N. A. Potapenko

Domain structure and ATP‐induced conformational changes in Escherichia coli protease Lon revealed by limited proteolysis and autolysis

Escherichia coli protease Lon (La) is an adenosine triphosphate (ATP)‐regulated homo‐oligomeric proteolytic complex responsible for the recognition and selective degradation of abnormal and unstable proteins. Each subunit of the protease Lon appears to consist of three functional domains: the C‐terminal proteolytic containing a serine active site, the central displaying the ATPase activity, and the N‐terminal with still obscure function. We have used limited proteolysis to probe the domain structure and nucleotide‐induced conformational changes in the enzyme. Limited proteolysis of the native protease Lon generated a low number of stable fragments roughly corresponding to its functional domains. Conformational changes in the wild‐type enzyme and its mutant forms in the presence or absence of adenine and guanine nucleotides were investigated by limited proteolysis. The nucleotide character was shown to play a key role for susceptibility of the protease Lon to limited proteolysis, in particular, for resistance of the ATPase functional domain. ATP and adenosine diphosphate displayed a protective effect of the ATPase domain of the enzyme. We suggest that these nucleotides induce conformational changes of the enzyme, transforming the ATPase domain from the most vulnerable part of the molecule into a spatially inaccessible one. Both limited proteolysis and autolysis demonstrate that the most stable part of the protease Lon molecule is its N‐terminal region. Obvious resistance of the protease Lon C‐terminus to proteolysis indicates that this region of the enzyme molecule including its substrate‐binding and proteolytic domains has a well folded structure.

Determination of the sidedness of the carboxy-terminus of the Na+/K+-ATPase α-subunit using lactoperoxidase iodination

The orientation of the carboxy-terminal pair of tyrosines of the Na+/K(+)-ATPase alpha-subunit with respect to the plane of the plasma membrane was determined. The approach was based on lactoperoxidase-catalysed radioiodination of the tyrosine residues accessible on the surface of the enzyme molecule in intact cells of a pig kidney embryonic cell line and those accessible in a broken plasma membrane fraction and in isolated membrane-bound Na+/K(+)-ATPase. The labeled alpha-subunit was isolated by SDS gel electrophoresis followed by electroblotting. Then the COOH-terminal amino acids were hydrolyzed by carboxypeptidases B and Y. Radioactivity and quantitative analysis of the protein and released amino acids showed that the COOH-terminal tyrosine residues of the alpha-subunit were only accessible to modification only when lactoperoxidase had access to the inner side of the plasma membrane. Therefore, the COOH-terminus of the Na+/K(+)-ATPase alpha-subunit is located on the cytoplasmic surface of the pump molecule and its polypeptide chain must have an even number of transmembrane segments.

State of nucleolar proteins B23/nucleophosmin and UBF in HeLa cells during apoptosis induced by tumor necrosis factor

The structural state of two major nucleolar proteins, UBF and B23/nucleophosmin (both monomeric and oligomeric forms), was for the first time established in HeLa cells treated with apoptosis inducers: tumor necrosis factor (TNF-α), emetine, and their combination. The treatment of the cells with either TNF-α or emetine did not induce apoptosis and affect the state of UBF and nucleophosmin (both monomers and oligomers). Apoptosis was rather pronounced only if HeLa cells were treated with a mixture of TNF-α and emetine. States of the UBF and B23 proteins were analyzed in samples containing 25, 45, and 100% of cells with apoptotic nuclei. It was shown by immunoblotting that TNF-α-induced apoptosis of HeLa cells was associated with proteolysis of UBF and production of a 76-kD fragment, the content of which increased in correlation with the fraction of apoptotically changed cells. The N-and C-terminal amino acid sequences of UBF and its 76-kD fragment were characterized, and the site of the apoptosis-induced specific proteolysis was identified. As differentiated from UBF, protein B23 did not undergo proteolytic degradation during the TNF-α-induced apoptosis of HeLa cells and its content was unchanged even in the cell fraction with fragmentation of virtually all nuclei. However, the ratio between the monomeric and oligomeric states of B23 protein was changed in apoptotic cells, and apoptosis-specific forms of nucleophosmin were detected.

Interaction between tubulin and Na+, K+-ATPase in brain stem neurons

Functionally active Na2+,K2+-ATPase isozymes containing three types of the catalytic subunits (α1, α2, and α3) were obtained from calf brain by two methods: selective removal of contaminating proteins according to Jorgensen (1974) and selective solubilization of the enzyme with subsequent reformation of the membrane structure according to Esmann (1988). All preparations were characterized with respect to ouabain-inhibition constants. The presence of the cytoskeleton protein tubulin (β3 isoform) in the high-molecular-weight complex of Na2+,K2+-ATPase α3β1 isozyme from brain stem axolemma and the junction between Na2+,K2+-ATPase α3 subunit and tubulin β3 subunit are shown for the first time.

State of oncomarker protein B23/nucleophosmin in HeLa cells

Western blot after SDS-PAGE for protein separation showed two immunoreactive bands corresponding to monomers (38–40 kDa) and oligomers (210–230 kDa) of nucleophosmin in HeLa cell lysates. Decreasing the buffer ionic strength during the incubation of cells and nuclei destabilized these oligomers. We also showed the existence of two B23/nucleophosmin pools in nuclei of HeLa cells with different sensitivity to hypotonic buffer treatment: one extractable from the nucleus and the other non-extractable and tightly bound to the nucleus. A detailed structural analysis of the extractable B23 pool was carried out: two closely related nucleophosmin isoforms (B23.1 and B23.2) were identified as a result of analysis of C-terminal amino acid sequences using carboxypeptidase hydrolysis; the N-termini of both isoforms are blocked by an acetyl group. As a result of sequencing of the deacetylated proteins, it has been established that the N-terminal amino acid sequence of nucleophosmin in these preparations is truncated by nine amino acid residues and the acetylated residue is Ser. The truncated monomer of nucleophosmin (represented only by the extractable part of the protein) on addition of magnesium ions to low ionic strength buffer or increase in buffer ionic strength was shown to form oligomers with molecular weights (210–230 kDa) similar to those revealed in the total cell lysate. It should be noted that the set of oligomers in this case differs from the one in total cell lysate. Our strategy of characterization of B23 forms for HeLa cells can be applied for other tumor cells.

