Autolysis of a novel multidomain subtilase-cold-adapted deseasin MCP-01 is pH-dependent and the surface loops in its catalytic domain, the linker, and the P_proprotein domain are susceptible to proteolytic attack

Cold-adapted deseasin MCP-01 is a novel type subtilase with a multidomain structure containing a catalytic domain, a linker, a P_proprotein domain, and a PKD domain. Its autolysis was pH-dependent due to its flexible structure. N-terminal sequence analysis of the autolytic peptides revealed four autolytic sites in the catalytic domain. Three of these are in the same loops as mesophilic subtilases and one is unlike anything previously reported. Two autolytic sites were deduced in its linker and three in its P_proprotein domain, indicating the linker and the P_proprotein domain are flexible and susceptible to proteolytic attacks. Therefore, during MCP-01 autolysis, the linker and the P_proprotein domain of MCP-01 were easily attacked by proteolysis, resulting in cleavage of the C-terminal region. At the same time, some autolytic sites in the surface loops of the catalytic domain were cleaved. This is the first report describing the autolytic mechanism of a multidomain subtilase.     

[14C]epicatechin and [14C]procyanidins from seed shells of Aesculus hippocastanum

Direct injection of sodium-[1-¹⁴C]acetate into growing fruits of horse chestnut provides a convenient route to [¹⁴C]labelled epicatechin and procyanidins.     

A sodium dodecyl sulfate--polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000

A discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described which provides superior resolution of polypeptides with molecular weights from approximately 2500 to 90,000. The system utilizes a relatively low-mobility acetate ion in the stacking gel and high-mobility strong anions, sulfate and chloride, as leading and trailing ions in the separating gel. The entire system is run at pH 7.8. The separating gel contains 8 M urea, and can be used at acrylamide concentrations from 5 to 18%, all with 5% crosslinker concentrations. Using a number of protein standards, the calibration curves obtained with this system are linear over the molecular weight range from 2500 to 90,000, regardless of acrylamide concentration. These studies indicate that by providing good resolution of small peptides, this system greatly extends the utility of one-dimensional peptide mapping techniques.     

Photolabeling of glucose-sensitive cytochalasin B binding proteins in erythrocyte, fibroblast and adipocyte membranes

(³H)Cytochalasin B has been photoincorporated into membrane fractions of the human erythrocyte, Rous sarcoma virus-transformed chicken embryo fibroblast and rat adipocyte. Identification of D-glucose sensitive cytochalasin B binding sites was achieved by photolyzing membranes with radioligand in the presence of 0.5–0.7M D- or L-glucose. In the erythrocyte the major labeled bands on SDS-polyacrylamide gels were at 55,000 and 46,000 daltons. In the virus-transformed fibroblasts a major labeled band was at 55,000 daltons, and in adipocyte microsomal membranes, peaks at 50,000 and 45,000 daltons were observed. Binding characteristics of these polypeptides suggest that they are the putative glucose transport proteins in these three cell types.     

Use of specific glycosidases to probe cellular interactions in the sea urchin embryo

We present an unusual and novel model for initial investigations of a putative role for specifically conformed glycans in cellular interactions. We have used α- and ß-amylase and α- and ß-glucosidase in dose-response experiments evaluating their effects on archenteron organization using the NIH designated sea urchin embryo model. In quantitative dose-response experiments, we show that defined activity levels of α-glucosidase and ß-amylase inhibited archenteron organization in living Lytechinus pictus gastrula embryos, whereas all concentrations of ß-glucosidase and α-amylase were without substantial effects on development. Product inhibition studies suggested that the enzymes were acting by their specific glycosidase activities and polyacrylamide gel electrophoresis suggested that there was no detectable protease contamination in the active enzyme samples. The results provide evidence for a role of glycans in sea urchin embryo cellular interactions with special reference to the possible structural conformation of these glycans based on the differential activities of the α- and ß-glycosidases.     

