Uroporphyrinogen decarboxylase. Purification, properties, and inhibition by polychlorinated biphenyl isomers

Uroporphyrinogen decarboxylase (EC 4.1.1.37) which converts uroporphyrinogen I or III into coproporphyrinogen I or III, respectively, was purified about 5,500-fold from chicken erythrocytes. Purification was accomplished by chromatography on DEAE-cellulose, ammonium sulfate fractionation, chromatography on Sephadex G-100, and chromatofocusing. The most purified preparation was homogeneous on polyacrylamide gel electrophoresis and had a specific activity of 1,420 units/mg of protein, the highest value so far reported. The molecular weight, as determined by Sephadex G-150 gel chromatography, is 79,000. The subunit molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 39,700, suggesting that uroporphyrinogen decarboxylase is dimeric in form. The purified enzyme had an isoelectric point of 6.2 and a pH optimum of 6.8. The SH reagents inhibited the enzyme activity, but neither metal ions nor cofactor requirements could be demonstrated. A new and simple method for the separation of free uroporphyrin, hepta-, hexa-, and pentacarboxylic porphyrins and coproporphyrin was developed using a high pressure liquid chromatograph equipped with a spectrofluorometric detector. Kinetic studies of the sequential decarboxylation of uroporphyrinogen with purified enzyme were performed. 3,4,3',4'-Tetrachlorobiphenyl and 3,4,5,3',4'5'-hexachlorobiphenyl which specifically induce delta-aminolevulinic acid synthetase also strongly inhibit uroporphyrinogen decarboxylase directly at two steps, i.e. first in the formation of hexacarboxylic porphyrinogen III from heptacarboxylic porphyrinogen III and second in the formation of heptacarboxylic porphyrinogen III from uroporphyrinogen III.     

Flavanol glucosides from rhubarb and Rhaphiolepis umbellata

Investigations of rhubarb and the bark of Rhaphiolepis umbellata led to the isolation of new flavan-3-ol glucosides. Their structures were elucidated on the basis of ¹H and ¹³C NMR analysis hydrolytic studies as (+)-catechin 5-O-β-d-glucopyranoside and (?)-catechin 7-O-β-d-glucopyranoside.     

The suppressive effect of peritoneal exudate macrophages on production of antibody to sheep erythrocytes in vitro

The effects of peritoneal exudate macrophages on antibody response to sheep erythrocytes (SRBC) were investigated in mice. Peritoneal exudate macrophages obtained from mice injected intraperitoneally with proteose peptone or Corynebacterium parvum 4 days earlier had stronger ability to phagocytize and degrade SRBC than normal resident macrophages. These macrophages suppressed antibody formation to SRBC in vitro as well as in vivo. This suppression was overridden by increasing the amount of SRBC and diminished completely by pretreatment of the macrophages with iodoacetate and partly by pretreatment with 2-deoxyglucose, both known to be inhibitors of phagocytosis, but not by addition of indomethacin to the in vitro culture. These results suggest that the suppression of antibody response by peritoneal exudate macrophages was due to the increased activity of these cells as scavenger cells, resulting in a reduced amount of effective antigenic stimulation, and that it was not mediated by a prostaglandin-dependent mechanism. The scavenger function of these macrophages may be due to Ia-negative macrophages.     

Immunohistochemical identification of neurons containing corticotropin-releasing factor in the rat hypothalamus

A specific rabbit anti-CRF serum and the immunoperoxidase technique were used to show that CRF-containing neurons are mainly distributed in the paraventricular and supraoptic nuclei of the rat hypothalamus. In addition, immunoreactive neurons are scattered in other hypothalamic regions. These neurons are 20--30 micrometers in diameter. From the present and previous investigations it may be concluded that the hypothalamic magnocellular nuclei, i.e., paraventricular and supraoptic, and other hypothalamic accessory nuclei, are the producing sites not only for vasopressin and oxytocin, but also for corticotropin-releasing factor.     

Combined method of fluorescence tracer technique and PAP immunohistochemistry for discrimination of the transplanted cells

Summary The retrograde fluorescence tracer, True Blue (TB), was injected into the forebrain septal area of neonatal rats. After 3 to 6 days the brains of these animals were carefully removed and placed in ice-cold sterilized physiological saline containing 1% glucose. Under the surgical microscope, one or two pairs of mesencephalic tissue samples, each containing a dorsal raphe nucleus, were punched out and transplanted into the third ventricle of a 5,6-DHT-pretreated adult rat. One month after transplantation, all animals were perfused and their brains sectioned using a cryostat. The sections were examined using a fluorescence microscope, and then processed for serotonin immunohistochemistry. The grafts were found to be successfully implanted and connected with the middle portion of the third ventricle. Four types of neurons, i.e., TB-labeled, serotonin-labeled, both TB-and serotonin-labeled, and non-labeled neurons, were detected in the grafts. This double-labeling method is considered to be a useful technique in characterizing the neurons in grafts which consist of a heterogeneous cell population.Supporied by grants from the Ministry of Education, Science and Culture of Japan     

Feeding habits of Japanese butterfyfishes (Chaetodontidae)

Synopsis Stomach content data from 32 species of Japanese butterflyfishes of the family Chaetodontidae were used to classify them into feeding groups and to determine their important food resources. Four major feeding groups were distinguished: (1) obligative coral feeders which prey exclusively or mostly on Scleractinian corals, (2) facultative coral feeders that take both corals and other benthic organisms, (3) noncoralline invertebrate feeders which consume benthic invertebrates other than corals, and (4) zooplankton feeders. Ten species representing 31% of the butterflyfishes belong to the first category. The second and third categories include 13 (41%) and 8 (25%) species, respectively. The fourth category is represented by only one species which picks individual zooplankters, especially calanoid copepods, in midwater above the reefs. Facultative coral feeders consumed varying quantities of scleractinians (from 2 to 74% of food volume), along with a variety of benthic organisms including algae, alcyonarians, sea anemones, sedentary polychaetes, sponges, hydroids, etc. Noncoralline invertebrate feeders, on the other hand, tend to have low diversified diets, predominated by one prey item such as sea anemones, zoanthideans, polychaetes, or colonial ascidians. These dietary data suggest that scleractinian corals are the most important food resource for the Japanese butterflyfishes, and next important are sea anemones, sedentary polychaetes, alcyonarians, and algae.     

Enzymic conversion of 11,12-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid. Purification of an epoxide hydrolase from the guinea pig liver cytosol

(11S,12S)-Epoxy-5,14-cis-7,9-trans-eicosatetraenoic acid (11,12-leukotriene A4) was nonenzymically converted to seven compounds: two diastereomers of (12S)-hydroxyeicosatetraeno-delta-lactones (major products), two diastereomers of (5,12S)-dihydroxyeicosatetraenoic acid and three stereoisomers of (11,12S)-dihydroxyeicosatetraenoic acid. Among these compounds, (11R,12S)-dihydroxy-5,14-cis-7,9-trans-eicosatetraenoic acid proved to be the only enzymic product. This hydrolysis activity was present in the cytosol fractions of various tissues of guinea pig such as liver, adrenal gland, small intestine, and brain. We purified the epoxide hydrolase to an apparent homogeneity from the guinea pig liver. The enzyme had a molecular weight of 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an isoelectric point of 7.3. The partial amino acid sequence was different from that of the microsomal enzyme. Km and Vmax values for 11,12-leukotriene A4 were 18 microM and 2.4 mumol/min/mg protein, respectively. These results indicate that 11,12-dihydroxyeicosatetraenoic acid is enzymically synthesized from 11,12-leukotriene A4 by the action of the cytosolic epoxide hydrolase in vitro.