Rabbit β-glucuronidase. Purification and properties, and the existence of multiple forms

1. beta-Glucuronidase (EC 3.2.1.31) was purified from rabbit liver by a procedure involving autolysis, (NH(4))(2)SO(4) fractionation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration, sedimentation in a sucrose gradient, and isoelectric focusing. 2. Electron microscopy revealed ferritin as the major contaminant in later stages of purification and also showed aggregates of enzyme molecules. Particular attention was paid to the removal of ferritin. 3. The purified enzyme was homogeneous in polyacrylamide-gel electrophoresis both in non-dissociating conditions and in the presence of sodium dodecyl sulphate, and in Ouchterlony gel diffusion and immunoelectrophoresis against polyspecific antisera. 4. Sedimentation in sucrose gradients gave a molecular weight of 300000, whereas gel filtration indicated 440000. 5. Subunits of 75000 molecular weight were observed in gel electrophoresis in the presence of sodium dodecyl sulphate and in gel filtration in the presence of urea. 6. The K(m) value for p-nitrophenyl beta-d-glucuronide was 0.6mm, and the enzyme was extremely sensitive to lactone inhibitors. It was also inhibited by Hg(2+) ions. 7. Multiple forms were observed in the pure enzyme by isoelectric focusing, with pI values of 4.5-5.8. Subunits showed similar heterogeneity. The origin of the multiple forms was investigated in detail, and the possibility of artifact generation largely excluded. Some of the forms of lowest pI disappeared after neuraminidase digestion. The nature of the residual heterogeneity remains to be elucidated.     

Evidence for the occurrence of two forms of β-lipotropin in rat pituitary

A peptide isolated from porcine gut according to its glucagon-like activity in liver (bioactive enteroglucagon) has been characterized immunologically, biologically and chemically: its potency relative to pancreatic glucagon in interacting with an antiglucagon antibody, hepatic glucagon-binding sites and hepatic adenylate cyclase was ～100%, 20% and 10%, respectively. In contrast, it is ～20-times more potent than glucagon in oxyntic glands, justifying the term ‘oxyntomodulin’. Chemically, it consists in the 29 amino acid-peptide glucagon elongated at its C-terminal end by the octapeptide Lys—Arg—Asn—Lys—Asn—Asn—Ile &;—Ala; accordingly, it is called ‘glucagon-37’     

Ultrastructural localization of corticotropin, β-lipotropin,and αand β-endorphin in the porcine anterior pituitary

Summary The peroxidase-antiperoxidase immunocytochemical technique was used to identify the ACTH/endorphin cells in the porcine pituitary at the ultrastructural level and to determine the precise subcellular localization of the pro-ACTH/endorphin fragments. The cells display different aspects: 1) large, regular shapes with numerous and large secretory granules; 2) small, irregular and angular shapes with small granules aligned along the periphery of the cell; and 3) intermediate forms. The presence of and -endorphin not only in the same cells but also in the same secretory granules that contain ACTH and -LPH clearly indicates that both the precursor or its fragments and the abovementioned peptides are stored in the same granules and released simultaneously by the corticotropic cells. The presence of FSH in some corticotropic cells is also discussed.Abbreviations used in this Article ACTH corticotropin - -MSH -melanotropin (ACTH I–I3) - CLIP corticotropin-like intermediate lobe peptide (ACTH 18–39) - -LPH -lipotropin - -MSH -melanotropin (-LPH 41–58); -endorphin (-LPH 61–91); -endorphin (-LPH 61–76)     

Avian β-endorphin: Alterations in immunoreactive forms in plasma and pituitary of embryonic and adult chickens

1. A heterologous radioimmunoassay for β-endorphin (β-endo) was established. Plasma and/or extracts of pituitaries from embryonic (days 14.5 and 17.5 of incubation), newly hatched, and adult chickens were chromatographed on a Sephadex G-75 column with 0.1 M acetic acid.2. Embryonic plasma had only a single immunoreactive peak that eluted similar to a β-lipotropin (β-LPH) standard. In contrast, adult plasma had 2 peaks, co-eluting with β-LPH (34%) and β-endo (66%).3. Chromatography of pituitary extracts demonstrated two immunoreactive peaks in both embryonic and adult birds. Although 70% of immunoreactivity eluted with β-endo for embryonic birds, 80% eluted with β-LPH from adults.4. The smaller proportion of β-endo in adult pituitaries may reflect a higher rate of secretion of this hormone into the blood.     

