Evidence for a heparin-induced conformational change on antithrombin III

The effect of heparin on the conformation of antithrombin III (AT-III) was investigated. Solvent perturbation difference spectroscopy shows that the binding of heparin to AT-III results in exposure of two tyrosine residues and a partial burial of a tryptophan residue. The occurrence of a conformational change suggested by this study is also substantiated by circular dichroism (CD) findings in the aromatic and peptide regions. The data in the peptide region show that heparin produces a decrease in the β-structure of AT-III, with a compensatory increase in random coil.     

Chelate effect and cooperativity effect in metal-ligand and macromolecule-ligand equilibria. I. Chemical potential changes and cooperativity-chelation parameters

Chelate and cooperativity effects both in the field of complexes formed in solution by metal ion with ligands and in the field of binding between protein and ligands were examined on the basis of thermodynamic arguments.The analysis was carried out by means of the formation function $\text{n}$ = ? ln Σ_M/? ln[A] where Σ_M is a partition function having free metal or macro-molecule as basis reference level and A is a ligand. The chemical potential changes due to cooperativity and chelation are calculated from differences between areas of the diagram $\text{n}$ = f(ln[A]). The chemical potentials are: Δμ°_γ = -RT ln K_γ (homotropic co-operativity), Δμ°_γ′ = -RT ln K_γ′ (heterotropic co-operativity), Δμ°_? = -RT ln K_? (homotropic chelation), Δμ°_?′ = -RT ln K_?′ (heterotropic chelation). The cooperativity and chelation parameters K_γ, K_γ′, K_?, K_?′ are related to each other by other parameters K_η = K_?·K_γ and K_η′= K_?′·K_γ′. All these dimensionless parameters are derived as ratios of experimental equilibrium constants. Therefore a corresponding consistent chemical potential scale can be obtained from experimental data for all these effects, leading to quantitative comparisons between cooperative and chelate effects, either homotropic or heterotropic.Thermodynamic function changes in metal-ligand complexes can also be compared on this same scale with the energetic changes in protein-ligand complexes.     

The effect of chronic ethanol consumption on temperature-dependent physical properties of liver mitochondrial membranes

We have reported that the monovalent ionophore monensin causes undersulfated chondroitin sulfate biosynthesis in cultured chondrocytes. In order to clarify the mechanism of this diminished sulfation, we have measured the rate of incorporation of sulfate into chondrocytes and assayed the cellular ATP levels. We have also measured sulfatase activity, the incorporation of ³⁵SO₄ into 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate and endogenous sulfotransferase activity in the cell-free extracts. We find that: (1) The incorporation of ³⁵SO₄ into the free sulfate pool in chondrocytes was not inhibited by monensin. (2) The ATP levels of monensin-treated chondrocytes were the same as control cells. (3) There was no sulfatase activity in both control and monensin-treated chondrocytes. (4) Enzymatic analyses revealed that ³⁵SO₄ incorporation into 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate and subsequent sulfotransferase activity were not inhibited in the presence of monensin. At present the most tenable hypothesis to account for monensin causing undersulfated chondroitin sulfate synthesis is that the ionophore impairs the access of proteoglycans to the sulfotransferases in the luminal walls of the Golgi structures.     

The essentiality of sulfhydryl groups to transport in Neurospora crassa.

Unfractionated Escherichia coli B tRNAs have been aminoacylated with selenocysteine by using homologous aminoacyl synthetases. Cochromatography of [³H]cysteyl-tRNA and [⁷⁵Se]selenocysteyl-tRNA on reverse-phase chromatography-5 columns revealed nearly coincident radioactive elution profiles for the two charged tRNAs. Acylation of a mixture of tRNAs with cysteine protected selenocysteine-acceptor activity from inactivation by periodate oxidation. Likewise, preacylation with selenocysteine protected cysteine acceptor from oxidation. Levels of charging with cysteine are reduced about 50% by the presence of a 40-fold excess of selenocysteine. These results indicate that selenocysteine is bound to cysteine-accepting tRNAs, although it does have considerably lower affinity for the ligase than cysteine. The ester linkage of selenocysteyl-tRNA was shown to be somewhat more stable than that of cysteyl-tRNA under the same conditions. These experiments show that selenocysteine can participate in the early steps leading to peptide-bond formation and provide a possible pathway for selenocysteine incorporation into protein.     

