Putative superoxide dismutase activity of iron-EDTA: a reexamination

It has been reported that iron-EDTA complexes mimic the action of superoxide dismutase, displaying 0.01% of the activity of the enzyme (Halliwell, B., 1975, FEBS Lett., 56, 34–38). This was purportedly directly confirmed by J. G. McClune, J. A. Fee, G. A. McCluskey, and J. T. Groves, 1977, J. Amer. Chem. Soc., 99, 5220–5222. A reexamination of the behavior of this compound has demonstrated that it does not catalyze the dismutation of O₂^?, but rather inferferes with assays for superoxide dismutation activity, which are based on the reductions of nitroblue tetrazolium or of cytochrome c. The sources of this interference have been examined. Investigators engaged in searching for mimics of superoxide dismutase are urged to be wary of similar artifacts.     

Carnosine binding: characterization of steric and charge requirements for ligand recognition.

The steric and charge requirements for binding of l-carnosine (β-alanyl-l-histidine) by bovine serum albumin were investigated with proton magnetic resonance (¹HMR) spectrometry. The histidinyl side chain of the dipeptide is responsible for primary recognition by the binding site. Furthermore, recognition is specific to a particular orientation of the histidinyl side chain that is determined by the other amino acid residue of the dipeptide. It was found that, although salts do not have a great effect on the binding of carnosine to bovine serum albumin, this binding cannot be measured by equilibrium dialysis in the presence of salt because of formation of a complex Donnan equilibrium. Carnosine, which has been postulated to have a role in olfaction, binds to the crude particulate fraction of nasal olfactory epithelium in the same steric orientation as it does to bovine serum albumin. Therefore, we have used the binding of carnosine to bovine serum albumin as a model system to test potential competitive inhibitors of carnosine binding that ultimately could be tested for activity in the olfactory pathway. It was found that the binding of carnosine to bovine serum albumin is a good model of nonspecific binding of carnosine to tissue preparations but not of the specific binding of carnosine to the nasal olfactory epithelium. In addition to requiring the proper conformation of the histidinyl residue, the binding to olfactory epithelium also appears to require recognition of the β-alanyl residue and of substituents on the imidazole ring. Evidence is provided that the carnosine binding by the nasal olfactory epithelium demonstrated by ¹HMR spectroscopy does not occur with the mature olfactory receptor neurons.     

Kinetics and mechanism of oxidation of some aldoses by vanadium(v) in perchloric acid medium

The kinetics of oxidation of some aldoses by vanadium(V) in perchloric acid media have been investigated. Each reaction is first order with respect to both [Vanadium(V)] and [Aldose]. The reactions are catalysed by acid. The addition of sodium perchlorate accelerates the rate of reaction. Kinetic evidence for the formation of an intermediate compound between vanadium(V) and aldoses is insignificant, and a mechanism is suggested in which vanadium(V) reacts with the aldoses by a fast step to form a transition state, followed by the decomposition of the latter to give the products of reaction in a slow step. The formation of free-radical intermediates has been demonstrated, and one-electron reduction of vanadium(V) by aldoses seems to be the most plausible mechanism. The oxidation rates follow the order: xyloses arabinose galactose mannose. The activation parameters are reported.     

The regulatory effect of macrophages on the antigen-induced lymph node cell proliferative response

Employing an antigen-induced T cell-dependent lymph node cell (LNC) proliferative assay in the mouse we observed differences in the capacity of unstimulated and thioglycollate-activated peritoneal exudate cells (PECs) to present antigen. Antigen-primed LNCs can be induced to proliferate by a brief (2 hr) antigen exposure and the addition of various numbers of thioglycollate PECs (thio-PECs) modifies the proliferative response depending on the ratio of $\text{PEC}\text{LNC}$ in cultures. With ratios of 3–12% both DNA and protein synthesis were enhanced, but at ratios greater than 12% suppression was significant. Treatment of thio-PECs with mitomycin C, irradiation (3000 rad), anti-Thy 1.2, or anti-Ia plus complement did not alter suppression, suggesting the possibility that the Ia negative macrophages present in the PECs were involved in the suppression. An enhancing effect on the proliferative response was noted following the addition of small numbers of thio-PECs. This was comparable to that seen with an equivalent concentration of supernatant from thio-PECs suggesting that soluble factors play an important role. Two enhancing fractions (separated on a G-75 column) which were themselves mitogenic were identified which eluted with approximate molecular weights of 15,000 and 60,000.     

Cetyltrimethylammonium bromide polyacrylamide gel electrophoresis: estimation of protein subunit molecular weights using cationic detergents.