Causes of the production of multiple forms of β-galactosidase by Bacillus circulans

The presence of multiple types of β-galactosidases in a commercial enzyme preparation from Bacillus circulans ATCC 31382 and differences in their transgalactosylation activity were investigated. Four β-galactosidases, β-Gal-A, β-Gal-B, β-Gal-C, and β-Gal-D, which were immunologically homologous, were isolated and characterized. The N-terminal amino acid sequences of all of the enzymes were identical and biochemical characteristics were similar, except for galactooligosaccharide production. β-Gal-B, β-Gal-C, and β-Gal-D produced mainly tri- and tetra saccharides at maximum yields of 20-30 and 9-12%, while β-Gal-A produced trisaccharide with 7% with 5% lactose as substrate. The Lineweaver-Burk plots for all of the enzymes, except for β-Gal-A, showed biphasic behavior. β-Gal-A was truncated to yield multiple β-galactosidases by treatment with protease isolated from the culture broth of B. circulans. Treatment of β-Gal-A with trypsin yielded an active 91-kDa protein composed of 21-kDa and 70-kDa proteins with characteristics similar to those for β-Gal-D.     

Localization of β1-Adrenergic Receptors in the Cochlea and the Vestibular Labyrinth

Sympathetic activation in a “fight or flight reaction” may put the sensory systems for hearing and balance into a state of heightened alert via β₁-adrenergic receptors (β₁-AR). The aim of the present study was to localize β₁-AR in the gerbil inner ear by confocal immunocytochemistry, to characterize β₁-AR by Western immunoblots, and to identify β₁-AR pharmacologically by measurements of cAMP production. Staining for β₁-AR was found in strial marginal cells, inner and outer hair cells, outer sulcus, and spiral ganglia cells of the cochlea, as well as in dark, transitional and supporting cells of the vestibular labyrinth. Receptors were characterized in microdissected inner ear tissue fractions as 55 kDa non-glycosylated species and as 160 kDa high-mannose-glycosylated complexes. Pharmacological studies using isoproterenol, ICI-118551 and CGP-20712A demonstrated β₁-AR as the predominant adrenergic receptor in stria vascularis and organ of Corti. In conclusion, β₁-AR are present and functional in inner ear epithelial cells that are involved in K⁺ cycling and auditory transduction, as well as in neuronal cells that are involved in auditory transmission.     

Characterization of multiple forms of α-glucan phosphorylase from Musa paradisiaca fruits

Three forms of α-glucan phosphorylase from mature banana fruit pulp separated by ammonium sulfate fractionation and DEAE-cellulose chromatography were anodic at pH 8·6 on starch gel electrophoresis. The three forms differed in sensitivity to the phenolics extracted from immature and mature banana fruit pulp. Only two forms of the enzyme were detected in immature banana fruit pulp.     

Ultrastructural localization of immunoreactive corticotropin, β-lipotropin, α- and β-endorphin in Cells of the human fetal anterior pituitary

Summary Cells immunoreactive with anti--(17–39) ACTH, -(1–24) corticotropin, -LPH, - and -EP were identified in the human fetal anterior pituitary at the ultrastructural level using the peroxidase-antiperoxidase complex method on ultrathin sections.Only one definite cell type was revealed by all these antisera. All granules of each individual immunostained cell reacted regardless of the antiserum used. The immunostained cells occurred in groups and were sometimes located in the wall of the follicle-like structures commonly observed in the fetal anterior pituitary. The cells revealed two main aspects: 1) The largest elements were rich in organelles, and their numerous secretory granules showed significant variations in size (250–500 nm in diameter), electron density of their content and stain-deposit intensity. The ergastoplasm, consisting of irregular tubules, was poorly developed. In the vicinity of the conspicuous Golgi apparatus, organelles related to the GERL complex were commonly observed. Multivesicular bodies were frequent. Some of these cells showed bundles of microfilaments (60 nm in thickness). 2) The smaller cells had an electron-lucent hyaloplasm with sparse organelles; they contained fewer granules and never showed microfilaments.The immunocytological results are consistent with the synthesis of a molecule similar to pro-opiocortin by this type of endocrine cell in human fetuses. Morphological evidence for the maturation process of this precursor and for the secretory activity of these cells and its possible regulation is presented and discussed.Abbreviations used ACTH corticotropin (39 amino acid polypeptide) - -MSH -melanotropin (ACTH [1–13]) - CLIP corticotropin-like intermediate lobe peptide (ACTH [18–39]) - -LPH -lipotropin (91 amino acid polypeptide) - -MSH -melanotropin (-LPH [41–58]) - -EP -endorphin (-LPH [61–91]) - -EP -endorphin (-LPH [61–76]) - PTA phosphotungstic acid Acknowledgements: The authors would like to thank Professors P. Magnin and J. Liaras, Hôpital Edouard Herriot; M. Dumont, Hôpital de la Croix-Rousse; A. Notter and R. Garmier, Hôtel Dieu; M. Bethenod, Hôpital Debrousse, Lyon, and the entire staff whose cooperation enabled samples to be taken under optimal conditions. The authors also thank Professor L. Graf, Research Institute for Pharmaceutical Chemistry (Budapest), and Professor R. Guillemin (Salk Institute, La Jolla) for their generous gift of antigensThis work was supported by a grant from I.N.S.E.R.M., ATP 46.77.78 (P.M. Dubois)     