Integration of the plasmid prophages P1 and P7 into the chromosome of Escherichia coli.

The prophages of the related temperate bacteriophages P1 and P7, which normally exist as plasmids, suppress Escherichia coli dnaA (ts) mutants by integrating into the host chromosome. The locations of the sites on the prophage used for integrative recombination were identified by restriction nuclease analysis and DNA-DNA hybridization techniques. The integration of P1 and P7 often involves a specific site on the host DNA and a specific site on the phage DNA; the latter is probably the end of the phage genetic map. When this site is utilized, the host Rec⁺ function is not required. In Rec⁺ strains, P1 and P7 may also recombine with homologous regions on the host chromosome; at least one of these regions is an IS1 element. In some integration events, prophage deletions are observed which are often associated with inverted repeat structures on the phage DNA. Thus, P1 and P7 may employ one of several different mechanisms for integration.     

Leakage of sucrose from phosphatidylcholine liposomes induced by interaction with serum albumin

Liposomes composed of rat-liver phosphatidylcholine rapidly lose entrapped sucrose when incubated in presence of blood or of solutions of bovine serum albumin. The phenomenon can not be ascribed to phospholipase A activity, since no such activity towards phosphatidylcholine substrates could be detected in various albumin preparations. Upon gel filtration on Sepharose 4B or Sephadex G-100 of incubated mixtures of radioactive liposomes and albumin, association of phosphatidylcholine with the albumin could be demonstrated. No measurable quantities of protein were found associated with liposomes. The albumin-associated phosphatidylcholine is hydrolyzed by pancreatic phospholipase A more slowly than free liposomal phosphatidylcholine, indicating a non-lamellar orientation of the associated phospholipid. The binding of phosphatidylcholine to albumin proceeds at a slow rate: increase of the amount of phosphatidylcholine bound continues over a period of several hours reaching a maximum at approx. 1 mol of phosphatidylcholine per mol of albumin. The process is reversible as indicated by transfer of albumin-associated radioactive phosphatidylcholine to unlabeled liposomes. The association between albumin and phosphatidylcholine is believed to be of the same type as described recently by Jonas) Jonas, A. (1976) Biochim. Biophys. Acta 427, 325–336)). The consequences of these observations are discussed with respect to the use of liposomes as carriers to introduce substance into cells.     

A kinetic model for bacterial transfection: the lambda DNA system.

A kinetic model describing the binding and uptake of free lambda phage DNA by the bacterium Escherichia coli is presented. The model is based on the assumption that adsorbed ‘helper’ phage particles serve as functional sites to which the lambda DNA specifically binds. When applied to experimental data, the model describes the reaction between cells and DNA as a rapid binding of DNA to helper phage attachment sites, followed by a slow, irreversible incorporation of bound DNA into the cells. Features of the model include a time-dependent exponential decay of functional sites required for DNA uptake and a minimum time for irreversibly bound DNA to enter the cell. We suggest that this model may be useful in studying processes involved in the active transport of DNA across a permeability barrier.     

Studies on Turbatrix aceti beta-N-acetylglucosaminidase. 2. Kinetic studies on the active site

The purified beta-N-acetylglucosaminidase isolated from Turbatrix aceti hydrolyzes both p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides. The enzyme had Km values of 0.28 and 0.23 mM, Vmax values of 104 and 69 mumol min-1 mg protein-1, and activation energies of 11.7 and 9.9 kcal/mol for the two substrates, respectively. Several lines of experimental evidence show that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same molecule at a single catalytic site. Substrate analogs were synthesized in which the acetamido group of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and galactopyranoside, and their 1-thio analogs was modified by replacement of the amido-carbonyl oxygen with sulfur. These substrate analogs competitively inhibited both enzymatic activities. Analysis of the inhibition data indicates that a single catalytic site of the enzyme is responsible for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities. Competition kinetics between the two substrates further confirm the presence of a single active site for both activities. The pH dependence of the hydrolysis of p-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and beta-D-galactopyranosides has been determined. pKe1 and pKe2 values of 4.7 and 5.2, determined from the dependence of log Vmax/Km on pH, suggest that two carboxyl groups are involved in the reaction mechanism. The heats of ionization of the groups further confirm the above results.     