Isolation of the protein B23/nucleophosmin from HeLa cell nuclei

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35–40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C-and N-terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.

Isolation and Characterization of Fragments of the ATP-Dependent Protease Lon from Escherichia coli Obtained by Limited Proteolysis

Conditions of limited proteolysis of the protease Lon from Escherichia coli that provided the formation of fragments approximately corresponding to the enzyme domains were found for studying the domain functioning. A method of isolation of the domains was developed, and their functional characteristics were compared. The isolated proteolytic domain (LonP fragment) of the enzyme was shown to exhibit both peptidase and proteolytic activities; however, it cleaved large protein substrates at a significantly lower rate than the full-size protease Lon. On the other hand, the LonAP fragment, containing both the ATPase and the proteolytic domains, retained almost all of the enzymatic properties of the full-size protein. Both LonP and LonAP predominantly form dimers unlike the native protease Lon functioning as a tetramer. These results suggest that the N-terminal domain of protease Lon may play a considerable role in the process of the enzyme oligomerization.

Structural Peculiarities of Na+,K+-ATPase Isozymes from the Calf Brain

Functionally active preparations of Na+,K+-ATPase isozymes from calf brain that contain catalytic subunits of three types (α1, α2, and α3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of their membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na+ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na+,K+-ATPase of the α1β1 type and minor amounts of isozymes of the α2β2(β1) and the α3β1(β2) type. The axolemma contains α2β1 and α3β1 isozymes. A carbohydrate analysis indicated that α1β1 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the β1 isoform. An enhanced sensitivity of the α3 catalytic subunit of Na+,K+-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR492 ↓ Y493 was localized (residue numbering is that of the human α3 subunit). This sequence corresponds to one of the regions of the greatest variability in α1-, α2-, α3-, and α4-subunits, but at the same time, it is characteristic of the α3 isoforms of various species. The presence of the β3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na+,K+-ATPase α3β1 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the α3 catalytic subunit was shown.

Isolation and Structural and Functional Analysis of New Types of Na,K‐ATPase Isozymes

Functionally active enzymes were obtained from microsomes from calf cerebral cortex gray matter, brain stem, and stem axolemrna by two different methods involving ( 1 ) the selective removal of contaminating proteins, according to Jorgensen, I and (2) the selective solubilization of the enzyme with subsequent reformation of the membrane structure, according to Esmann.2 The protein components of the isolated preparations were separated by polyacrylamide gel electrophoresis, transferred to an immobilon membrane by electroblotting, and subjected to structural analysis. To carry out preliminary structural analysis of the protein components of the enzymes, the preparations were dansylated? Semiquantitative analysis of the proteins was possible using comparison of their NH2-terminal amino acid residues directly from immobilon, because dansylated proteins with different M, transferred to immobiIon membranes at about the same rate. Brain gray matter Na,K-ATPase was characterized by biphasic kinetics with respect to ouabain inhibition (Ki 1.5 . lop8 M) and was comprised of a set of isozymes with subunit compositions of a l p l , a2pm, and a3pm (where m = 1 andlor 2 ) , with the a l p 1 form clearly predominating. Na,K-ATPase from the brain stem and axolemma consisted mainly of the mixture of isozymes a2pl and a3j31, which had identical ouabain inhibition constants (K, lo-’ M). Catalytic subunit a3 within the native enzyme complex exhibited increased sensitivity to endogenous proteolysis. Specific proteolysis was localized to the region of the polypeptide chain that is unique to all a3 type isofor&: PNDNR492 1 (Y493) (according to the numbering of human a 3 subunit). As shown for the first time, two other proteins were present in all enzyme preparations containing a2 and a3 isoforms isolated by both methods: p5 chain of tubulin and glyceraldehyde-3-phosphate dehydrogenase. The biological meaning of this association is still unclear. However, the influence not only of subunit composition, but also of cytoskeletal structure and other plasma membrane-associated proteins might be taken into account in functional peculiarities of Na,K-ATPase isozymes. Many diseases and pathological changes in the organism occur directly or indirectly from dysfunction of the sodium pump. The Na,K-ATPase in ischemic

Determination of sidedness of COOH-terminus of Na+/K+ ATPase alpha subunit by vectorial labeling

Extensive studies have been performed on structure-function relationship of the Na+ /K+-ATPase. Nevertheless, current ideas on basic processes underlying coupled ATP hydrolysis, energy transduction and cation transport are deficient in molecular details. This is mainly due to the lack of reliable data on the spatial protein structure (1). The three-dimensional structure of the Na+/K+-ATPase cannot yet be solved by X-ray analysis. However, significant information on the spatial organization of the protein molecule may be derived by other experimental approaches such as limited proteolysis, affinity modification, labeling by permeable and impermeable reagents, analysis with specific antibodies and spectroscopic methods. A combination of these techniques has been used to identify functional domains and to probe the folding of subunit polypeptide chains in the membrane. It is generally accepted that the beta subunit spans the membrane only once (1,4). In contrast, the transmembrane folding of the catalytic alpha subunit has been the subject of considerable controversy. Several topological models differ in the number and location of the membrane-spanning segments within the COOH-terminal half of the polypeptide chain and therefore the orientation of the COOH-terminus (2,3,5,7).