Lec15 cells transfer glucosylated oligosaccharides to protein.

B4-2-1 cells (Lec15 cells) are Chinese hamster ovary cells deficient in mannosylphosphoryldolichol synthase activity. They synthesize the truncated lipid intermediate Man5GlcNAc2-P-P-dolichol rather than the Glc3Man9GlcNAc2-P-P-dolichol synthesized by wild-type cells. In this report we present evidence that these cells did synthesize glucosylated Man5GlcNAc2-P-P-dolichol, but this species represented only a minor fraction of the labeled oligosaccharide-lipid. On the other hand, glucosylated oligosaccharides were a major species transferred to protein in these cells, showing that in vivo, glucosylated oligosaccharides are preferentially transferred to protein. The truncated oligosaccharides found in B4-2-1 cells were removed from the protein by N-glycanase treatment, since they were resistant to both endo-beta-N-acetylglucosaminidase H and F activity. B4-2-1 cells processed the glucosylated, truncated oligosaccharides transferred to G protein of vesicular stomatitis virus, leading to infectious virus.     

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.     

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.