Polyacrylamide gel electrophoresis in the presence of the cationic detergent, cetyltrimethylammonium bromide (CTAB), has been previously used to obtain more accurate estimates of the molecular weight of certain highly charged and membrane protein subunits that exhibit anomalous electrophoretic behavior in the presence of sodium dodecyl sulfate (SDS). The improved method reported herein is comparable to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) method in simplicity, time, and quality of gels, but the CTAB-PAGE method appears to have a wider range of application for diverse types of proteins. The technique may also be used for verification of molecular weight data and thus detection of possible anomalous results obtained using the anionic SDS-PAGE method. The described method eliminates the precipitates formed between ammonium persulfate and cationic detergents during gel polymerization and between cationic detergents and the protein dyes during staining that have complicated previous methods. The reliability of the technique is indicated by the high correlation coefficient (?0.97) between R_f and molecular weight. Data are presented to indicate that the method can be used to estimate the subunit molecular weight of unknown proteins with a 95% level of confidence.     

Preparation and characterization of met apo hemocyanin: a single copper (II) active site.

A new derivative of $\text{Busycon canaliculatum}$ hemocyanin has been prepared for which one copper has been removed from the binuclear active site of the holoprotein and the remaining copper has been oxidized with a variety of small molecule oxidizing agents. This met apo derivative [( )…Cu(II)] binds a number of ligands; EPR spectra of several forms are reported and compared to those obtained for a singly oxidized (half met-L) derivative [Cu(I)…Cu(II)L]. The site of the oxidized copper for both forms is found to be quite similar in structure but shows large differences in ligand binding ability.     

Human epidermal transglutaminase: stimulation by trypsin, organic solvents, and chaotropic salts.

The activity of pure human epidermal transglutaminase was enhanced 3- to 10-fold by various treatments. Incubation with trypsin caused a time-dependent enhancement in activity, up to 3 times the initial activity, with no apparent change in electrophoretic mobility as detected by disc and sodium dodecyl sulfate electrophoresis, and with no apparent change in immunological properties. This enhancement was specific for trypsin among the several enzymes tested. Preincubation of transglutaminase with 0.1 to 2.0 m potassium thiocyanate or potassium iodide, and with 10 to 50% solutions of alcohols and other organic solvents caused a time-dependent enhancement of activity up to 10-fold over control. The presence of calcium was required for the observed enhancement. Kinetic studies suggest that the K_m values of the substrates putrescine and casein determined for the native enzyme are similar to those for the stimulated forms of the enzyme. These in vitro methods of altering enzyme activity may be indications of potential in vivo controls of transglutaminase activity.     

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.     

A substrate for the fluorogenic assay of exo-beta-N-acetylmuramidase: synthesis and purification of 4-methylumbelliferyl N-acetylmuramide.

A fluorogenic substrate for exo-β-N-acetylmuramidase from Bacillus subtilis B was synthesized. 4-Methyl-2-oxo-1,2-benzopyran-7-yl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranoside was prepared from 4-methyl-2-oxo-1,2-benzopyran-7-yl 2-acetamido-2-deoxy-β-d-glucopyranoside, condensed with dl-2-chloropropionic acid, the benzylidene residue removed by acetolysis and the 4-methyl-2-oxo-1,2-benzopyran-7-yl 2-amino-3-O-(d-1-carboxyethyl)-2-deoxy-β-d-glucopyranoside purified by chromatography on silica gel and Sephadex G-10 and by high-voltage paper electrophoresis. The identity of the product was confirmed by pmr studies, acid hydrolysis followed by chromatography of the products, and enzymic digestion.     

Isolation and characterization of a tryptic fragment containing the thioesterase segment of fatty acid synthetase from the uropygial gland of goose.

Trypsin treatment of purified fatty acid synthetase from the uropygial gland of goose released a 33,000 molecular weight peptide from the 270,000 molecular weight synthease. A combination of ammonium sulfate precipitation, Sephadex G-100 gel filtration, anion-exchange chromatography with QAE-Sephadex, and cation-exchange chromatography with cellulose phosphate gave rise to the first homogeneous preparation of the 33,000 molecular weight fragment containing fatty acyl-CoA thioesterase activity. Amino acid composition of this peptide was quite similar to that of the intact fatty acid synthetase except for a lower valine content; a partial specific volume of 0.734 was calculated for the thioesterase fragment. The pH optimum for the thioesterase was near 7.5 and the enzyme showed a high degree of preference for CoA esters of fatty acids with 16 or more carbon atoms. Palmitoyl-CoA inhibited the enzyme and therefore the rate of hydrolysis was not proportional to the amount of protein at low concentrations. Inclusion of bovine serum albumin in the reaction mixture prevented this inhibition. Disregarding the substrate inhibition, an apparent K_m of 5 × 10^?5m and a V of 340 nmol/min/mg were calculated. The thioesterase was inhibited by active serine-directed reagents such as phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate as well as by SH-directed reagents as p-chloromercuribenzoate and N-ethylmaleimide. The isolated thioesterase fragment generated antibodies in rabbits and the antithioesterase inhibited the enzymatic activity of fatty acid synthetase. The antithioesterase showed immunoprecipitant lines with fatty acid synthetase from the uropygial gland and the synthetase from the liver of goose. Anti-fatty acid synthetase prepared against the enzyme from the gland cross-reacted with the thioesterase segment. Even though the synthetase from the uropygial gland synthesizes multimethyl-branched fatty acids in vivo, the thioesterase segment of this synthetase appears to be quite similar to that isolated from the rat.     

