Kinetics of the removal of ferric ion from transferrin by aminoalkylphosphonic acids

The kinetics of ion removal at 25 degrees C in 0.1 M Tris, pH 7.4 by a series of phosphonic acids have been evaluated. The initial rate of iron removal is first order in ferric-transferrin, but shows a hyperbolic dependence on the concentration of the phosphonate ligand. At high ligand concentrations the reaction is clearly biphasic, and the data are interpreted in terms of nonequivalent rate constants for iron removal from the two transferrin iron-binding sites. Rate constants for three phosphonic acid ligands are approximately 0.025 min-1 and approximately 0.007 min-1 for the faster and slower binding sites. The results are discussed in relation to the conformational change mechanism for iron removal from transferrin proposed by Coward et al. [21].     

Developmental patterns of copper and zinc concentrations in mouse liver and brain: evidence that the gene crinkled (cr) is associated with an abnormality in copper metabolism

An abnormality in copper metabolism during both the prenatal and postnatal (preweaning) periods was found to be associated with the autosomal recessive gene ”crinkled“ (cr) in mice. Liver copper concentration was significantly lower in crinkled mice (cr/cr) than in littermate controls (+/?) from 18 days of gestation to 20 days after birth. Crinkled mice older than 20 days of age had liver copper concentrations similar to those of littermate controls. Liver zinc and brain copper and zinc were similar in crinkled and noncrinkled mice at all times tested. In both crinkled and noncrinkled mice, brain copper concentration increased during the suckling period, and liver copper concentration decreased.     

Effect of sample preparation on analysis of superoxide dismutase activity and isoenzymes

The effect of superoxide dismutase (SOD) activity and isoenzyme pattern of detergents, incubation time, and sonication in the preparation of rat liver samples was investigated. The activity of the manganese form of the enzyme (Mn-SOD) was found to decrease significantly after 4 hr of incubation at room temperature, and activity of the copper, zinc form of the enzyme (Cu, Zn-SOD) was not changed significantly even after 24 hr, although levels were somewhat decreased. Sonication of the sample did not affect Cu, Zn-SOD activity, but total Mn-SOD activity was increased. Addition of detergents did not increase Mn-SOD activity when homogenates were sonicated, indicating that Mn-SOD is not membrane bound. Detergents also had no effect on Cu, Zn-SOD activity. None of the treatments investigated altered the isoenzyme patterns, providing evidence that these isoenzymes are not degradation products.     

Isolation of a low molecular weight zinc binding ligand from human milk

A low molecular weight zinc binding compound from human milk has been purified by ultrafiltration, gel filtration, and ion-exchange chromatography. Evidence is provided that this compound is citrate. A higher amount of citrate-bound zinc was found in human milk than in cow's milk. It is suggested that the therapeutic value of human milk for patients with the genetic disorder of zinc metabolism acrodermatitis enteropathica (AE) derives from a greater content of bioavailable zinc citrate in human than in cow's milk.     

Triplet state properties of the methylmercury(II)-tyrosine complex

CH₃Hg(II)OH forms complexes at pH 8 with tyrosine and with tyrosine ethyl ester (TEE) that are detected by ultraviolet difference absorption spectra. With K_f defined by CH₃HgOH + HB

CH₃HgB + H₂O, we find log K_f = 3.61 (tyrosine) and 3.36 (TEE). A heavy-atom effect is observed in frozen glasses of the complexes; this indicates a close interaction between Hg and the chromophore. No UV difference spectrum or heavy-atom effect is observed with N-acetyl tyrosine ethyl ester, indicating that complexing at the phenol O does not occur, and suggesting that binding occurs at the amine N. Zero field optically detected magnetic resonance (ODMR) measurements of the CH₃Hg(II)-tyrosine triplet state give (D, E) = (0.129, 0.047) or (0.134, 0.041) cm^?1 depending upon assignment of transitions. D of tyrosine is relatively unaffected, but E is reduced by CH₃Hg(II) complexing. Low-temperature kinetic measurements show that the shortest lived sublevel of the complex is T_z, where z lies along the phenol long axis in tyrosine. A dominant 11.6-msec component in the 77 K decay of the phosphorescence is consistent with the individual sublevel lifetimes obtained by ODMR.     