Studies on base-exchange reactions of phospholipids in rat brain particles and a "solubilized" system.

A filtration-method on Millipore-membranes for the assay of the base-exchange reaction was described. Its advantage over the usual procedure based upon the extraction and the washing of lipids was discussed with the viewpoint of processing many samples, which would be indispensable for purifying the enzyme.The reaction showed an absolute dependency for calcium ion with different optimal concentrations for each of the three bases, a sensitivity to inhibition by high ionic strength, and a pH optimum around 9.0. Exogenously added phospholipid, asolectin, gave a slight stimulation for ethanolamine and l-serine incorporation at a low concentration while choline incorporation was essentially inhibited at all concentrations examined. In heat-denaturation experiments with the particulate and soluble the incorporation of choline into lipid was more sensitive than that of ethanolamine and l-serine. A developmental study showed that brain particles sedimenting between 10,000 and 35,000g prepared from rats aged 22–27 days readily incorporated ethanolamine, l-serine, and choline into their corresponding phosphatidyl compound.Several procedures for solubilization of the “base-exchange” enzyme were examined. The most effectively solubilized preparation was obtained by the use of an ionically balanced detergent, Miranol H2M. This preparation showed a marked dependency on exogenously added phospholipids for its maximal enzymic activity, had a pH optimum at around 7.2, and had an absolute requirement for Ca²⁺. This particular detergent at a concentration of 1% ($\text{w}\text{v}$) solubilized approximately 50% of the protein, and about 30% of the phospholipids, 40% of the cerebrosides, and only 11% of the cholesterol originally present in the particles. The relative proportions of different phospholipids solubilized by the detergent were, however, similar to those present in the original particles.The base-exchange reaction catalyzed by the solubilized enzyme was found to be highly sensitive to ionic strength, and the inhibitory effect of a specific monovalent cation paralleled its ionic size. Substantial differences in the K_m value for each of the substrates with only slight differences in V were observed.The choice of solubilizing agents in relation to these properties and to the maintenance of the activity of the base-exchange reaction was discussed.     

Organic derivatives of aluminium. Part I. Reactions of alkanolamines with aluminium isopropoxide

Reactions of alkanolamines [R₁R₂NXOH; R₁ = H, CH₃, C₂H₅; R₂ = H, CH₃, C₂H₅ and X = -CH₂CH₂-, -CH₂CH₂CH₂-, -CH₂CHCH₃, -C₆H₄CH₂CH₂-] with aluminium isopropoxide in different molar ratios (1 to 3) yield compounds of the type Al(OPrⁱ)_3?n(OXNR₁R₂)_n, where ‘n’ can be 1, 2 and 3. Most of the derivatives are distillable liquids, soluble in common organic solvents and susceptible to hydrolysis even by atmospheric moisture. The new derivatives are characterized by elemental analysis, IR and ¹H NMR spectra. Molecular weight measurements of Al(OPrⁱ)_3?n(OXNR₁R₂)_n reveal them to be tetrameric in nature.     

Biosynthesis of patulin. Dehydrogenase and dioxygenase enzymes of penicillium patulum

Two aromatic dehydrogenases catalyzing the reversible conversions of gentisyl alcohol and m-hydroxybenzyl alcohol to their corresponding aldehydes have been partially purified. These partially purified dehydrogenases were shown to require NADPH. In the case of the gentisyl alcohol-gentisaldehyde interconversion, a 46-fold purification was achieved using POLYCLAR AT and DEAE-cellulose chromatography.A cell-free system capable of converting gentisaldehyde to patulin was prepared with a pH optimum of 8.0. The system was dependent on O₂ and NADPH, was stimulated by ATP and inhibited by the Fe²⁺ chelators, α, α-dipyridyl and o-phenanthroline. These results suggest a dioxygenase mechanism for patulin synthesis from gentisaldehyde.     

