Direct visualization of the calmodulin subunit of phosphorylase kinase via electron microscopy following subunit exchange.

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme, (alphabetagammadelta)4. To introduce specifically labeled calmodulin into the phosphorylase-b kinase complex for its eventual visualization by electron microscopy, we have developed a method for rapidly exchanging exogenous calmodulin for the intrinsic delta subunit. This method exploits previous findings that low concentrations of urea in the absence of Ca(2+) ions cause the specific dissociation of only the delta subunit from the holoenzyme [Paudel, H. K., and Carlson, G. M. (1990) Biochem. J. 268, 393-399]. In the current study, phosphorylase-b kinase was incubated with excess exogenous calmodulin and a threshold concentration of urea to promote exchange of its delta subunit with the exogenous calmodulin. Size exclusion HPLC was then used to remove the excess calmodulin from the holoenzyme containing exchanged delta subunits. Using metabolically labeled [35S]calmodulin to allow quantification and optimization of exchange conditions, we achieved exchange of approximately 10% of all delta subunits within 1 h, with the exchanged holoenzyme retaining full catalytic activity. Calmodulins derivatized with Nanogold for visualization by scanning transmission electron microscopy were then exchanged for delta, which for the first time allowed localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex. The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzyme's catalytic gamma subunit.

Multimeric structure of the secreted meprin A metalloproteinase and characterization of the functional protomer.

Meprin A secreted from kidney and intestinal epithelial cells is capable of cleaving growth factors, extracellular matrix proteins, and biologically active peptides. The secreted form of meprin A is a homo-oligomer composed of alpha subunits, a multidomain protease of 582 amino acids coded for near the major histocompatibility complex of the mouse and human genome. Analyses of the recombinant homo-oligomeric form of mouse meprin A by gel filtration, nondenaturing gel electrophoresis, and cross-linking (with disuccinimidyl suberate or N-(4-azido-2,3,5,6-tetraflourobenzyl)-3-maleimidylpropionamide) indicate that the secreted enzyme forms high molecular weight multimers, with a predominance of decamers. The multimers are composed of disulfide-linked dimers attached noncovalently by interactions involving the meprin, A5 protein, receptor protein-tyrosine phosphatase mu (MAM) domain. The active protomer is the noncovalently linked dimer. Linkage of active protomers by disulfide-bonds results in an oligomer of approximately 900 kDa, which is unique among proteases and distinguishes meprin A as the largest known secreted protease. Electron microscopy revealed that the protein was present in two states, a crescent-shaped structure and a closed ring. It is concluded from this and other data that the covalent attachment of the protomers enables noncovalent associations of the native enzyme to form higher oligomers that are critical for hydrolysis of protein substrates.

The cytokine portion of p43 occupies a central position within the eukaryotic multisynthetase complex.

Multicellular eukaryotes contain a macromolecular assembly of nine aminoacyl-tRNA synthetase activities and three auxiliary proteins. One of these, p43, is the precursor of endothelial monocyte-activating polypeptide II (EMAP II), an inflammatory cytokine involved in apoptotic processes. As a step toward understanding this paradoxical association, the EMAP II portion of p43 has been localized within the rabbit reticulocyte multisynthetase complex. Immunoblot analysis demonstrates strong reaction of anti-EMAP II antiserum with p43, as well as cross-reactivity with isoleucyl-tRNA synthetase. Electron microscopic images of immunocomplexes show two antibody binding sites. The primary site is near the midpoint of the multisynthetase complex at the intersection of the arms with the base. This site near the lower edge of the central cleft is assigned to the C-terminal cytokine portion of p43. The secondary site of antibody binding is in the base of the particle and maps the location of isoleucyl-tRNA synthetase. These data allow refinement of the three-domain model of polypeptide distribution within the multisynthetase complex. Moreover, the central location of p43/EMAP II suggests a role for this polypeptide in optimizing normal function and in rapid disruption of essential cellular machinery when apoptosis is signaled.