Localization of β2-microglobulin in the term villous syncytiotrophoblast

The non-covalent association of beta 2-microglobulin with MHC class I molecules and MHC class I-type molecules such as FcRn or the hemochromatosis protein (HFE) is of major importance for their function, i.e., antigen presentation, IgG transport, and regulation of iron uptake, respectively. In the human hemochorial placenta, the syncytiotrophoblast forms a continuous epithelial layer covering the villous trees, where it directly contacts maternal blood and, among many other functions, mediates uptake of maternal IgG and iron. The villous syncytiotrophoblast lacks MHC class I molecules but expresses FcRn and HFE. Since data on beta 2-microglobulin synthesis and localization in the term villous syncytiotrophoblast were contradictory, we investigated the subcellular localization of beta 2-microglobulin by immunoelectron microscopy. Synthesis in the trophoblast is demonstrated by colocalization of beta 2-microglobulin with protein disulfide isomerase, a marker protein of the endoplasmic reticulum. The presence of beta 2-microglobulin at the apical plasma membrane corresponds to the recently observed association of beta 2-microglobulin with HFE and FcRn. Localization of beta 2-microglobulin in late endosomes/lysosomes, labeled with antibodies to lysosome membrane antigen LAMP 2, suggests also a degradative route of beta 2-microglobulin internalized by fluid-phase from the maternal blood.     

Functional symmetry of the β-galactoside carrier in escherichia coli

Cytoplasmic membrane vesicles with either normal or inverted orientation were prepared from Escherichia coli. The lactose transport activity of these vesicle preparations was compared. The parameters measured were net efflux, counterflux, and K⁺/valinomycin-induced active uptake of lactose. With membrane vesicles derived from both wild-type and cytochrome-deficient strains the right-side-out and inverted membrane preparations showed similar rates of lactose flux in all assays. According to these criteria, the activity of the β-galactoside transport protein is inherently symmetrical.One major difference was observed between the native and inverted vesicle preparations: the inverted vesicles had approximately twice the specific activity of native vesicles in the counterflux and K⁺/valinomycin-induced uptake assays. This difference can be largely ascribed to the presence in the normal vesicle preparation of vesicles with a high passive permeability to lactose. Such vesicles are apparently absent from the inverted vesicle preparations.     

Purification and characterisation of β-N-acetylhexosaminidase from the butter clam, saxidomus purpuratus

A β-N-acetylhexosaminidase [EC 3.2.1.30] has been purified ～98-fold from an extract of the digestive organs of Saxidomus purpuratus by using ammonium sulfate fractionation, and chromatography on Toyopearl HW-50, CM-cellulose, and Sepharose 4B. The purified enzyme, the molecular weight of which was estimated to be ～66,000 by gel filtration, was composed of two sub-units of molecular weight 30,000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The purified enzyme had a pH optimum of 3.8 and an optimum temperature of 55°, and its activity was enhanced ～2-fold in the presence of 0.1m sodium chloride. The Michaelis constants toward p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucoside and -galactoside were 1.2 × 10^?4 and 1.3 × 10^?4m, respectively.     

Localization and synthesis of α-fetoprotein in the chicken

Summary The localization and sites of synthesis of -fetoprotein in chick embryos throughout development have been investigated using the combined techniques of immunofluorescence microscopy and tissue culture in the presence of radiolabelled amino acids, followed by immunoautoradiographic analysis.Alpha-fetoprotein is present in a range of embryonic tissues and especially concentrated in the yolk sac, liver and connective tissue. Analysis of culture fluids revealed that the yolk sac is the major site of -fetoprotein synthesis with smaller, but significant quantities being produced by the liver.These results are discussed in relation to mammalian -fetoprotein, and the merits of the chick embryo for studies on the biological function of AFP are considered.Supported by an award from the Science Research Council, to whom grateful acknowledgement is made     

Characterization of cyanogenic glucosides and β-glucosidases in Triglochin maritima seedlings

In addition to triglochinin, taxiphyllin has been detected as a cyanogenic glucoside in seedlings of Triglochin maritima. Taxiphyllin at first increases during seedling development and then decreases, whereas tri-glochinin increases to a level higher than that ever reached by taxiphyllin and remains there during further seedling development. Two β-glucosidases have also been characterized in these seedlings. One of these shows a distinct specificity for triglochinin, whereas taxiphyllin appears to be the preferred substrate of the other.     

