Amino acid sequence homologies in alfa-sarcin, restrictocin and mitogillin

The NH₂-terminal amino acid sequence of the three anti-tumor proteins, alfa-sarcin, mitogillin and restrictocine, has been determined for 20 cycles by automated sequencing procedure. A high degree of sequence homology was observed in this region of the molecule. In addition, extensive sequence homology, ranging from 65 to 100% was found in three other carboxymethylcysteine-containing peptides isolated and sequenced from each molecule.     

Primary structure of heat-stable enterotoxin produced by Yersinia enterocolitica

A heat-stable enterotoxin was isolated and purified from the culture supernatant of $\text{Yersinia enterocolitica}$ by reversed-phase high-performance liquid chromatography. The amino acid sequence of the purified toxin was determined to be as follows: Gln-Ala-Cys(X)-Asp-Pro-Pro-Ser-Pro-Pro-Ala-Glu-Val-Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys (X: not determined). The C-terminal sequence containing 6 half-cystine residues was highly homologous to that of heat-stable enterotoxin of enterotoxigenic $\text{Escherichia coli.}$     

Iron-sulfur clusters and cysteine distribution in a ferredoxin from Azotobactervinelandii

In 80% dimethyl sulfoxide/H₂O, $\text{Azotobacter}$ ferredoxin FeS clusters can be extruded with benzene thiol. The extruded clusters have an absorption spectra maximum at 458 nm which is characteristic of 4Fe4S centers. The amino terminal sequence of the $\text{Azotobacter}$ ferredoxin has 7 of the 8 Cys residues at residue numbers 8, 11, 16, 20, 24, 39 and 42. Except for Cys 24, all of these residues can be correlated to homologous Cys residues in other bacterial ferredoxins. Although two thirds of the first 45 residues are identical to or conservative replacements for the first 43 residues of other bacterial ferredoxins, the insertion of Cys-24 indicates a major change in the environment of one of the two 4Fe4S clusters.     

Thermodynamic studies on synthetic lac operator core

The nine base pairs long central region of the $\text{lac}$ operator gene forms a stable double helix. A comparison of melting temperatures with other biologically useful oligonucleotides indicates the importance of specific base sequence. Binding constants measured with ethidium bromide (1.7 × 10⁵ M^?1), tyrosine (4.0 × 10³ M^?1), and glutamine (1.5 × 10³ M^?1), are interpreted in terms of the involvement of a relatively small number of amino acids in the $\text{lac}$ operator-repressor interaction.     

Pharmacology of excitatory amino acid receptors mediating the stimulation of rat cerebellar cyclic GMP levele invitro

L-glutamate and related amino acids produced dose-related increases in cyclic GMP concentrations in cerebellar slices from 8-day old rats. The effects of L-glutamate, L-aspartate, and N-methyl-D-aspartate were antagonised in part by glutamate diethylester, while D-α-aminoadipate and DL-α-aminosuberate were ineffective. Kainic acid, although producing an effective stimulation of cyclic GMP, failed to achieve the same maximal response as glutamate, suggesting different sites of action. Cyclic GMP levels were also increased, although to a lesser extent, by L-glutamate in slices from hippocampus, medulla, hypothalamus, cortex, striatum and spinal cord of young animals. In the adult, however, this response was no longer observed.     

The bioadhesive of Mytilus byssus: A protein containing L-DOPA

L-DOPA was identified in hydrolysates of $\text{Mytilus}$ byssal adhesive discs and is present at about $\text{10}\text{res}\text{1000}$. The compound was isolated and purified by ion exchange on cellulose phosphate and Biogel P-2 gel filtration. Identity with standard DOPA was demonstrated using thin-layer chromatography, the effect of pH on UV absorbance, fluorescence spectrophotometry, amino acid analysis, and the preparation of ethylenediamine derivatives. Contrary to earlier reports, dityrosine was not detected. A sodium dodecylsulfate-insoluble protein containing $\text{48}\text{res}\text{1000}$ of DOPA was isolated from the gland that secretes the disc adhesive. This protein is presumed to be a precursor of the adhesive.     

Identification of a new glutathione S-transferase from rat liver cytosol

A new glutathione $\text{S}$-transferase has been purified to homogeneity from 105,000 × g supernatant of Sprague-Dawley rat liver homogenates. The purified enzyme exhibited specific activities of approximately 1.8, and 0.12 μmoles. min^?1. mg^?1 toward 1-chloro 2,4-dinitrobenzene and cumene hydroperoxide respectively. The SDS gel electrophoresis data on subunit composition revealed that the new transferase is composed of two subunits with an identical M_r of 24,400 (Y_α Family). Our $\text{in}\text{vitro}$ translation experiments with rat liver poly(A) RNAs and substrate specificity data suggest that this subunit is different from the previously reported Ya, Yb and Yc subunits of rat liver glutathione $\text{S}$-transferases. Comparatively, the new isozyme showed significant activity toward 1,2 epoxy-3-(P-nitrophenoxy)-propane, ethacrynic acid and P-nitrophenyl acetate, 0.4, 0.34 and 0.18 μ moles. min^?1. mg^?1 respectively.     