Biosynthesis of polyketides. Purification and inhibition studies of 6-methylsalicylic acid synthase

6-MSA³ synthase has been purified 190-fold with 33% yield. The purification was found to be dependent on the presence of glycerol. The acetylenic inhibitors 3-pentynoyl- and 2-hexynoyl-NAC completely inhibit 6-MSA production at concentrations in which fatty acid synthesis, TAL production as well as NADPH oxidation are only partially affected. These results confirm earlier studies on the specificity of inhibition by acetylenic inhibitors and support a mechanism wherein the NADPH-mediated reduction step occurs on a 6-carbon rather than on an 8-carbon intermediate.     

Author index for volume 171

Kinetic analyses of the irreversible inhibition of l-tyrosine and l-phenylalanine transport in Bacillus subtilis by phenylalanine chloromethyl ketone revealed that the inhibition was due to an affinity labeling process. Phenylalanine chloromethyl ketone is a competetive inhibitor of l-tyrosine and l-phenylalanine transport. The K_i values for irreversible inhibition of l-tyrosine and l-phenylalanine transport were 194 and 177 μm, respectively, and the first order rate constants for the alkylation reaction leading to inactivation of transport of l-tyrosine and l-phenylalanine were 0.016 and 0.012 min^?1, respectively. The similarity of these constants are consistent with the involvement of the same functional site for l-phenylalanine and l-tyrosine transport. A second effect of phenylalanine chloromethyl ketone was inhibition of the uptake of neutral, aliphatic amino acids; transport of basic and acidic amino acids was unaffected by it. Since high concentrations of any amino acid did not reduce the inhibitory effects of phenylalanine chloromethyl ketone on transport of neutral, aliphatic amino acids, an independent effect, not due to an affinity labeling process was inferred. A procedure for selective labeling of the l-tyrosine/l-phenylalanine transport system was demonstrated that should be applicable to the introduction of a radioactive label into the transport protein(s).     

Calcium inhibition of adenylate cyclase: Studies in turkey erythrocyte and S49 CYC− cell membranes

We studied the mechanism of calcium inhibition of adenylate cyclase using partially purified components of the enzyme complex and computer analysis of free metal and substrate concentrations. A sigmoidal relationship was observed between percentage maximal adenylate cyclase activity with 1-isoproterenol/guanylyl-β,γ-imidodiphosphate and the calculated free calcium. Fifty percent inhibition occurred at 2.5 × 10^?4m free calcium. This inhibition appeared to be independent of calmodulin. Calcium inhibited the holocatalytic enzyme in a manner indentical to that of the native enzyme, but did not affect the ability of 1-isoproterenol and guanylyl-β,γ-imidodiphosphate to promote the formation of the holocatalytic state. There was no effect of calcium on the conformation of the activated G unit nor on the holocatalytic enzyme as determined by sedimentation velocity analysis. Calcium did not cause detectable dissociation of the activated G unit from the catalytic unit, nor convert activated G unit to an inactive form. Calcium inhibition of the catalytic unit of adenylate cyclase was studied in S49 CYC^? lymphoma cell membranes. High concentrations of calcium inhibited manganese-stimulated CYC^? enzyme, but this could be explained by competition between calcium and manganese for ATP. With addition of forskolin, CYC^? adenylate cyclase utilized MgATP^2? as substrate and was shown to have a separate binding site for free magnesium. Calcium inhibited forskolin-stimulated CYC^? enzyme by competing with free magnesium for its regulatory site. Calcium inhibition was noncompetitive with respect to MgATP^2?. We conclude that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit.     

Immunological reconstitution of T-cell-deprived mice. I. Inability of thymosin to restore spleen cell mitogen and tumor allograft response.

