Triacylglycerol and phospholipid hydrolysis in human plasma lipoproteins: role of lipoprotein and hepatic lipase.

To explore the interactions of triacylglycerol and phospholipid hydrolysis in lipoprotein conversions and remodeling, we compared the activities of lipoprotein and hepatic lipases on human VLDL, IDL, LDL, and HDL2. Triacylglycerol and phospholipid hydrolysis by each enzyme were measured concomitantly in each lipoprotein class by measuring hydrolysis of [14C]triolein and [3H]dipalmitoylphosphatidylcholine incorporated into each lipoprotein by lipid transfer processes. Hepatic lipase was 2-3 times more efficient than lipoprotein lipase at hydrolyzing phospholipid both in absolute terms and in relation to triacylglycerol hydrolysis in all lipoproteins. The relationship between phospholipid hydrolysis and triacylglycerol hydrolysis was generally linear until half of particle triacylglycerol was hydrolyzed. For either enzyme acting on a single lipoprotein fraction, the degree of phosphohydrolysis closely correlated with triacylglycerol hydrolysis and was largely independent of the kinetics of hydrolysis, suggesting that triacylglycerol removed from a lipoprotein core is an important determinant of phospholipid removal via hydrolysis by the lipase. Phospholipid hydrolysis relative to triacylglycerol hydrolysis was most efficient in VLDL followed in descending order by IDL, HDL, and LDL. Even with hepatic lipase, phospholipid hydrolysis could not deplete VLDL and IDL of sufficient phospholipid molecules to account for the loss of surface phospholipid that accompanies triacylglycerol hydrolysis and decreasing core volume as LDL is formed (or for conversion of HDL2 to HDL3). Thus, shedding of whole phospholipid molecules, presumably in liposomal-like particles, must be a major mechanism for losing excess surface lipid as large lipoprotein particles are converted to smaller particles. Also, this shedding phenomenon, like phospholipid hydrolysis, is closely related to the hydrolysis of lipoprotein triacylglycerol.     

Refined structure of monomeric diphtheria toxin at 2.3 A resolution.

The structure of toxic monomeric diphtheria toxin (DT) was determined at 2.3 A resolution by molecular replacement based on the domain structures in dimeric DT and refined to an R factor of 20.7%. The model consists of 2 monomers in the asymmetric unit (1,046 amino acid residues), including 2 bound adenylyl 3'-5' uridine 3' monophosphate molecules and 396 water molecules. The structures of the 3 domains are virtually identical in monomeric and dimeric DT; however, monomeric DT is compact and globular as compared to the "open" monomer within dimeric DT (Bennett MJ, Choe S, Eisenberg D, 1994b, Protein Sci 3:0000-0000). Detailed differences between monomeric and dimeric DT are described, particularly (1) changes in main-chain conformations of 8 residues acting as a hinge to "open" or "close" the receptor-binding (R) domain, and (2) a possible receptor-docking site, a beta-hairpin loop protruding from the R domain containing residues that bind the cell-surface DT receptor. Based on the monomeric and dimeric DT crystal structures we have determined and the solution studies of others, we present a 5-step structure-based mechanism of intoxication: (1) proteolysis of a disulfide-linked surface loop (residues 186-201) between the catalytic (C) and transmembrane (T) domains; (2) binding of a beta-hairpin loop protruding from the R domain to the DT receptor, leading to receptor-mediated endocytosis; (3) low pH-triggered open monomer formation and exposure of apolar surfaces in the T domain, which insert into the endosomal membrane; (4) translocation of the C domain into the cytosol; and (5) catalysis by the C domain of ADP-ribosylation of elongation factor 2.     

Sodium in gramicidin: an example of a permion.

The reaction path and free energy profile of Na+ were computed in the interior of the channel protein gramicidin, with the program MOIL. Gramicidin was represented in atomic detail, but surrounding water and lipid molecules were not included. Thus, only short range interactions were investigated. The permeation path of the ion was an irregular spiral, far from a straight line. Permeation cannot be described by motions of a single Na+ ion. The minimal energy path includes significant motion of water and channel atoms as well as motion of the permeating ion. We think of permeation as motion of a permion, a quasi-particle that includes the many body character of the permeation process, comparable with quasi-particles like holes, phonons, and electrons of solid-state physics. Na+ is accompanied by a plug of water molecules, and motions of water, Na+, and the atoms of gramicidin are highly correlated. The permion moves like a linear polymer made of waters and ion linked and moving coherently along a zigzag line, following the reptation mechanism of polymer transport. The effective mass, free energy, and memory kernel (of the integral describing time-dependent friction) of short range interactions were calculated. The effective mass of the permion (properly normalized) is much less than Na+. Friction varies substantially along the path. The free energy profile has two deep minima and several maxima. In certain regions, the dominant motions along the reaction path are those of the channel protein, not the permeating ion: there, ion waits while the other atoms move. At these waiting sites, the permion's motion along the reaction path is a displacement of the atoms of gramicidin that prepare the way for the Na+ ion.     

Retrieval of cryostat section for comparison of histochemistry and quantitative electron microscopy in a muscle fiber.

A method is presented that can be used to perform histochemical and morphometric analyses on the same muscle fiber. Freshly dissected fibers from medial gastrocnemius muscle of adult guinea pig were kept at a resting length and rapidly frozen. Serial frozen cross-sections were cut and reacted for myofibrillar adenosine triphosphatase and succinic dehydrogenase. The adjacent section, while still frozen, was immersed into 20 degrees C glutaraldehyde fixative to which EGTA was added to minimize artifactious contraction. The fixed section was processed for electron microscopy and the section rotated before thin sectioning to give longitudinal sections enabling study of sarcomeres. Ultrastructure was well-preserved despite slight disorganization of the contractile filaments and some vesiculation of the sarcoplasmic reticulum. The Z line width was measured and the mitochondrial volume fraction estimated by point counting morphometry from 89 fibers. The fibers with dark myofibrillar adenosine triphosphatase staining have Z widths of 547 +/- 165 A (n=69) and thoshosphatase staining have Z widths of 547 +/- 165 A (n=69) and those with light stain have 1023 +/- 113 A (n=20). The density of the succinic dehydrogenase reaction product in the fibers was divided into dark and light and the mitochondrial volume fractions were foud to be 4.3 +/- 2.1% (n=52) and 1.0 +/- 1.1% (n=37), respectively.     

The kinetic mechanism of action of an uncoupler of oxidative phosphorylation

Summary The chemiosmotic hypothesis predicts that the mechanism by which weak acids uncouple oxidative phosphorylation in mitochondria is identical to the mechanism by which they transport hydrogen ions across artificial bilayer membranes. We report here the results of a kinetic study of uncoupler-mediated hydrogen ion transport across bilayer membranes. We made electrical relaxation measurements on black lipid membranes exposed to the substituted benzimidazole 5,6-dichloro-2-trifluoromethylbenzimidazole. The simplest model consistent with our experimental data allowed us to deduce values for adsorption coefficients and rate constants. Our major conclusions are that the back diffusion of the neutral species is the rate limiting step for the steady state transport of hydrogen ions, that both the neutral and charged forms of the uncoupler adsorb strongly to the interfaces, and that the reactions at the membrane-solution interfaces occur sufficiently rapidly for equilibrium to be maintained. Independent measurements of the adsorption coefficients of both the neutral and anionic forms of the weak acid and also of the permeability of the membrane to the neutral form agreed well with the values deduced from the kinetic study.     

The role of gene A protein and E. coli rep protein in the replication of phi X 174 replicative form DNA

The gene A protein of bacteriophage phi X 174 initiates replication of super-twisted RFI DNA by cleaving the viral (+) strand at the origin of replication and binding to the 5' end. Upon addition of E. coli rep protein (single-stranded DNA dependent ATPase), E. coli single-stranded DNA binding protein and ATP, complete unwinding of the two strands occurs. Electron microscopic analyses of intermediates in the reaction reveal that the unwinding occurs by movement of the 5' end into the duplex, displacing the viral strand in the form of a single-stranded loop. Since unwinding will not occur in the absence of either gene A protein or rep protein, it is presumed that the rep protein interacts to form a complex with the bound gene A protein. Single-stranded DNA binding protein facilitates the unwinding by binding to the exposed single-stranded DNA. Further addition of the four deoxyribotriphosphates and DNA polymerase III holoenzyme to the reaction results in synthesis of viral (+) single-stranded circles in amounts exceeding that of the input template. A model describing the role of gene A protein and rep protein in duplex DNA replication is presented and other properties of gene A protein discussed.     

Limited proteolysis of glutamine synthetase is inhibited by glutamate and by feedback inhibitors.

Limited proteolysis of glutamine synthetase from Escherichia coli has been studied under nondenaturing conditions (pH 7.6, 20 degrees C). Trypsin cleaves the polypeptide chain of glutamine synthetase into two principal fragments, Mr = about 32,000 and 18,000. The covalently bound AMP group is attached to the larger fragment and its presence does not affect cleavage. Although the cleaved polypeptide chain does not dissociate under nondenaturing conditions, catalytic activity is lost. Chymotrypsin and Staphylococcus aureus protease produce similar cleavages in glutamine synthetase. The substrate L-glutamate retards tryptic as well as chymotryptic digestion. Tryptic digestion is also retarded by some of the feedback inhibitors of glutamine synthetase including CTP, L-alanine, L-serine, L-histidine, and glucosamine 6-phosphate. An implication of these findings is that there is a region of the glutamine synthetase polypeptide chain that is particularly susceptible to proteolysis. Either the glutamate and inhibitor sites are formed partly by this suceptible peptide or the binding of glutamate and some inhibitors induces conformational changes within the E. coli glutamine synthetase molecule in the region of the susceptible peptide.