Effect of changes in thyroid state on metabolism of thyroxine by rat placenta

We studied the effect of the state of the thyroid on T4 monodeiodination in the rat placenta, and it was compared with those in the liver and kidney. The tissues, maternal serum, and amniotic fluid were obtained from pregnant rats. The tissues were homogenized in cold 50 mM Tris-HCl buffer, pH 7.5. The homogenate (1 mg protein) was incubated at 37 degrees C for 60 min with 1 microgram T4 in the presence of 5 mM DTT. The T3 and reverse T3 generated in the reaction mixture were extracted into cold ethanol and measured by RIAs. The conversion of T4 to reverse T3 in rat placenta was not significantly changed in MMI-induced hypothyroidism or T4 induced hyperthyroidism. On the other hand, conversion of T4 to T3 in the liver and kidney were changed in parallel with the thyroid state. The concentration of reverse T3 in the amniotic fluid was increased in accordance with the increase in the maternal serum T4 concentration. These results indicate that the placental T4 inner ring deiodination is not affected by the thyroid state, and that the change in the amniotic fluid reverse T3 concentration in this study is mainly dependent upon the change in maternal thyroid function.     

Four- and five-coordinate species in nickel-reconstituted hemoglobin and myoglobin: Raman identification of the nickel-histidine stretching mode

Nickel(II)-reconstituted hemoglobin (NiHb) and myoglobin (NiMb) and model Ni porphyrins have been investigated by Soret-resonance Raman difference spectroscopy. Two sets of frequencies for the oxidation-state and core-size marker lines in the region from 1300 to 1700 cm-1 indicate two distinct sites in NiHb. Only one of these sites is evident in the Raman spectra of NiMb. This result is consistent with the UV-visible absorption spectrum of NiHb, which shows two Soret bands at 397 and 420 nm and one Soret at 424 nm for NiMb. Excitation at the blue Soret component of NiHb with 406.7-nm laser radiation preferentially enhances the set of Raman marker lines typical of Ni-protoporphyrin IX [Ni(ProtoP )] in noncoordinating solvents. The wavelength of the blue Soret component and the Raman spectrum indicate four-coordination for this site in NiHb. Laser excitation in the red Soret band enhances a set of lines whose frequencies are compatible with neither four- nor six-coordinate frequencies but are intermediate between the two. The red Soret band of the proteins is also considerably less red shifted than six-coordinate Ni-porphyrin models. These results suggest that Ni in the second site possesses a single axial ligand. Raman spectra of 64Ni-reconstituted and natural abundance Ni-reconstituted hemoglobins, obtained simultaneously in a Raman difference spectrometer, have identified the Ni-ligand stretch at 236 cm-1. The line shifts to 229 cm-1 for the 64Ni-reconstituted Hb. For a pure Ni-ligand stretch a 10-cm-1 shift would be predicted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ compartments in saponin-skinned cultured vascular smooth muscle cells

A method for saponin skinning of primary cultured rat aortic smooth muscle cells was established. The saponin-treated cells could be stained with trypan blue and incorporated more 45Ca2+ than the nontreated cells under the same conditions. At low free Ca2+ concentration, greater than 85% of 45Ca2+ uptake into the skinned cells was dependent on the extracellularly supplied MgATP. In the intact cells, both caffeine and norepinephrine increased 45Ca2+ efflux. In the skinned cells, caffeine increased 45Ca2+ efflux, whereas norepinephrine did not. The caffeine-releasable 45Ca2+ uptake fraction in the skinned cells appeared at 3 X 10(-7) M Ca2+, increased gradually with the increase in free Ca2+ concentration, and reached a plateau at 1 X 10(-5) M Ca2+. The 45Ca2+ uptake fraction, which was significantly suppressed by sodium azide, appeared at 1 X 10(-5) M Ca2+ and increased monotonically with increasing free Ca2+ concentration. The results suggest that the caffeine-sensitive Ca2+ store, presumably the sarcoplasmic reticulum, plays a physiological role by releasing Ca2+ in response to norepinephrine or caffeine and by buffering excessive Ca2+. The 45Ca2+ uptake by mitochondria appears too insensitive to be important under physiological conditions.     

Isolation, and catalytic and immunochemical properties of cathepsin D-like acid proteinase from rat erythrocytes

An erythrocyte membrane-associated cathepsin D-like acid proteinase, termed "EMAP," was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80,000-82,000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgCl2, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.     

A method for direct incorporation of radiolabeled cholesteryl ester into human plasma low-density-lipoproteins: preparation of tracer substrate for cholesteryl ester transfer reaction between lipoproteins

[14C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density greater than 1.006 at 37 degrees C in the presence of dithionitrobenzoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose (LA01) (Arteriosclerosis 4, 276-282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.     

Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers

A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation.     

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).     

Characteristics of a Urate-Degrading Diamine Oxidase-Peroxidase Enzyme System in Soybean Radicles

The cadaverine content of soybean radicles showed a maximumpeak 3–4 days after planting. The variation coincidedwith radicle uricase activity during seed germination. The uricase activity could not be fractionate when the bufferpH for the extraction was at 6.0. The addition of 1 M KCl orNaCl to the buffer allowed the extraction of the uricase activity,but an addition of 1 M MgCl₂ or BaCl₂ inhibited this enzyme'sactivity. The urate-degrading enzyme system was purified 248-fold permilligram of protein from soybean radicles. The respective K_mvalues of the diamine oxidase activity for cadaverine and ofthe urate-degrading activity for hydrogen peroxide and uratewere 1.25, 2.93 and 50.3 µM. Analysis by gel electrophoresisof the partially purified enzyme fraction revealed that theurate-degrading enzyme system consisted of a peroxidase thatdegrades urate with hydrogen peroxide and a diamine oxidasethat releases hydrogen peroxide. These data are evidence that a urate-degrading diamine oxidaseand peroxidase system exists in soybean radicles and that thereaction rate of urate-degradation is controlled by the concentrationof cadaverine. (Received November 28, 1984; Accepted April 8, 1985)