Ultraviolet light (uv)-induced tumors appear to possess unique and common determinants, both of which can function as rejection antigens in appropriate in vivo tumor transplantation assays. We postulate that the common tumor antigens can play an important role in anti-tumor immunity. We have previously reported on the phenomenon of ultraviolet light generated suppressor lymphocytes which mediate susceptibility to transplanted uv-induced murine skin tumors. We now report that these uv generated suppressor cells are capable of specifically inhibiting effector responses directed against the common tumor antigens. A hypothesis is presented to explain how the presence of suppressor cells might account for the observed differences between in vivo and in vitro assays of tumor immunity.     

Cholesterol-lipid interactions: An infrared and raman spectroscopic study of the carbonyl stretching mode region of 1,2-dipalmitoyl phosphatidylcholine bilayers

Infrared and Raman spectra were obtained for the 1690–1770 cm^?1 carbonyl stretching mode region for 1,2-dipalmitoyl phosphatidylcholine (DPPC) bilayers in the anhydrous, partially hydrated and completely hydrated states. Spectral features at approx. 1740 and 1721 cm^?1 are assigned to CO stretching modes associated with the 1- and 2-chain carbonyl groups, respectively. Splittings of the primary transitions at 1743, 1738, ～1731 and ～1721 cm^?1 are attributed to rotational isomers involving the entire chain. Hydrogen bond formation between the fatty acid carbonyl and 3βOH cholesterol groups was investigated for anhydrous DPPC bilayers. Examination of frequencies, intensities and half-widths of the carbonyl bands indicates that no hydrogen bonding occurs at either of the two carbonyl sites. However, the addition of cholesterol to completely hydrated DPPC dispersions reduces the conformational inequivalence between the two fatty acid carbonyl groups by specifically perturbing the 2-chain. For cholesterol containing systems the carbonyl stretching mode transitions were also used to monitor lattice effects within the interface region as water binds to the bilayer head groups. Specifically, the addition of approx. 2 molecules of water per lipid molecule orders the lipid lattice and increases the bilayer packing density, while the subsequent addition of 4 molecules of water per lipid molecule releases the packing constraints within the interface region and thereby decreases the packing density.     

Coenzyme B12-dependent diol dehydrase: purification, subunit heterogeneity, and reversible association.

A purification procedure for diol dehydrase (dl-1,2-propanediol hydro-lyase, EC 4.2.1.28) of Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 has been developed which gives the highest specific activity for this enzyme obtained so far. The purified enzyme is homogeneous by the criteria of ultracentrifugation (s_20,w = 8.9 S) and disc gel electrophoresis in the presence of substrate. The molecular weight of approximately 230,000 was obtained by gel filtration and ultracentrifugal sedimentation equilibrium. The enzyme is composed of components F and S whose molecular weights were determined to be approximately 26,000 and 200,000, respectively, by gel filtration. The incubation of both components F and S with the substrate leads to complete reassociation of the components. Disc gel electrophoresis in the presence of sodium dodecyl sulfate and terminal amino acid analyses indicate that component S consists of at least four nonidentical subunits. The reversible association and heterogeneity of the subunits were also demonstrated with the crude enzyme by immunoelectrophoresis.     

Preparation of highly purified 3 alpha- and 3 beta-hydroxysteroid dehydrogenases from Pseudomonas sp.

A method is described for preparing highly purified 3α- and 3β-hydroxysteroid dehydrogenases (EC 1.1.1.50 and EC 1.1.1.145, respectively), essentially uncontaminated with one another, from extracts of a steroid-induced Pseudomonas species. These enzymes are suitable for the microanalysis of 3α-hydroxy-, 3β-hydroxy-, and 3-ketosteroids.     

Preparation and properties of hydrated uranium(III) complex chlorides. Part I. Uranium trichloride heptahydrate

The synthesis of uranium trichloride heptahydrate together with some of its structural, spectroscopic and magnetic properties is reported. The compound possesses the triclinic lattice of LaCl₃·7H₂O (space group P$\text{1}$). Controlled vacuum thermal dehydration of the substance enabled the preparation of the anhydrous trichloride in gram quantities. Magnetic susceptibilities of polycrystalline samples were measured by the Faraday method in the 6.5–295 K range. The uranium trichloride heptahydrate follows in this region the Curie-Weiss law with C = 1.0839 emu K mol^?1 and θ = ?32.7 K.