Response of plasma and tissue levels of enteroglucagon immunoreactivity to intestinal resection,lactation and hyperphagia

Elevation of plasma enteroglucagon has been described in the presence of intestinal hyperplasia in both animals and man. In order to investigate whether enteroglucagon plays a role in the stimulation of small intestinal growth, fasting plasma and small intestinal tissue levels of enteroglucagon immunoreactivity were measured in control rats and in 3 different rat models of intestinal adaptation: 5 weeks after proximal and distal small bowel resection, on the 12th day of lactation and following 5 weeks of cold acclimation induced hyperphagia. Plasma enteroglucagon levels increased significantly from the control value of 89±(SEM) 13.1 fmol/ml to 147±13.6 after proximal resection (p < 0.005) and to 207±32.6 following 5 weeks of hyperphagia (p < 0.001). During lactation ileal tissue enteroglucagon increased by 183% from 58.1±8.6 pmol/g tissue in controls to 163.9±20.4 (p < 0.001). Ileal tissue enteroglucagon levels were also elevated by 60% following proximal resection (p < 0.005) and by 91% following the hyperphagia of cold-acclimation (p < 0.005). In contrast, jejunal tissue enteroglucagon levels of cold-acclimated rats were only increased by 55% when compared to the control value of 27±2.9 pmol/g tissue (p < 0.02). These results confirm that in the rat, ileal enteroglucagon levels are significantly greater than those found in the jejunum (p < 0.005). Enteroglucagon levels were also significantly elevated in the 2 groups of rats with hyperphagia, namely the lactating and cold-acclimated groups. The data lend further support to the proposal that enteroglucagon may play a trophic role in producing intestinal growth.     

The role of plastoquinone and β-carotene in the primary reaction of plant Photosystem II

Extraction of Triton Photosystem II chloroplast fragments with 0.2% methanol in hexane for 3 h results in the removal of 90 to 95% of the plastoquinone in the original preparation. The extracted fragments (chlorophyll : plastoquinone ratio, 900 : 1) showed no P-680 photooxidation at 15 K after a single laser flash. The extracted fragments also showed no light-induced C-550 absorbance change at 77 K. Reconstitution of the primary reaction of Photosystem II, as evidenced by restoration of low-temperature photooxidation of P-680, could be obtained by the addition of plastoquinone A but not by the addition of β-carotene. The addition of β-carotene plus plastoquinone A restored the C-550 absorbance change. These results indicate that plastoquinone functions as the primary electron acceptor of Photosystem II and that β-carotene does not play a direct role in the primary photochemistry but is required for the C-550 absorbance change.     

Activation of the contractile system in crustacean muscle: Ultrastructural evidence for the role of the T system

Freeze-fracture and thin sections of lobster abdominal fast flexor muscle were used to study the morphology of the sarcoplasmic reticulum (SR) and T system of crustacean muscle. Tannic acid mordanting, which can result in a dense black deposit in the T system lumen, was used to distinguish T system from SR membranes. Ferritin was also used as an extracellular tracer to confirm the tannic acid method. The T system consists of an extensive network of flattened sacs which fills most of the space between the mycfibrils and is in close contact with them. The SR also appears as flattened sacs, sometimes with fenestrations. There is extensive junctional contact between the SR and T system. Quantitative estimates of the volume and surface area of the membranes show that the T system has about 50 % more surface area than the SR. The intramembrane particle (IMP) density of the PF face of the T system is about 1100/ μm² membrane, while the IMP density of the PF face of the SR is about 4800/ μm² membrane. In morphology, extent, and IMP density, the T system of lobster abdominal fast flexor muscle appears (AFF) adapted to provide at least part of the Ca²⁺ for muscle activation and the transport system for relaxation.     