Derivative cyclic voltabsorptometry of cytochrome c

The recently reported method of derivative cyclic voltabsorptometry has been applied to horse heart cytochrome $\text{c}$ at fluoride doped tin oxide optically transparent electrodes. The advantages of the derivative cyclic voltabsorptometric method compared to voltammetric methods in analytical, kinetic and mechanistic studies are discussed.     

Energy-dependence of nitrate reductase induction in Candidautilis

The requirement of a suitable energy source during the induced synthesis of nitrate reductase in $\text{Candida}\text{utilis}$ was investigated. The levels of nitrate reductase induced were shown to be energy-dependent, and to vary in response to the type of carbon source provided. Glycerol, fructose, ethanol, glucose, and sucrose served as efficient energy sources. Growth rate of the yeast and the induced level of nitrate reductase were dependent on the ratio of carbon to nitrogen in the induction medium, and ratio of 2 being optimal. Induction of nitrate reductase was inhibited by uncouplers, 2,4-dinitrophenol (DNP), dicumarol and carbonyl cyanide $\text{p}$-trifluoromethoxy phenyl hydrazone (CCCP), and by cyanide and azide, indicating an absolute energy-dependency. The facilitation of induction of a high level of nitrate reductase by exogenously added ATP as sole source of energy confirmed the obligate requirement of ATP for the synthesis of nitrate reductase in $\text{Candida}$$\text{utilis}$.     

Gonadotropin releasing hormone (GnRH) is not degraded by intact pituitary tissue in vitro

The possible degradation of GnRH by intact pituitary tissue was investigated. Trypsin-, collagenase- and mechanically dispersed pituitary cells in culture and fresh pituitaries cut into eight segments were incubated with specifically tritiated GnRH or unlabeled GnRH which were detected by HPLC and RIA in incubation media. Degradation of GnRH could not be detected by either method during incubations with any of the pituitary cell cultures or fresh tissue. The results suggest that pituitary degradation is not involved in the regulation of circulating levels of GnRH.     

In vitro liver clearance of tritiated estradiol-17β in the female rat after retrochiasmatic transection and ovariectomy

It was reported in a previous study that serum estradiol-17β (E₂) was elevated in rats after retrochiasmatic transection (FC). Serum E₂ was also higher in estradiol cypionate treated, ovariectomized (ovx) rats that had been subjected to FC than in those that had not. This suggested that increased secretion of E₂ was not the only factor responsible for elevated serum E₂ after FC. To ascertain the contribution of decreased metabolism of E₂ to this response, liver tissue slices were incubated with 3H-estradiol-17β, and the rates of ³h uptake and conversion to water-soluble conjugates were measured.The rate of uptake of ³H by the tissue was not indicative of the rate of conjugate formation. Livers of rats with high serum E₂ exhibited lower rates of ³h uptake than those of rats with low serum E₂. Even so, the formation of ³H conjugates was greater in liver of rats with high serum E₂. As hypothesized, livers of rats with FC formed conjugates at a significantly lower rate than those of similarly treated rats without FC. Thus, FC of an intact rat leads to an increase in serum E₂ by increasing the secretion of E₂, and apparently also by decreasing the rate of E₂ metabolism by the liver.     

Preparation and characterization of a stable half met derivative of type 2 depleted Rhus laccase: Exogenous ligand binding to the type 3 site

We report the preparation and characterization of a stable half met (Cu(II)Cu(I)) type 2 copper depleted derivative of $\text{Rhus}$ laccase. Anion binding studies to this mixed valent type 3 protein form indicate no tight binding of anions nor group 1 - group 2 ligand behavior. This suggests that, in contrast to the well-characterized hemocyanins and tyrosinase coupled binuclear sites, exogenous ligands do $\text{not}$ appear to bridge the type 3 binuclear copper ions in laccase.     

An oxygen-18 tracer investigation of the mechanism of myo-inositol oxygenase

In the conversion of $\text{myo}$-inositol to D-glucuronic acid catalyzed by $\text{myo}$-inositol oxygenase only one atom of ¹⁸O from ¹⁸O₂ is incorporated into the product, and it is found exclusively in the carboxyl group. Control experiments indicate that under the reaction conditions no exchange of solvent oxygens with D-glucuronate occurs. To avoid exchange during isolation and analysis the oxygenase product was enzymically reduced to L-gulonate and isolated in that form. The results eliminate one possible mechanism for the oxygenase reaction, but are consistent with two others which seem chemically reasonable.     

Effects of thiamine and pyridoxine on the content and composition of sterols in Saccharomyces carlsbergensis 4228

The level of sterols in $\text{S. carlsbergensis}$ 4228 cells grown aerobically on a synthetic medium fortified with thiamine was significantly low compared with that in the control cells. The levels of free and esterified sterols in the thiamine-cells were 60% and 10% of the corresponding sterol levels in the control cells, respectively. Analysis by gas-liquid chromatography of non-saponifiable lipids extracted from the cells revealed that the amounts of squalene, lanosterol and two unidentified sterols were higher than those in the control cells and that ergosterol and zymosterol, major sterols in the control cells, were not present. These effects of thiamine on the content and composition of sterols were abolished by the addition of pyridoxine to the medium.     

Chromatographic resolution of two DNA polymerase activities from bloodstream forms of Trypanosomabrucei: Differential responses to exogenous polyamine addition

A single peak of DNA polymerase activity from extracts of $\text{T.}\text{brucei}$, obtained by DEAE-cellulose and phosphocellulose ion-exchange chromatography, was resolved into two peaks differing in KCl concentration necessary to elute them from a DNA-agarose column. Peak I (eluting at 0.2 M KCl) and Peak II (eluting at 0.4 M KCl), differed in response to increasing KCl concentrations, although both functioned optimally with Mg²⁺ as divalent cation when DNA synthesis was directed either by activated DNA or poly (dC)·(dG)_12–18. Due to the potential significance of polyamines in the metabolism of $\text{T.}\text{brucei}$, the effect of exogenous polyamine on rates of DNA synthesis by the peak I and II enzymes was compared with that of murine DNA polymerase alpha. Only the peak I enzyme was significantly stimulated (up to 4-fold) by the biologically active polyamines spermine and spermidine at physiological concentrations. The response of the peak I enzyme resembled that of the alpha polymerase. This result suggests a possible functional difference between peak I and II enzymes, as well as a potential target site for trypanocidal drug development.     

Application of CNDO2 theoretical calculations to interpretation of the chemical reactivity and biological activity of the syn and anti diolepoxides of benzo[a]pyrene

$\text{CNDO}\text{2}$ molecular orbital theoretical calculations performed on the anti and syn diolepoxides (1 and 2) of the potent carcinogen benzo[a]pyrene provide insight into the molecular structure and reactivity of these mutagenic and carcinogenic hydrocarbon metabolites. Hydrogen-bonded interaction between the 7-HO proton and the epoxide oxygen atom of 2 is shown to be absent in the normal semichair conformation of the tetrahydro ring, (H…O bond distance = 2.7 Å), but is energetically favored in a somewhat distorted puckered structure (H…O bond distance = 1.7 Å). Unexpectedly, internal H-bonding alters the relative electron density at C₉ and C₁₀, leading to prediction of the former as the more electrophilic center. Since all reactions of 2 take place exclusively at C₁₀, transannular H-bonding is concluded not to contribute significantly to the structure of 2. Diolepoxide reactions with both weak and strong nucleophiles and with DNA are discussed and the mechanisms interpreted in terms of molecular structure as determined by the theoretical calculations.     

Specific in vivo binding of 77Br-p-bromospiroperidol in rat brain: A potential tool for gamma ray imaging

The binding of the gamma labeled neuroleptic, ⁷⁷Br-$\text{p}$-bromosprioperidol, in the rat brain was examined $\text{in vivo}$. This binding parallels the binding of ³H-spiroperidol, in that binding is especially high in dopaminergically innervated areas, is saturable, and is displaced by high doses of unlabeled spiroperidol (1–5). Thus, ⁷⁷Br-$\text{p}$-bromospiroperidol is a suitable ligand for use in gamma ray imaging techniques for $\text{in vivo}$ monitoring of receptor binding.     

Identification of specific amino acids in diabetic glomerular basement membrane collagen subject to non-enzymatic glucosylation in vivo

Glomerular basement membrane was purified from normal and streptozotocin-diabetic rats, and the insoluble collagenous fraction isolated following reduction and alkylation. Amino acid analysis of diabetic samples revealed three unusual peaks eluting near glucosamine. Two of these peaks had respective elution times identical to those of synthetic glucitol-hydroxylysine and glucitol-lysine. These findings identify lysine-derived amino acids in glomerular basement membrane collagen as sites of excess non-enzymatic glucosylation $\text{in vivo}$ in hyperglycemic diabetes.     

3-Hydroxy-3-methylglutaryl CoA reductase of Hevea latex: The occurrence of a heat-stable activator in the C-serum

The effect of the C-serum (the cytosol) on the activity of 3-hydroxy-3-methylglutaryl CoA reductase in the latex of $\text{Hevea}\text{brasiliensis}$ was investigated. Depending on the clone from which the latex was obtained, the C-serum was found to depress or activate or have little effect on the enzyme activity. Boiling the C-serum however, resulted in a consistent activation effect in all the clones examined. Optimal activation was obtained with 20 μl boiled C-serum. Dialysis or EDTA (40 mM) treatment of the boiled C-serum did not diminish the activation effect. Although not essential, dithiothreitol complemented the activation effect of the boiled C-serum and the optimal concentration was 10 mM. Trypsin digestion of the boiled C-serum resulted in the complete loss of the activation effect. The activator in the boiled C-serum was salted out by ammonium sulphate at 25 – 100% saturation. Hevein had no effect on reductase activity.