Immunoelectron microscopic localization of glutamyl-/ prolyl-tRNA synthetase within the eukaryotic multisynthetase complex.

A high molecular mass complex of aminoacyl-tRNA synthetases is readily isolated from a variety of eukaryotes. Although its composition is well characterized, knowledge of its structure and organization is still quite limited. This study uses antibodies directed against prolyl-tRNA synthetase for immunoelectron microscopic localization of the bifunctional glutamyl-/prolyl-tRNA synthetase. This is the first visualization of a specific site within the multisynthetase complex. Images of immunocomplexes are presented in the characteristic views of negatively stained multisynthetase complex from rabbit reticulocytes. As described in terms of a three domain working model of the structure, in "front" views of the particle and "intermediate" views, the primary antibody binding site is near the intersection between the "base" and one "arm." In "side" views, where the particle is rotated about its long axis, the binding site is near the midpoint. "Top" and "bottom" views, which appear as square projections, are also consistent with the central location of the binding site. These data place the glutamyl-/prolyl-tRNA synthetase polypeptide in a defined area of the particle, which encompasses portions of two domains, yet is consistent with the previous structural model.

Ultrastructure of the eukaryotic aminoacyl-tRNA synthetase complex derived from two dimensional averaging and classification of negatively stained electron microscopic images.

Several aminoacyl-tRNA synthetases in higher eukaryotes are consistently isolated as a multi-enzyme complex for which little structural information is yet known. This study uses computational methods for analysis of electron microscopic images of the particle. A data set of almost 2000 negatively stained images was processed through reference-free alignment and multivariate statistical analysis. Interpretable structural information was evident in five eigenvectors. Hierarchical ascendant classification extracted clusters corresponding to distinct image orientations. The class averages are consistent with rotations around and orthogonal to a central particle axis and provide particle measurements: approximately 25 nm in height, 30 nm at the widest point and 23 nm thick. The results also provide objective evidence in support of the working structural model and demonstrate the feasibility of obtaining the three dimensional structure of the multisynthetase complex by single particle reconstruction methods.

Structural analysis of the multienzyme aminoacyl-tRNA synthetase complex: a three-domain model based on reversible chemical crosslinking.

A subset of eukaryotic aminoacyl-tRNA synthetases (a-RS) are contained in a multienzyme complex for which little structural detail is known. Three reversible chemical crosslinking reagents have been used to investigate the arrangement of polypeptides within this particle as isolated from rabbit reticulocytes. Identification of the crosslinked protein pairs was accomplished by two-dimensional SDS diagonal gel electrophoresis. Seventeen neighboring protein pairs have been identified. Eight are seen with at least two reagents: K-RS:p38, D-RS:K-RS, R-RS dimer, K-RS dimer, K-RS:Q-RS, E/P-RS:K-RS, E/P-RS:I-RS, and Q-RS with one of the nonsynthetase proteins. Nine more are observed with one reagent: D-RS dimer, R-RS:p43, D-RS:Q-RS, D-RS:M-RS, K-RS:L-RS, I-RS:R-RS, D-RS:E/P-RS, I-RS:Q-RS, I-RS:L-RS. One trimeric association is seen: E/P-RS:I-RS:L-RS. The observed neighboring protein pairs suggest that the polypeptides within the aminoacyl-tRNA synthetase complex are distributed in three structural domains of similar mass. These can be arranged in a U-shaped particle in which each "arm" is considered a domain and the third forms the "base" of the structure. The arms have been termed domain I (D-RS, M-RS, Q-RS) and domain II (K-RS, R-RS), with domain III (E/P-RS, I-RS, L-RS) assigned to the base. The smaller proteins (p38, p43) may bridge the domains. This proposed spatial relationship of these domains, as well as their compositions, are consistent with earlier studies. Thus, this study provides an initial three-dimensional working model of the arrangement of polypeptides within the multienzyme aminoacyl-tRNA synthetase complex.

Proximal regions of the catalytic gamma and regulatory beta subunits on the interior lobe face of phosphorylase kinase are structurally coupled to each other and with enzyme activation.

Phosphorylase kinase from skeletal muscle is a hexadecameric enzyme with the subunit composition (alphabeta gammadelta)4 and a mass of 1.3 x 10(6) Da. The catalytic gamma subunit and the remaining regulatory subunits are packed as a tetrahedral structure composed of two elongated, opposing (alphabeta gammadelta)2 octameric lobes. We show by immunoelectron microscopy with subunit-specific monoclonal antibodies that a portion of the beta subunit occurs on the interior face of the lobes at a region of inter-lobal interactions, and that at a proximal position slightly more central and distal on the interior lobe face lies the base (residues 277 to 290) of the helical domain of the catalytic core of the gamma subunit. Activation of the kinase by a variety of means caused similar increases in the binding to the holoenzyme of the monoclonal antibodies against these two regions of the beta and gamma subunits. Moreover, monovalent fragments of the antibodies against both regions stimulated the activity of the non-activated holoenzyme. Thus, the epitopes of the beta and gamma subunits recognized by the monoclonal antibodies are structurally coupled to each other and with the activation of phosphorylase kinase. Activation of the holoenzyme apparently involves the repositioning of the base of the catalytic domain of the gamma subunit and a proximal region of the beta subunit within the identified area on the interior face of the lobes of the tetrahedral phosphorylase kinase molecule.

Novel isolation method and structural stability of a eukaryotic chaperonin: the TCP-1 ring complex from rabbit reticulocytes.

In the course of removing a contaminant from preparations of aminoacyl-tRNA synthetase complexes, a novel purification method has been developed for the eukaryotic cytoplasmic chaperonin known as TRiC or CCT. This method uses only three steps: ammonium sulfate precipitation, pelleting into a sucrose cushion, and heparin-agarose chromatography. As judged by electrophoresis, sedimentation, and electron microscopy, the preparations are homogeneous. The particle is identified as a chaperonin from electrophoretic polypeptide pattern, electron microscopic images, direct mass measurement by sedimentation velocity analysis, amino-terminal sequencing, and ATP-dependent refolding of rhodanese and actin. Further investigation of the biochemical and physical properties of the particle demonstrates that its constituent polypeptides are not glycosylated. The particle as a whole binds strongly to polyanionic matrices. Of particular note is that negatively stained images of chaperonin adsorbed to a single carbon layer are distinctly different from those where it is sandwiched between two layers. In the former, the "characteristic" ring and four-stripe barrel predominate. In the latter, most images are round with a highly reticulated surface, the average particle diameter increases from 15 to 18 nm, and additional side, end, and substrate-containing views are observed. The particle structure is strikingly resistant to physical forces (long-term storage, repeated cycles of freezing and thawing, sedimentation), detergents (Triton, deoxycholate), salts (molar levels of KCl or LiCl), and pH changes (9-6). Only a strongly chaotropic salt (NaSCN) and extremely acidic conditions (pH 4.5) cause aggregation and dissociation of TRiC, respectively. However, treatment with KCl or deoxycholate reduces TRiC folding activity.

Structure of phosphorylase kinase. A three-dimensional model derived from stained and unstained electron micrographs.

Phosphorylase kinase, the first protein kinase discovered, is a key regulatory enzyme in glycogen metabolism. Although its biochemical properties are well characterized, details of its three-dimensional structure and subunit topology are yet to be elucidated. This study describes four characteristic views of the hexadecameric holoenzyme (alpha 4 beta 4 gamma 4 delta 4) as observed in both negatively stained and unstained electron micrographs. The predominant views are the widely reported "butterfly" with two wing-like lobes connected by thin bridges, and the previously described "chalice", composed of "cup" and "stem" segments. Two additional views, a "cube", similar to the previously reported "tetrad", and a "cross" or "X" are less common, but illustrate the overall geometry of the particle. Based on these images, the first three-dimensional model of the enzyme has been constructed. It is composed of four identical protomers that associate with D2 symmetry to form the two major structural elements (the two lobes). Two protomers in a head to head arrangement make up each symmetrical lobe; to complete the holoenzyme, one lobe is inverted and placed perpendicular to the other. Thus, the overall structure has three 2-fold axes of symmetry, and the arrangement of the four protomers approximates a tetrahedron. Each lobe of the model corresponds to a wing of the butterfly projection. Two projections form the chalice: in the intra-lobe orientation, one lobe forms the cup and the other forms the stem, and in the inter-lobe view, one-half of each lobe contributes to each segment of the image. The cube and cross projections result from 90 degrees rotations from the butterfly orientation. In the cube, the distal portions of each lobe are projected separately. In the cross, one lobe is crossed over and is above the other. This model both accounts for and predicts all of the observed microscopic images.

An epitope proximal to the carboxyl terminus of the alpha-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer.

An epitope of the alpha-subunit of phosphorylase kinase from fast-twitch skeletal muscle was localized to the tips of the bilobal kinase molecule by two types of immunoelectron microscopy. This is the first direct evidence identifying the location of any of the enzyme's 16 subunits within the phosphorylase kinase molecule. Negatively stained complexes of phosphorylase kinase with an immunoglobulin G monoclonal antibody specific for the alpha-subunit (mAb 157) were observed by conventional transmission electron microscopy, and complexes of the unstained enzyme with undecagold-labeled Fab' fragments derived from mAb 157 were visualized by scanning transmission electron microscopy. Images from both techniques indicate a symmetrical arrangement of the epitope, consistent with a "head-to-head" packing arrangement of the four alpha-subunits. In Western blots, mAb 157 crossreacted with comigrating fragments obtained by digesting non-denatured phosphorylase kinase with a variety of proteases, suggesting that the epitope for the anti-alpha mAb is contained within a protease-resistant domain. Partial sequencing of a 24.1 kDa immunoreactive chymotryptic fragment narrowed the epitope to somewhere within the carboxyl-terminal one-sixth of the alpha-subunit. Studies of the crossreactivity of mAb 157 with the holoenzyme in the presence of calmodulin, after phosphorylation or with different isoforms (all with known alpha-subunit sequence targets or differences), suggest that the epitope is even more proximal to the carboxyl terminus. This epitope was not implicated in any known function or activity of the enzyme, suggesting that the region proximal to the carboxyl terminus of the alpha-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.

Isolation of an acute-phase phosphorylcholine-reactive pentraxin from channel catfish (Ictalurus punctatus).

1. Channel catfish (Ictalurus punctatus) serum contains a protein that precipitates pneumococcal C-polysaccharide (CPS) in a calcium-dependent fashion. 2. The serum titer of this protein follows an acute-phase pattern in catfish injected with turpentine. 3. A non-glycosylated, phosphorylcholine (PC)-reactive protein (PRP) with molecular mass ca 100 kDa, was isolated from channel catfish acute-phase sera by affinity chromatography on PC-Sepharose 4B. 4. Contaminating proteins with molecular masses ca 700 kDa and ca 20 kDa that co-eluted with PRP from PC-Sepharose appear to be aggregated and native low-molecular weight factors (LMFs), respectively. 5. Purified PRP has gamma mobility but in serum samples PRP has gamma-beta mobility. 6. Electron microscopy confirmed that PRP has planar, pentagonal symmetry. 7. The amino terminus of PRP is blocked, but based on comparison of amino-acid compositions channel catfish PRP is clearly similar to human CRP and is most like CRPs from the dogfish (Mustelus canis) and rainbow trout (Oncorhynchus mykiss).

Structural analysis of the high molecular mass aminoacyl-tRNA synthetase complex. Effects of neutral salts and detergents.

The effects of a variety of detergents and neutral salts on the structure of the eukaryotic high molecular mass aminoacyl-tRNA synthetase complex have been directly determined by observing alterations in the composition, sedimentation behavior, and electron microscopic appearance of the rabbit reticulocyte complex. The intact complex is shown to exhibit the enzymatic activities, polypeptide composition, relative stoichiometry, and morphological features that are characteristic of this eukaryotic multienzyme particle. The structure of the high molecular mass aminoacyl-tRNA synthetase complex is seen to be resistant to both ionic and nonionic detergents. However, both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and deoxycholate induce formation of large protein aggregates. In contrast, the chaotropic salts LiCl and NaSCN both selectively remove individual polypeptides from the high molecular mass aminoacyl-tRNA synthetase complex and promote formation of specific particulate subcomplexes which have distinct sizes, polypeptide compositions, and structural features. These data support the view that many of the protein interactions within the high molecular mass amino-acyl-tRNA synthetase complex are hydrophobic in nature. This study also provides direct evidence that the complex contains a core of tightly interacting synthetases onto which the remaining polypeptides are arrayed. The structural alterations observed here may account for the ability of these reagents to markedly inhibit several enzymatic activities within the complex.

Isolation and electron microscopic characterization of the high molecular mass aminoacyl-tRNA synthetase complex from murine erythroleukemia cells.

A high molecular mass aminoacyl-tRNA synthetase complex has been isolated from a murine erythroleukemia cell line. This multienzyme complex contains activities for the arginyl-, aspartyl-, glutamyl-, glutaminyl-, isoleucyl,- leucyl-, lysyl-, methionyl-, and prolyl-tRNA synthetases. This enzyme composition, the polypeptide pattern observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the relative stoichiometry of the component polypeptides are characteristic of high molecular mass complexes of aminoacyl-tRNA synthetases isolated from a variety of mammalian tissues and cell types. Negatively stained preparations of native complex and of glutaraldehyde-treated material have been examined by electron microscopy. In both cases, a distinctive particle is observed which appears in several orientations. The most common views are of two different projections of a squarish particle that measures approximately 27 x 27 nm. Other commonly observed views are of a "U" shape, a rectangle, and a triangle. All of these views are seen in both gradient-purified samples and those prepared directly from material as isolated. These data are consistent with a model for the multienzyme aminoacyl-tRNA synthetase complex as a "cup" or elongated U structure. These studies demonstrate that the high molecular mass complex of eukaryotic aminoacyl-tRNA synthetases does have a coherent structure that can be visualized by electron microscopy.

Structure and stability of Z* DNA.

The structure and stability of the left handed Z* DNA aggregate was examined by spectroscopic methods and by electron microscopy. Poly(dGdC), upon heating in the presence of Mn++, forms a large aggregate which may be sedimented at 12,000 X g, with a circular dichroism spectrum characteristic of left handed DNA. Aggregation gives rise to turbidity changes at visible wavelengths, providing a convenient means of monitoring the transition in solution. The wavelength dependence of turbidity is consistent with the scattering behavior of a long thin rod. Electron microscopy shows that Z* DNA is a large fibrous structure of indeterminant length, with a uniform diameter of approximately 20 nm. The results obtained in solution and under the requisite conditions for electron microscopy are mutually consistent. Poly(dGdC) preparations with average lengths of 60, 240, 500, and 2000 base pairs all form Z* DNA. Poly(dGm5dC) forms Z* DNA in the presence of Mn++ without heating, but poly(dAdC)-poly(dGdT) and calf thymus DNA cannot be induced to the Z* form under any conditions tried. Kinetic studies, monitored by turbidity changes, provide evidence that the formation of Z* DNA proceeds by a nucleated condensation mechanism. Dissolution of the Z* aggregate results from the chelation of Mn++ or by the addition of the intercalator ethidium bromide. The allosteric conversion of Z* DNA to an intercalated, right handed form by ethidium is demonstrated by kinetic studies, equilibrium binding studies and circular dichroism spectroscopy. Electron microscopy provides a striking visualization of the dissolution of the Z* aggregate by ethidium.