Abundance and tissue distribution of selenocysteine-containing proteins in the rat

The form and distribution of selenium (Se) in proteins from selected tissues of the rat were studied by measuring 75Se radioactivity in animals provided for 5 months with [75Se]selenite as the main dietary source of Se. Equilibration of the animals to a constant specific activity of 75Se allowed the measurement of 75Se to be used as a specific elemental assay for Se. Skeletal muscle, liver and blood accounted for 73% of the whole-body Se and 95% of the total Se-dependent glutathione peroxidase activity. Over 80% of the whole-body Se was in protein in the form of the selenoamino acid, selenocysteine. All other forms of Se that were measured accounted for less than 3% of the whole-body Se. The Se in protein was distributed in seven subunit sizes and nine chromatographic forms. The Se in glutathione peroxidase accounted for one-third of the whole-body Se. These results show that the main use of dietary Se, as selenite, in rats is for the synthesis of selenocysteine-containing proteins. Furthermore, the presence of two-thirds of the whole-body Se in nonglutathione peroxidase, selenocysteine-containing proteins suggests that there may be other important mammalian selenoenzymes besides glutathione peroxidase.     

Reversible inhibition by interferon of the maturation of human peripheral blood monocytes to macrophages

We demonstrated that interferon delays the maturation of human monocytes to macrophages in vitro as assessed by morphologic, histochemical, and biochemical parameters. After exposure to interferon, monocytes were slightly smaller, less stretched out, and had a delayed loss of granules with peroxidase positive reactivity, as compared with control, noninterferon-treated cells. Also, interferon prevented the increase of the specific activity of three lysosomal enzymes (β-galactosidase, β-glucuronidase, and β-N-acetylglucosaminidase) in the monocytes, and enzyme activities were 30–40% of that observed in untreated cells. Both human leukocyte and human fibroblast interferons were effective in delaying the maturation process. The effects of the interferon were species specific and reversible and were neutralized by antiinterferon serum. These studies describe a new nonantiviral effect of interferon, unique in that it involves the delay of maturation of cells in a system which is not associated with cell proliferation. Thus interferon could potentially effect host defense mechanisms and aspects of the immune response which are dependent on the maturation of monocytes to macrophages.     

Maintenance of the shore-level size gradient in the marine snail Tegula funebralis (A. Adams): Importance of behavioral responses to light and sea star predators

The intertidal gastropod, Tegula funebralis (A. Adams) exhibits a shore-level size gradient with mean shell size increasing in a down-shore direction. Snails transferred to zones where they do not usually occur migrated back towards their original zone, thus re-establishing a size gradient and implying differential movement among size classes. Both large (≥2.1 cm shell width) and small (≤ 1.77 cm) snails were photonegative on a horizontal surface and geonegative in the laboratory; there were no statistical differences between size classes. Light, however, inhibited upward, or caused downward, movement of large snails on vertical surfaces. Small snails were unaffected, ranging higher on illuminated vertical surfaces than large snails. Both sizes exhibited similar distributions in the dark. In an experimental chamber providing both emersed and immersed surfaces, T. funebralis established vertical size gradients when the chamber was illuminated from above. It is suggested that light is an important factor in the formation and maintenance of Tegula's shore-level size gradient.In response to water-borne chemicals derived from the sea star Pisaster ochraceus (Brandt), large snails moved up vertical surfaces in greater proportion than small. In response to contact with the predator, large snails moved away faster than small and individuals collected from crevices in the field moved away slower than those collected from open rock faces. Although predation may select for a size gradient in Tegulafunebralis, it is unlikely that responses to predatory sea stars directly and proximally cause or maintain them over the short term.     