Multiple forms of β-glucuronidase in rat liver lysosomes and microsomes

Multiple forms of β-glucuronidase have been demonstrated using sucrose gradient and polyacrylamide gel isoelectric focusing techniques in 6 m urea. Microsomal β-glucuronidase, a membrane-bound enzyme, was solubilized from lysosome-free, Ca²⁺-precipitated microsomes by detergents and isolated by chromatography on columns of rabbit anti-rat preputial gland β-glucuronidase antibody bound to Sepharose. The enzyme has a pI of 6.7. Polyacrylamide gel isoelectric focusing resolves the microsomal enzyme into three components, each of which is protease sensitive. The protease-modified microsomal enzyme is very similar to several forms of β-glucuronidase in lysosomes. The lysosomal β-glucuronidase, isolated from osmotically shocked lysosomes, is very heterogeneous after isoelectric focusing over the range pI 5.4–6.0. The lysosomal enzyme can be resolved into 10–12 bands by polyacrylamide gel isoelectric focusing. The more acid forms of the lysosomal enzyme are neuraminidase sensitive, suggesting they may be sialoglycoproteins.     

N,O versus O,O coordination in β-imino diketonato complexes: Role of the metal center and of the imino substituent

β-Imino carbonyl enolato metal(II) complexes of general formula [M((RCO)(R′CO)CC(R″)NH)₂] (M = Mn, Fe, Co, Ni, Cu, Zn, Pd, R = R′ = Me, R″ = CCl₃; M = Cu, Pd, R = R′ = Me, R″ = PhCO; R = Me, R′ = Ph, R″ = PhCO; R = R′ = Ph, R″ = PhCO) are easily synthesized by the reaction of metal(II) acetates with the proper β-enaminodiones in 1/2 molar ratio in ethanol at room temperature. In all the cases the trifunctional NOO β-imino carbonyl enolate ligand acts as bidentate to give ML₂ complexes, whose structure depends on the metal center and on the nature of the substituent R″ at the imino carbon. With R″ = CCl₃ an O,O coordination is observed for all the metal centers but one, in fact palladium(II) exhibits an N,O coordination through the imino nitrogen and one keto oxygen. By contrast with R″ = PhCO the ligand is always coordinated through the imino nitrogen and one keto oxygen atom.     

Multiple molecular forms of β-amylase in seeds and vegetative tissues of barley

The molecular forms of -amylase present in developing, mature, germinating and malted grains of barley (Hordeum vulgare L.), and in vegetative tissues, have been studied using Western-blot analyses and isoelectric focusing of isoenzymes. Five isoforms with different relative molecular masses (M_rs) could be recognised. The major isoform present in the mature grain, called isoform B, had an M_r of about 60 000. This was converted on malting or germination to two lower-M_r forms called C and D. Previous work (R. Lundgard and B. Svensson, 1986, Carlsberg Res. Commun. 51, 487–491) has shown that these result from partial proteolysis of isoform B. Isoenzyme analyses showed complex patterns of bands, with pIs between about 5.0 and 6.0. Two allelic types were present in the eight lines. A number of new bands with a range of pIs appeared during germination and malting.An isoform with the same M_r as D and a minor low-M_r isoform (E) were present in young developing whole caryopses (8–12 d after anthesis), but not in older developing endosperms (14–21 d after anthesis). Isoenzyme analyses also showed different patterns of bands in these two tissues, while hybrid-dot analyses indicated the presence of separate populations of mRNAs. It is suggested that the early endosperm isoforms (D and E) are green -amylases present in the pericarp and-or testa of the young caryopses.Roots but not shoots or leaves also contained an isoform with the same M_r as D, although the pattern of isoenzymes differed from that present in the seed tissues.The fifth isoform, A, was a diffuse high-M_r form present in small amounts in all seed and vegetative tissues, and may correspond to a constitutively expressed form.These multiple molecular forms of -amylase are discussed in relation to the recent report that -amylase is encoded by two structural loci, with a total copy number of two to three per haploid genome (Kreis et al, 1988, Genet. Res. Camb. 51, 13–16).Abbreviations M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis