The mechanism of action of 4-hydroxynonenal in cell injury

The effect of the C9 ketoaldehyde, 4-hydroxynonenal (HNE), a cytotoxic product of lipid peroxidation, on DNA, RNA and protein synthesis has been investigated in cells in culture. Macromolecular synthesis is powerfully inhibited by this agent which readily enters the lipid-rich membranes and is considerably more toxic than the polar ketoaldehyde, methyl glyoxal (MG). The entry of HNE into membranes lowers their glutathione GSH content. This is associated with an increased lipid peroxidation measured in vitro which is blocked by added GSH or alpha-tocopherol. It is proposed that this latter sequence of events is the underlying cause of the cytopathic effect of HNE in cells in culture.     

Lipid/myelin basic protein multilayers. A model for the cytoplasmic space in central nervous system myelin

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins . The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic protein and lipids. Cholesterol may enhance binding by opening gaps between diacyl-lipid headgroups.     

Human testis-specific histone TH2B: Fractionation and peptide mapping

The presence of the nonionic detergent Triton X-100 was shown to improve the resolution of the human TH2B on gel electrophoresis and on gel filtration. Total histones of human testis, including TH2B, were resolved by electrophoresis in 15% polyacrylamide gels containing 0.4% Triton X-100, 1.5 m urea, and 0.9 n acetic acid. Gel filtration on Bio-Gel P-200 in 0.4% Triton X-100, 5.0 m urea, and 0.01 n HCl permitted the purification of human TH2B from human testis and sperm in preparative amounts. The structure of human TH2B so prepared was compared to that of rat TH2B, human H2B, and rat H2B by tryptic peptide mapping. The results showed some similarities between all four proteins, but closer similarity was observed within the germ cell histone (TH2B) group and within the somatic histone (H2B) group than between histones of the same species. In addition, human TH2B and rat TH2B each contained one unique peptide absent from other histones.     

Thymosin beta arg10, a major variant of thymosin beta 10 in rabbit tissues

Two homologous peptides, designated thymosin beta 4 and thymosin beta 10, respectively, have been shown to be widely distributed in mammalian cells and tissues (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker (1983) Arch. Biochem. Biophys. 221, 570-576; S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B.L. Horecker, (1983) Arch. Biochem. Biophys. 225, 407-413). In the rabbit, thymosin beta 4 is replaced by a variant, thymosin beta ala4, that contains alanine in place of serine at the blocked NH2-terminus. It is reported that in rabbit tissues thymosin beta 10 is also replaced by a variant, designated thymosin beta arg10, that contains an additional amino acid, arginine, inserted following lysine-38. The rabbit tissues analyzed also differ from those of other mammals in the relative quantities of thymosin beta ala4 and beta arg10, which are nearly equal, compared to tissues from other mammals where the quantities of thymosin beta 10 are only one-third to one-tenth those of thymosin beta 4.     

The inhibition of acetylcholinesterase by arsenite and fluoride

The effect of fluoride on the rate of reaction of acetylcholinesterase with arsenite, on the rate of dissociation of the enzyme-arsenite complex, and on the equilibrium between enzyme and arsenite was studied. Fluoride decreases the rate of the reaction between acetylcholinesterase and arsenite and changes the apparent equilibrium dissociation constant between the enzyme and arsenite, but even at concentrations as high as 0.2 M has no effect on the rate of dissociation of the enzyme-arsenite complex. The binding of fluoride and arsenite with the enzyme is highly anticooperative and may well be mutually exclusive. These results are consistent with a model in which the binding sites overlap and in which the same functional groups are involved.     

Interaction of nitrogenase with nucleotide analogs of ATP and ADP and the effect of metal ions on ADP inhibition

The interaction of a large number of ATP and ADP analogs with nitrogenase from Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianum has been examined. Only 1,N6-etheno-ATP and 2'-deoxy-ATP served as substrates for acetylene reduction. Other triphosphates including GTP, ITP, 8-Br-ATP, alpha,beta-methylene ATP, beta,gamma-methylene ATP, 6-chloropurine riboside triphosphate, and AMP-PNP were inert, showing less than 50% inhibition at levels up to two- to fivefold greater than ATP. Xanthosine triphosphate behaved simply as a chelator of magnesium, activating the enzyme at low levels but strongly inhibiting at high levels. When nucleotide diphosphates were tested as inhibitors with enzyme from A. vinelandii, GDP, dGDP, and 6-chloropurine riboside diphosphate were ineffective, XDP was three- to fivefold less effective, and dADP and 1,N6-etheno-ADP were about equally as effective as ADP. With enzyme from C. pasteurianum, dADP was twofold less effective than ADP, XDP was fivefold less effective, and IDP and 1,N6-etheno-ADP appeared to be ineffective. Results with enzyme from K. pneumoniae were very similar to those obtained with A. vinelandii. Different metal ions were tested in the presence of both ATP and ADP to determine whether preferential binding to one nucleotide or the other might alter the ADP/ATP ratio needed for 50% inhibition of activity. Magnesium and manganese gave the same ratio, while with Fe and Co, slightly less ADP was required for equivalent inhibition. Nickel appeared to reduce the sensitivity of A. vinelandii nitrogenase to ADP inhibition while increasing that of C. pasteurianum, but both effects were less than twofold. Calcium, strontium, and aluminum ions were inert with enzymes from these organisms. Cd and Zn were also ineffective with K. pneumoniae. Two isomers of ATP beta S were prepared by enzymatic synthesis from ADP beta S. The A form was a more potent inhibitor of A. vinelandii nitrogenase.     

Relationship between the cytoplasm and the vacuole phosphate pool in Acer pseudoplatanus cells

The Pi concentration of Acer pseudoplatanus cells in the two major intracellular compartments, the cytoplasm and the vacuole, has been studied using 31P NMR. For sycamore cells containing approximately 2 mM of total Pi, the cytoplasmic Pi and the vacuolar Pi concentrations were approximately 6 and 1.5 mM, respectively. When the cells were transferred to a phosphate-deficient medium, the vacuolar Pi decreased rapidly while the cytoplasmic Pi decreased slowly during the first 48 h, indicating that Pi in the cytoplasm was maintained at the expense of the vacuolar Pi. When the Pi-starved cells (i.e., those containing less than 0.5 mumol of total Pi/g wet wt) were transferred to a medium containing 300 microM Pi, Pi entered the cells rapidly and accumulated in the cytoplasm. Once the cytoplasmic Pi pool was filled, Pi was taken up in the vacuole until the vacuole Pi pool was filled. On the contrary when the non-Pi-starved cells were transferred to a phosphate-rich medium (i.e., containing 45 mM Pi), Pi entered the cells slowly by diffusion and accumulated in the vacuole but not in the cytoplasm. These results demonstrate that the Pi content of the cytoplasm is maintained at the expense of the vacuolar Pi pool when sycamore cells are transferred to either a phosphate-deficient or a phosphate-rich medium.     

Inhibition of sugar uptake by ascorbic acid in Escherichia coli

The uptake of glucose by the glucose phosphotransferase system in Escherichia coli was inhibited greater than 90% by ascorbate. The uptake of the nonmetabolizable analog of glucose, methyl-alpha-glucoside, was also inhibited to the same extent, confirming that it was the transport process that was sensitive to ascorbate. Similarly, it was the transport function of mannose phosphotransferase for which mannose and nonmetabolizable 2-deoxyglucose were substrates that was partially inhibited by ascorbate. Other phosphotransferase systems, including those for the uptake of sorbitol, fructose and N-acetylglucosamine, but not mannitol, were also inhibited to varying degrees by ascorbate. The inhibitory effect on the phosphotransferase systems was reversible, required the active oxidation of ascorbate, was sensitive to the presence of free-radical scavengers, and was insensitive to uncouplers. Because ascorbate was not taken up by E. coli, it was concluded that the active inhibitory species was the ascorbate free radical and that it was interacting reversibly with a membrane component, possibly the different enzyme IIB components of the phosphotransferase systems. Ascorbate also inhibited other transport systems causing a slight reduction in the passive diffusion of glycerol, a 50% inhibition of the shock-sensitive uptake of maltose, and a complete inhibition of the proton-symport uptake of lactose. Radical scavengers had little or no effect on the inhibition of these systems.     

Effect of Mn2+ on fructose 2,6-bisphosphate inhibition of mouse liver, intestinal, and muscle fructose-1,6-bisphosphatases

Fructose 2,6-bisphosphate inhibited all three fructose-1,6-bisphosphatases from the liver, intestine, and muscle of the mouse. The sensitivity of the liver enzyme to the inhibitor was significantly diminished when Mg2+ was replaced by Mn2+ as the activating cation. Inhibition of the liver enzyme by fructose 2,6-bisphosphate decreased as the concentration of the metal activator, Mn2+ or Mg2+, increased. The respective I50 values obtained by extrapolation of metal ion concentrations to zero were 40 microM with Mn2+ and 0.25 microM with Mg2+. The extent of desensitization to either fructose 2,6-bisphosphate or AMP inhibition by Mn2+ decreased in the order of the liver, intestine, and muscle enzyme. Only in the case of the liver enzyme was the substrate cooperativity induced by fructose 2,6-bisphosphate in the presence of Mg2+. In all three isoenzymes from the mouse, fructose 2,6-bisphosphate greatly potentiated the AMP inhibition of the enzyme in the presence of either Mg2+ or Mn2+. The liver enzyme with Mn2+ in addition to Mg2+ was still active in the presence of less than 1 microM fructose 2,6-bisphosphate, even though AMP was present at 100-200 microM.     

Unusual heterogeneity in the glycosylation of the G protein of the hazelhurst strain of vesicular stomatitis virus

The asparagine-linked oligosaccharides of the G protein of the Hazelhurst subtype of the New Jersey serotype of vesicular stomatitis virus (VSV) have been compared with the oligosaccharides from the G protein of the well-characterized Indiana serotype of VSV, with baby hamster kidney cells in monolayer culture as the host for both viruses. [3H]Glucosamine- and [3H]mannose-labeled glycopeptides from the G protein of purified virus were analyzed by the combined techniques of endo-beta-N-acetylglucosaminidase H (ENDO-H) digestion, concanavalin A and lentil lectin affinity chromatography, and Bio-Gel P-4 chromatography. Although almost all of the Indiana G protein oligosaccharides were acidic-type structures, as expected from previous studies; the Hazelhurst G protein contained a mixture of acidic-type, hybrid-type containing sialic acid, and neutral-type (predominantly Man5-6GlcNAc2-Asn) structures. The vast majority of acidic-type oligosaccharides from both the Hazelhurst and Indiana G proteins were diantennary structures, with less than half containing fucose linked to the innermost N-acetylglucosamine. Additional analysis of the Hazelhurst G protein by ENDO-H digestion and gel electrophoresis suggested that some of the mature G polypeptides contained acidic-type structures at both glycosylation sites, whereas the remainder contained an ENDO-H-resistant, acidic-type structure at one site and an ENDO-H-sensitive, hybrid- or neutral-type structure at the other site.