoplasma acidophilum, consists of two subunits (M(r) 58,000 and 60,000) which assemble into a cylindrical complex of pseudo eight-fold ...rotational symmetry. The sequences of the two subunits are approximately 60% identical to each other and to TF55 from Sulfolobus shibatae, and are 30-40% identical to the subunits of the TCP1 containing ring complex (TRiC) from the eukaryotic cytosol. A dendrogram of this family of chaperonins contains eight eukaryotic branches of TRiC subunits and one archaebacterial branch of thermosome subunits. Alignment of thermosome/TRiC sequences with eubacterial and eukaryotic Hsp60 sequences reveals a statistically significant similarity in two large N- and C-terminal blocks of sequence. Based on this alignment and on the recently published crystal structure of GroEL, we propose that subunits of the thermosome/TRiC family of chaperonins have a similar equatorial domain and overall domain topology as GroEL but differ in the structure of the apical domain.
Single cystals of proteasomes from the archaebacterium Thermoplasma acidophilum were obtained using the hanging-drop vapor diffusion method. The crystals diffract to better than 3.0 A and belong to ...the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 308.9 A, b = 208.8 A and c = 116.9 A. There is one molecular complex in the asymmetric unit. Two potentially useful heavy-atom derivatives have been obtained. The self-rotation function of the native Patterson map shows local sevenfold symmetry, consistent with the low-resolution structure obtained by electron microscopic techniques. The unit cell dimensions and crystal symmetry together with the shape and size of the proteasome suggest a packing arrangement of proteasome molecules in the unit cell, with their cylinder axis nearly parallel to the crystallographic a-axis.
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