Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification

Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1 alpha revealed that the protein was EF-1 alpha. Copurification of rabbit EF-1 alpha and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1 alpha protein sequence deduced from a human EF-1 alpha cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine.     

Nonlocal interactions stabilize compact folding intermediates in reduced unfolded bovine pancreatic trypsin inhibitor.

To further our understanding of the protein folding process, it is desirable to examine the structural intermediates (equilibrium and kinetic) that are populated between the statistical coil state and the folded molecule. X-ray crystallography and NMR structural studies are unable to determine long-range distances in proteins under denaturing solution conditions. Nonradiative (F?rster) energy transfer, however, has been shown to be a spectroscopic ruler for the measurement of distance distributions and diffusion between selected sites in proteins under a range of different solution conditions. The distributions of distances between a donor probe at the N-terminal residue and an acceptor attached to one of the four lysine residues (15, 26, 41, 46) of reduced and unfolded (in 6 M guanidine hydrochloride and 20 mM dithiothreitol) bovine pancreatic trypsin inhibitor (BPTI) were measured as a function of temperature. Even in strong denaturant and reducing agent, BPTI does not exist as a statistical coil polypeptide. It appears that nonlocal (long-range) interactions are already beginning to "fold" the protein toward a more compact, native conformation. As the temperature is increased under these conditions, hydrophobic interactions lead to an even more compact structure consistent with the predictions of phase diagrams for globular proteins.     

A study of correlates of hand preferences in squirrel monkeys (Saimiri sciureus)

The purpose of this study was to test the influence of sex, age, social rank, matriline membership, posture, and visual and tactual motor control on manual preferences inSaimiri sciureus. A well-established social group of 12 squirrel monkeys, aged 2 to 14 yrs and consisting of two matrilines with social rank known for each animal, was presented with four different food-reaching tasks and assessed for hand preferences with a minimum of 100 reaches per animal. Frequency of occurrence of hand preferences at the group level and degree of hand preferences at the individual level depended on posture and on whether the reaching act took place under visual or tactual guidance. Sex, age, social rank, and matriline membership were not found to determine frequency of occurrence, direction or degree of hand preferences with the exception of one task in which a significant negative correlation between the degree of hand preference and age was found. Nine out of 12 monkeys showed task-dependent changes in the hand they used preferentially while only three animals preferred the same hand in all four tasks. Significant preferences for the use of right or left hand on a given task were distributed almost equally between individuals. Thus, the results of this study suggest task-specific demands like posture and/or whether reaching was visually or tactually guided to be the major correlates of hand preferences in food-reaching tasks in squirrel monkeys.     

Comparative analysis of ribonuclease P RNA structure in Archaea.

Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

The influence of the cytosolic oncotic pressure on the permeability of the mitochondrial outer membrane for ADP: implications for the kinetic properties of mitochondrial creatine kinase and for ADP channelling into the intermembrane space

Summary Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed.     

Electron microscopic structural analysis of Photosystem I,Photosystem II,and the cytochromeb6/f complex from green plants and cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochromeb6/f complex are discussed.     

Crystallization and preliminary crystallographic studies of the precursor and mature forms of a neutral lipase from the fungus Rhizopus delemar

A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X-ray studies. It is monoclinic (C2, a = 92.8 Å, b = 128.9 Å, c = 78.3 Å, β = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P2₁2₁2₁, with a = 79.8 Å, b = 115.2 Å, c = 73.0 Å) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form. © 1994 John Wiley & Sons, Inc.