Reduction kinetics of bacterial cytochromes c2.

In a continuing effort to understand the mechanism of electron transfer by c-type cytochromes we have extended our investigations of the oxidation and reduction of Rhodospirillum rubrum cytochrome c₂. We have utilized the oxidant, oxidized azurin, and the reductants SO₂^?, S₂O₄^2?, sodium ascorbate, and reduced azurin. The results of these studies demonstrate that, as found previously with the iron hexacyanides, electron transfer apparently takes place at the exposed heme edge. Furthermore, we report studies on the reduction of ferricytochrome c₂ from Rhodopseudomonas sphaeroides, Rhodopseudomonas capsulata, Rhodomicrobium vannielii, and Rhodopseudomonas palustris by potassium ferrocyanide. Based on the amino acid sequence homology between the various cytochromes c₂ and presumed structural homology, the observed rates of electron transport are analyzed in terms of the structure in the region of the exposed heme edge.     

alpha-linolenic acid biosynthesis in Cyanidium caldarium.

Although α-linolenic acid is nearly absent from Cyanidium caldarium cultured at 53 °C, it is the most abundant unsaturated fatty acid in 20 °C-grown cells. A sudden growth temperature shift of 55 to 25 °C does not stimulate the immediate biosynthesis of α-linolenic acid. However, after an induction period of 48 h, synthesis of α-linolenic acid from acetate can be detected, and the fatty acid accumulates in phosphatidyl choline and sulfolipid. The newly synthesized α-linolenic acid appears to be formed primarily by de novo synthesis and to a much lesser extent from the elongation of a previously formed hexadecatrienoic acid precursor. On the other hand, when a cell-free algal preparation was presented with a hexadecatrienoic acid precursor in the presence of [¹⁴C] malonyl-CoA, the α-linolenic acid formed demonstrated a synthesis by elongation of the precursor. While the cell appears enzymatically capable of α-linolenic acid biosynthesis by both the de novo and elongation processes, de novo synthesis of α-linolenic acid appears to be the more significant mode of synthesis.     

Light-dependent phosphorylation of thylakoid membrane polypeptides.

The phosphorylation of thylakoid membrane proteins was studied using isolated chloroplasts from $\text{Euglena gracilis}$. We have found, using [³²P] labelling, that this phenomenon was light-driven, reversible in the dark, and completely inhibited by Carbonyl cyanide m-chlorophenyl-hydrazone (CCCP). Polyacrylamide gel electrophoresis containing SDS has revealed five main bands which have been found to be proteins. Amino acid analysis of the bands has shown that [³²P] is incorporated into phosphothreonine.     

Reactions and interconversion of met and dimer hemocyanin.

The regenerable methemocyanin of $\text{Busycon canaliculatum}$ is shown to be EPR-nondetectable. The small EPR signal present in met preparations is a nonregenerable binuclear cupric unit accounting for ～ 6% of the active sites. A variety of anions are found to bind to met with the reactions being complicated by both reduction and competitive binding of buffer. The metastable dimer hemocyanin is shown to rearrange either directly to EPR-nondectable met or through an EPR-detectable regenerable binuclear cupric form obtained on reaction of dimer hemocyanin with azide. These results combined with previous results on half met derivatives are used to support the presence of an endogenous protein bridge between the two coppers in hemocyanin.     

The action of ribonuclease T1 on reovirus double-stranded RNA.

A small number of nucleotides are released from highly purified reovirus double-stranded RNA by ribonuclease T₁ in the presence of 0.3 m NaCl. These nucleotides include ppGp, which is quantitatively released from the RNA, and lesser amounts of ApUpGp, Gp, and ApGp. The same products are released from each of the three size classes of double-stranded RNA segments. In experiments involving specific labeling of termini, the only demonstrable sites of hydrolysis were at the 5′ termini of the minus strands. The limited extent of ribonuclease T₁ hydrolysis and localization of its action at the 5′ termini of the minus strands are compatible with a perfect duplex structure for the double-stranded RNA segments wherein the secondary structure of the termini is less stable than that of internal regions.     

