Crystallographic studies of glutamate dehydrogenase. Preliminary crystal data.

Native and pyridoxal phosphate modified rat liver glutamate dehydrogenase crystals have been obtained and used for a preliminary x-ray crystallographic analysis. The space group is P6222 (P6422) having unit cell dimensions a = b = 101 A, c = 724 A and gamma = 120 A. The unit cell contains 36 subunits (six hexameric molecules) of molecular weight 56,000 and there is one half-molecule, i.e. three subunits, in the asymmetric unit. Packing considerations suggest that the glutamate dehydrogenase molecule has the point group symmetry 32 and that each subunit can be represented as a particle with approximate dimensions of 45 x 45 x 60 A.     

Molecular symmetry of fructose-1,6-diphosphatase by X-ray diffraction analysis.

Large single crystals of rabbit liver fructose-1,6-diphosphatase suitable for a high resolution structure analysis have been grown from polyethylene glycol. The space group of these crystals is I222 with a = 75 A, b = 81 A, and c = 132 A and there are 2 tetrameric molecules in the unit cell. These crystals have one protein subunit as the crystallographic asymmetric unit and establish point group symmetry 222 as the molecular symmetry.     

Characterization of crystals of xylose isomerase from Streptomyces violaceoniger

Crystals of the tetrameric xylose isomerase from Streptomyces violaceoniger have been examined by x-ray analysis. Octahedral crystals with a maximum dimension of 0.7 mm were grown from ammonium sulfate solution. They possess the symmetry of P4(1)2(1)2 or P4(3)2(1)2 space groups, which are crystallographically indistinguishable. The unit cell dimensions are a = b = 140 A and c = 134 A. There is one tetramer of molecular weight 160,000 per asymmetric unit. The crystals diffract to 2.2 A.     

Crystalline alkaline form fructose-1,6-diphosphatase. A simple purification procedure and preliminary x-ray diffraction analysis.

A simple and rapid procedure has been found for the preparation of higly pure alkaline form fructose-1,6-diphosphatase from turkey liver. The protein is tetrameric and is composed of subunits comprised of a 29,000 molecular weight core fragment and an associated 7,000-dalton S-peptide. Large single crystals of the protein, suitable for x-ray diffraction analysis, are produced in the course of the preparation. These are of space group R3 and have rhombohedral cell dimensions of a = 153 A and delta = 163 degrees (equivalent hexagonal dimensions a = 303 A, c = 75 A). There is one entire tetramer of 144,000 daltons in the asymmetric unit. The enzymatic properties of this enzyme appear to be very similar to those reported for the protein from other organisms, but its unique propensity to crystallize opens the way for a full three-dimensional structural analysis.     

Crystallization and preliminary X-ray investigation of uridine phosphorylase from Escherichia coli

Crystals of uridine phosphorylase from Escherichia coli K12 have been grown from solutions of polyethylene glycol 4000. The crystals are trigonal, space group R3; the hexagonal axes are a = 154.4 A and c = 49.4 A. The crystals are quite stable to x-rays and diffract beyond 2.6 A resolution. It appears that the molecule is a hexamer with a subunit molecular weight of 27,500 and utilizes the 3-fold symmetry of the space group, resulting in two subunits/asymmetric unit.     

A soybean trypsin inhibitor. Crystallization and x-ray crystallographic study

Five trypsin and alpha-chymotrypsin inhibitors which have low molecular weights (ranging from 6800 to 8600) and are present in soybean seeds of the Tracy variety have been isolated and purified, and single crystals which give x-ray diffraction data beyond 3-A spacings have been obtained from one of them. The trypsin inhibitor crystallizes in a monoclinic unit cell of symmetry P2(1) and dimensions a = 25.919(7) A, b = 43.23(1) A, c = 19.905(5) A, and beta = 103.63(2) degrees. The assymmetric unit contains 1 molecule of molecular weight 6800. The crystal, which has been found to be unusually stable to x-radiation, has solvent content of approximately 26% by volume.     

Preliminary X-ray data for aspartate aminotransferase from Escherichia coli

Crystals of the aspartate aminotransferase from Escherichia coli (aspC gene product) have been examined by X-ray analysis. The crystals grow as elongated rectangular prisms, with the symmetry of space group C2221. Unit cell dimensions are a = 156 A, b = 87.6 A, c = 80.6 A and alpha = beta = gamma = 90 degrees. There is one protein subunit of molecular weight 43,600 per asymmetric unit.     

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum.

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.     