Fluorescent thin-layer peptide mapping for protein identification and comparison in the subnanomole range

Conditions and simple precautions are presented for carrying out highly reproducible and sensitive peptide mapping by thin-layer chromatography and subsequent electrophoresis of subnanomole amounts of tryptic digest on silica gel G or GHL plates. The fluorogenic reagent “fluorescamine” is employed for visualization under long-wavelength ultraviolet illumination. Permanent photorecording of high-contrast images, using readily available filters, is substituted for subjective hand scoring of plates. Contrast reversal is used to produce peptide maps suitable for half-tone reproduction.     

Effects of plumbous ion on guanine metabolism.

The enzyme guanine aminohydrolase (guanase) is inhibited by low levels of Pb2+. The inhibition is noncompetitive and the Ki is 3.0 X 10(-6) M. The only other heavy metals that are inhibitory at low concentrations are Ag+, which is 36% more, and Hg2+, which is about 50% less inhibitory than Pb2+. The inhibition of guanase by Pb2+ and Hg2+ is synergistic and the inhibition of the enzyme was readily reversed by EDTA. The relationship of these studies with guanase and to the etiology and treatment of saturnine gout, which appears in humans suffering from lead poisoning, is discussed.     

Effects of castration on sexual behavior of tropical male goats.

This study was undertaken because comparative information regarding the role of gonadal androgen in sexual behavior of adult male mammals is notably deficient in data from ungulate species. After a series of preoperative tests, eight Red Sokoto male goats were castrated and tested for sexual behavior with receptive females at 1 to 2 wk intervals for 52 postoperative wk. Only one animal was judged to have lost the ejaculatory response; this was after 18 wk of postoperative testing. Compared with observations on other species, an unusually high percentage of these goats showed a long-term retention of sexual activity after castration. Even with this long-term retention, however, there was a significant decrement in frequency of ejaculatory responses within one week after castration. The flehmen response, which some believe to be related to detection of excreted urinary pheromones, also declined in frequency after castration.     

Equilibrium ligand binding assays using labeled substrates: nature of the errors introduced by radiochemical impurities

The effects of radiochemical impurities in a labeled substrate on the characteristics of the experimental equilibrium binding plots were examined. The protein (receptor) was assumed to be a monomer or an oligomer composed of identical, noninteracting subunits. The substrate was assumed to be chemically and radiochemically impure (Case I) or just radiochemically impure (Case II). In both cases, the apparent free substrate concentration required for half-saturation of the protein, [S]_0.5,app, increases linearly with increasing total protein concentration. Reciprocal plots and Scatchard plots are nonlinear. The curvature of both plots is opposite to that observed for the heterogeneity of binding sites. Hill plots are curved with average slopes >1 in the region of half-saturation. If the radiochemical impurity goes undetected, the experimental data might lead an investigator to suggest a number of unnecessarily complicated binding models. The plots obtained in the presence of a radiochemical impurity are very similar to those seen when the receptor protein is a dissociable dimer and K_s (monomer) <K_s (dimer). However, in the dimer model the variation of [S]_0.5 with total protein concentration is nonlinear. The most direct way of assessing the radiochemical purity of a labeled substrate is to vary the binding protein concentration at a fixed concentration of S¹. If all of the radioactivity resides in S¹, the concentration of the PS¹ complex will approach [S¹]_t as [P]_t increases, while the free unbound radioactivity will be driven toward zero. If the labeled substrate is radiochemically impure, “saturating” protein will not bind all of the label. This procedure will detect some types of major impurities missed by paper chromatography (e.g., ³H₂O and nonreactive isomers of S¹).     

Fatty acid and complex lipid composition of a Saccharomyces cerevisiae mutant unable to synthesize delta-aminolevulinic acid.

The lipid composition of a Saccharomyces cerevisiae mutant (GL 1–38) lacking δ-aminolevulinic acid synthase (EC 2.3.1.37) was investigated. This mutant is unable to synthesize heme compounds and, as a consequence, cannot make unsaturated fatty acids or ergosterol. The mutant cells were grown (i) in medium supplemented with δ-aminolevulinic acid or (ii) in medium supplemented with Tween 80 (as a source of oleate) and ergosterol. After growth in the presence of δ-aminolevulinic acid, the fatty acid composition of total lipids and mitochondrial lipids was the same as that of the corresponding wild-type strain. After growth in the presence of Tween 80 and ergosterol, the mutant cells contained increased levels of oleate and greatly decreased levels of palmitoleate. The ratio of unsaturated to saturated fatty acids in these cells was still close to that of the wild type but much lower than that of the medium. The sphingolipids accounted for 5.2% of the lipid phosphate in the wild type and, after growth in Tween 80 and ergosterol, for 12.7% in the mutant. Changes in other phospholipids were too small to be considered significant.     

Sequential conversion of the glutathionyl side chain of slow reacting substance (SRS) to cysteinyl-glycine and cysteine in rat basophilic leukemia cells stimulated with A-23187

In rat basophilic leukemia (RBL-1) cells stimulated with A-23187, the major slow reacting substance (SRS) species contain glutathione, cysteinyl-glycine, or cysteine in their side chains, corresponding or closely related to leukotrienes LTC₄, LTD₄, and LTE₄, respectively.³ Evidence is presented that most of the SRS produced during the first few minutes of stimulation by the ionophore has a glutathionyl side chain which is sequentially converted to cysteinyl-glycine and cysteine.     

Maternal meal quality alters fetal rat brain serotonin concentrations

The quality of food eaten by pregnant animals may influence fetal neurotransmitter biosynthesis by altering the relative availability of precursor amino acids. Fetal brain concentrations of tryptophan and the neurotransmitter serotonin increase following the consumption of a carbohydrate-fat meal or after the injection of insulin in fasting animals; in contrast, the fetal concentrations of these compounds are reduced in animals whose mothers eat a meal containing casein protein or amino acids which compete with tryptophan for uptake from maternal blood into the placenta and fetus.     

Selective 7-O-methylation of salsolinol in rat brain and heart in vivo

Intracerebroventricular and intraperitoneal injections of salsolinol and subsequent analyses of rat brain and heart revealed that 7-O-methylsalsolinol (7-OMe-Sal) is the principal O-methylated product formed in vivo. Production of 7-OMe-Sal was blocked by pretreatment of the rats with pyrogallol, an inhibitor of catechol-O-methyltransferase. 7-OMe-Sal and 6-O-methylsalsolinol (6-OMe-Sal) injected intraventricularly or prepared as external standards were distinguished by gas chromatography as peaks with different relative retention times compared to salsolinol as an internal standard. These substances were chromatographed as their corresponding pentafluoropropionic anhydride derivatives on a JXR column. Similar experiments performed with 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline revealed that this substance was similarly selectively converted to its 7-O-methylated product in rat brain.     

The thermodynamics of lanthanide ion binding to adriamycin

We have examined the thermodynamics of lanthanide ion binding to adriamycin by monitoring the effects of variations in temperature on the dissociation constants of various lanthanide ion complexes of the drug. These constants were obtained by analyzing the extent of quenching of the fluorecence of adriamycin in the presence of lanthanide ions in terms of an equilibrium binding process. Our binding model included the following features, all of which are supported by evidence derived from previous published reports, vide infra. The lanthanides form 1:1 complexes with adriamycin. The binding is dependent on the pH of the solution, indicating that only the nonprotonated amine form of the drug participates in lanthanide ion binding. And finally the drug self-associates in solution to for a dimeric species. Our present results indicate that the binding process is almost completely independent of temperature, indicating that the enthalpy of complex formation is extremely small. The entropy terms are consistent with the formation of a complex in which the adriamycin acts as a bidentate ligand. Our results suggest that the lanthanide complexes are isostructural, at least as far as the adriamycin is concerned, throughout the lanthanide series.