Transport of C4-dicarboxylic acids in salmonella typhimurium.

C₄-Dicarboxylic acids are transported into Salmonella typhimurium by stereospecific systems of both high and low affinity. Succinate and l-malate are accumulated in a tricarboxylic acid cycle mutant as was d(+)-malate in induced wild-type cells. Accumulated dicarboxylates are exchangeable with exogenous dicarboxylates. The trichloroacetic acid cycle dicarboxylates are the best inducers of their own transport. Specific mutants devoid of dicarboxylate transport activity (dct) were isolated and differed from tricarboxylate transport mutants (tct) with respect to growth and transport. A mutant devoid of α-ketoglutarate dehydrogenase was unable to transport dicarboxylic acids but citrate transport remained unaffected. Tricarboxylic acid cycle mutants were markedly dependent on an exogenous energy source for the transport of succinate, proline, or leucine. Dicarboxylate transport was largely inhibited by various metabolic inhibitors but could only be inhibited by N,N'-dicyclohexylcarbodiimide anaerobically. ATPase mutants were unimpaired in their ability to transport succinate or proline aerobically.     

Effect of ribosome stripping procedures on antigenicity and conformation of endoplasmic reticulum membrane.

The effects of 3 different procedures for stripping ribosomes from membranes on theantigeniticity and conformation of isolated rough and smooth endoplasmic reticulum from rat liver were examined by microcomplement fixation and circular dichroism. Some of the blocked antigenic binding sites in rough endoplasmic reticulum became available after stripping of ribosomes. None of the 3 methods used is capable of stripping ribosomes completely from rough endoplasmic reticulum without the concomitant removal of protein from the membrane. Such loss of membrane protein by the stripping treatments is probably involved in the observed changes in rough endoplasmic reticulum, since a marked reduction in complement fixing capacity and in ellipticity of circular dichroism is observed also in smooth endoplasmic reticulum after similar treatments.     

Chemical and spectroscopic conformation of an exogenous ligand bridge in half met hemocyanin.

Half $\text{met-N}{}_3{}^{\mathrm{?}}$ hemocyanin is shown to undergo a unique change at the Cu(II)?Cu(I) active site with temperature, exhibiting class II mixed valent properties at low temperature (The appearance of an intense near IR intervalence-transfer transition and a delocalized EPR spectrum). This requires a Cu(II)NNNCu(I) bridging geometry. The effects of CO coordination to half $\text{met-N}{}_3{}^{\mathrm{?}}$, combined with the presence of a low energy $\text{N}{}_3{}^{\mathrm{?}}\text{→ Cu(II)}$ charge transfer transition, demonstrate that azide is also bridging at room temperature. Finally, half $\text{met-N}{}_3{}^{\mathrm{?}}$ is found to be capable of coordination of a second $\text{N}{}_3{}^{\mathrm{?}}$ at the copper(II) site.     

Construction of a paralytic shellfish toxin analyzer and its application

A simple and rapid liquid chromatographic-fluorometric analyzer for quantitating paralytic shellfish toxins is described. Its capability and limitations are discussed.     

Citrate transport in Salmonella typhimurium.

Citrate was rapidly metabolized in wild-type cells of Salmonella typhimurium but actively accumulated in both aconitase mutants and fluorocitrate-poisoned cells. In aconitase mutants citrate was transported by a single high affinity system (K_m 23 μm, V_max 27.2 nmol min^?1 mg^?1), characterized by a single pH optimum of 7.0 and a Q₁₀ of 3.0, and was stimulated by Na⁺. cis-Aconitate, tricarballylate, trans-aconitate, and dl-fluorocitrate were weak competitive inhibitors of citrate transport whereas various other tricarboxylic acid cycle intermediates and carboxylates were ineffective. Spontaneous citrate transport mutants were unable to oxidize citrate, cis-aconitate, or tricarballylate. Such mutants were specific for citrate and transported dicarboxylates normally whereas dicarboxylate transport mutants transported and oxidized citrate normally. In whole cells of an aconitase mutant citrate transport was strongly dependent on an energy source. d(?)-Lactate dehydrogenase mutants were singularly defective in energization by d(?)-lactate. Membrane vesicles of wild-type cells were capable of energized transport by d(?)-lactate or ascorbate-phenyl-methyl sulfonate. Citrate transport in whole cells was primarily energized aerobically, and ATPase deficient mutants were still able to transport citrate in whole cells.