Preliminary X-ray data for a D-amino acid amino-transferase from a novel thermophilic Bacillus

Crystals of the D-amino acid aminotransferase (D-ATA) from a novel thermophilic Bacillus species (Escherichia coli pICT113 cloned gene product) have been examined by X-ray analysis. The crystals grow as hexagonal prisms, with the symmetry of space group P61 or P65 (indistinguishable crystallographically). The cell dimensions are a = b = 135 A, c = 53 A, alpha = beta = 90 degrees, and gamma = 120 degrees. The unit cell has a volume of 850,000 A3 with six asymmetric units per unit cell. There is one dimer of molecular weight 62,000 per asymmetric unit, and the crystals diffract to 2.7 A.     

Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.     

Preliminary crystallographic data and quaternary structural implications of the central subunit of the multi-subunit complex transcarboxylase

The hexameric central subunit (Mr = 360,000) of the multi-subunit complex transcarboxylase has been crystallized by bulk dialysis against 250 mM-sodium acetate (pH 5.5). The crystals are cubic, a = 193.1 A, space group P4(1)32 or enantiomorph. The number of molecules per unit cell is four and was deduced from the density of the crystals (1.10 g cm-3) and the mother liquor (1.01 g cm-3) and the specific volume of the protein calculated from molecular dimensions obtained from electron microscopy studies. Four molecules per cell requires the central subunits to lie on 3-fold axes, which are perpendicular to 2-fold rotation axes, so that the molecules satisfy 32 symmetry giving one subunit as the asymmetric unit. Of the four possible models that have been considered for the quaternary structure of transcarboxylase, only that with antiparallel subunits, two sets of isologous binding sites and D3 symmetry is in agreement with the symmetry requirements of the cubic crystals.     

Subunit structures of AMP deaminase isozymes in rat

AMP deaminases A and B have been purified to apparent homogeneity from rat muscle and liver, respectively. The molecular weights of 286,000 and 351,000 were obtained for the native muscle and liver enzymes, respectively, by sedimentation equilibrium studies. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the muscle preparation exhibited a single polypeptide band with a molecular weight of 72,000; the liver preparation, a molecular weight of 85,000. The data indicate that each enzyme has a tetrameric structure.     

Three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts

Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to a resolution of 3.7 A on a hexagonal reciprocal lattice. The unit cell in projection (a = 127.0 A) and the symmetry of the diffraction pattern (6 mm) suggested that the hexagonal plates were highly ordered stacks of two-dimensional crystals suitable for structure analysis by electron microscopy and image processing. Crystals of a second form grew as dark green octahedra measuring roughly 0.5 mm across. Low-resolution X-ray diffraction patterns suggested a large cubic unit cell (a = 390 A). SDS/polyacrylamide gel electrophoresis of single octahedral crystals showed the same polypeptide composition as the starting solution, one major band at 24,000 apparent molecular weight and two satellite bands of 23,000 and 23,500 apparent molecular weight.     

A protein inhibitor of the mitochondrial adenosine triphosphatase complex of rat liver. Purification and characterization.

A heat-stable protein has been purified from rat liver mitochondria which inhibits the ATP hydrolytic activity of both the soluble and membrane-bound mitochondrial F1-ATPase. The overall purification is about 2400-fold with the major purification step consisting of Sephadex "affinity" chromatography. The purified rat liver inhibitor is homogeneous as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 12,300. Amino acid analysis reveals a high content of glutamic acid, lysine, and arginine and the absence of cysteine, proline and methionine. Whether tested with the rat liver or bovine heart ATPase, the liver inhibitor is equally as potent and specific as the heart inhibitor preparation of Pullman and Monroy (Pullman, M.E., and Monroy, G.C. (1963) J. Biol. Chem. 238, 3762-3769). Although the results presented show that the rat liver ATPase inhibitor resembles closely the ATPase inhibitors from other tissues with respect to specific activity and reaction specificity, it is important to note that the rat liver inhibitor is almost 2000 daltons larger than the bovine heart inhibitor, about 5000 daltons larger than ATPase inhibitors of yeast, and contains significantly more lysine residues than both the bovine heart and yeast inhibitors.     

Crystallization and preliminary X-ray analysis of complexes of peptide inhibitors with human recombinant and mouse submandibular renins.

Inhibitor-complexed crystals of mouse and human renins suitable for X-ray analysis have been prepared. The mouse renin is complexed with a non-hydrolysable decapeptide analogue of rat angiotensinogen containing a hydroxyethylene isostere in place of the scissile bond. The crystals are monoclinic, space group P2(1) with cell dimensions a = 78.3 A, b = 117.8 A, c = 85.9 A, beta = 101.18 degrees containing four molecules per asymmetric unit. The human renin is fully glycosylated and complexed with a tetrapeptide containing norstatine. The complex crystallises in the cubic space group P2(1)3 with a = 143.1 A and has two molecules in the asymmetric unit. The rotation function of the mouse renin complex indicates pseudo 222 symmetry while that of human renin indicates a pseudo 2-fold axis. Full structural analyses of the two complexes are underway.     

Molecular weights of subunits of acetyl CoA carboxylase in rat liver cytoplasm

Monomeric [14C] methyl avidin was shown to bind to sodium dodecyl sulfate-denatured biotinyl proteins and remain bound through polyacrylamide gel electrophoresis which allowed their detection by fluorography. This method was used to show that purified rat liver acetyl CoA carboxylase contained two high molecular weight forms of the enzyme (MR = 241,000 and 252,000) while rapidly prepared, crude rat liver cytoplasm contained two larger molecular weight (MR = 257,000 and 270,000) forms. Thus, the enzyme had undergone substantial proteolysis during purification. The crude enzyme preparation also contained a smaller biotinyl protein (MR = 141,000) which is likely a proteolytic product of the larger forms of acetyl CoA carboxylase.     

Crystallization and preliminary X-ray analysis of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.     

The functional and physical form of mammalian cytochrome c oxidase determined by gel filtration, radiation inactivation, and sedimentation equilibrium analysis

When solubilized in laurylmaltoside, cytochrome oxidases from beef heart and rat liver mitochondria exist as monodisperse populations that are stable, highly active, and have apparent molecular weights of 300,000 to 350,000, as measured by gel filtration. To determine whether these are monomeric (2 heme A, 2 Cu) or dimeric forms of the enzyme, we performed radiation inactivation and sedimentation equilibrium analyses. From radiation inactivation experiments under two different sets of conditions, we obtained estimates for the functional molecular weight of beef heart cytochrome oxidase of 114,000 and 99,000, much less than a dimer and significantly smaller than a 200,000 molecular weight monomer containing one copy of each of the 12 subunits normally present in the complex. The same functional size is obtained for a rat liver oxidase preparation depleted of subunit III. The physical molecular weight of cytochrome oxidase was determined by sedimentation equilibrium measurements in solvents of different densities using mixtures of H2O and D218O. Estimates of Mr = 194,000 +/- 9,000 for the beef heart oxidase and Mr = 152,000 +/- 6,000 for the rat liver enzyme were obtained, consistent with the size predicted for monomers of their subunit composition. From these results we conclude that mammalian cytochrome oxidases from beef heart and rat liver exist in laurylmaltoside as monomers capable of high rates of electron transfer and normal substrate binding. Further, these functions appear to be associated with a subset of the peptides present in the monomer, mainly composed of subunits I and II.     

Electron microscopy of alpha-latrotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus]

Two-dimensional crystals of alpha-latrotoxin from the venom of black widow spider (Latrodectus mactans tredecimguttatus) were studied by the negative staining electron microscopy. Two-dimensional crystals were obtained by adsorption of the protein solution with a high Mg2+ concentration on carbon-coated electron microscopy grids. The crystals were about 0.4 mkm in size, had the unit cell parameters: a = b = 15.55 nm, gamma = 90 degrees, p4 plane group symmetry. The contour map of a stain-excluding region of such crystals was calculated by the Fourier-filtering procedure at about 4 nm resolution. The calculation of molecular weight of the unit cell, with the symmetry p4 taken into account, showed that alpha-latrotoxin particles, revealed by negative staining, consisted of 4 or 8 protomers.     

Mitochondrial localization and oligomeric structure of HClpP, the human homologue of E. coli ClpP.

A bacterially expressed recombinant HClpP protein, the human homologue of Escherichia coli ClpP protease, was used to obtain specific polyclonal antibodies. Those antibodies identify a 26 kDa polypeptide in mitochondrial subcellular fractions of rat and human liver. Immunofluorescence and electron microscopic studies demonstrate that the mammalian homologue of ClpP is located in the mitochondrial matrix with a tendency to be found in association with the inner mitochondrial membrane. An HClpP recombinant protein with a truncated NH2terminus (missing the first 58 amino acid residues) shows a molecular mass of 26 kDa under denaturing conditions. This N-truncated HClpP recombinant protein shows a native molecular mass of 340 kDa that is identical with the native molecular mass of the partially purified protein from rat liver mitochondria. Electron microscopy shows that the N-truncated recombinant HClpP has a ring shape with seven identical morphological units in the periphery, exhibiting a 7-fold symmetry. The native molecular mass and the electron microscopic studies suggest that mitochondrial ClpP is composed of two heptameric rings with 7-fold symmetry, similar to E. coli ClpP.