Intracellular binding of lead in the kidney: The partial isolation and characterization of postmitochondrial lead binding components

Gel chromatography of kidney postmitochondrial fractions from control rats 2 hr after injection of ²⁰³Pb or after in vitro incubation with ²⁰³Pb disclosed the presence of two fractionated Pb-binding components plus binding in the void volume and total volume regions. The binding of Pb to the two components, with molecular weights of 11,500 and 63,000 daltons, was markedly decreased in Pb-pretreated rats. Sodium dodecyl sulfate-gel electrophoresis and autoradiography showed the presence of one major ²⁰³Pb band with an estimated molecular weight of 60,000 daltons. The 11,500-dalton peak did not incorporate ¹⁴C-leucine nor did concomitant administration of cycloheximide with the ²⁰³Pb inhibit incorporation of ²⁰³Pb activity, suggesting that the component is a preformed constituent of the kidney. In vitro incubation of brain, liver and lung postmitochondrial supernatants with ²⁰³Pb disclosed that these two binding components were also present in brain but not in liver or lung, suggesting a target tissue-specific localization for these Pb-binding macromolecules.     

Characterization of a zebrafish estrogen-sulfating cytosolic sulfotransferase: inhibitory effects and mechanism of action of phytoestrogens

Cytosolic sulfotransferases (STs) are generally thought to be involved in detoxification of xenobiotics, as well as homeostasis of endogenous compounds such as thyroid/steroid hormones and catecholamine hormones/neurotransmitters. We report here the identification and characterization of a zebrafish estrogen-sulfating cytosolic ST. The zebrafish ST was bacterially expressed, purified, and examined for enzymatic activities using a variety of endogenous compounds as substrates. Results showed that the enzyme displayed much higher activities toward two endogenous estrogens, estrone (E(1)) and 17beta-estradiol (E(2)), in comparison with thyroid hormones, 3,3',5-triiodothyronine (T(3)) and thyroxine (T(4)), dopamine, dihydroxyphenylalanine (Dopa), and dehydroepiandrosterone (DHEA). The kinetic parameters, K(m), and V(max), with estrogens and thyroid hormones as substrates were determined. The calculated V(max)/K(m) for E(1), E(2), T(3), and T(4) were, respectively, 31.6, 16.7, 1.5, and 0.8 nmol min(-1) mg(-1) microM(-1), indicating clearly the estrogens being preferred physiological substrates for the enzyme. The inhibitory effects of isoflavone phytoestrogens on the sulfation of E(2) by this zebrafish ST were examined. The IC(50) determined for quercetin, genistein, and daidzein were 0.7, 2.5, and 8 microM, respectively. Kinetic analyses revealed that the mechanism underlying the inhibition by these isoflavones to be of the competitive type.     

The lack of movement of [14C]-phenylalanine and [14C]-cinnamate after administration to leaves of Polygonum and wheat

U-¹⁴C-phenylalanine and 3-¹⁴C-cinnamic acid were fed to detached Polygonum leaves through the cut petioles and to the bases of detached wheat leaves. After feeding, the leaves were divided into basal, middle and terminal segments; for each treatment of each plant more than 80% of the total radioactivity incorporated remained in the basal segment. The distribution of radioactivity between ethanol-soluble and insoluble fractions in each segment was examined. The basal segments contained more insoluble radioactivity than the terminal ones; the differences were far more marked for both plants when cinnamate rather than phenylalanine was administered. In view of the gross differences in distribution of radioactivity between the basal and terminal segments of each leaf, it is concluded that basal infusion of precursors is not the most suitable technique for studies of phenolic biosynthesis.     

Retinoids increase the incorporation of D-[3H]galactose into epidermal glycoproteins

All-trans retinoic acid increased the incorporation of D-[³H]galactose into particulate and soluble glycoproteins in the epidermis of cultured pig skin slices nearly two-fold. Increased incorporation of D-[³H]galactose was not blocked by tunicamycin. This effect was specific for D-[³H]galactose since the incorporation of D-[³H]glucosamine and L-[¹⁴C]leucine into epidermal glycoproteins was unaffected by all-trans retinoic acid. All-trans retinoic acid and 13-cis retinoic acid had quantitatively similar effects on D-[³H]galactose incorporation. All-trans retinyl acetate and an aromatic retinoic acid analogue (‘Etretinate’) were less effective. SDS polyacrylamide gel electrophoresis and fluorography showed increased incorporation of D-[³H]galactose into all epidermal glycoproteins in the presence of all-trans retinoic acid. There was no evidence for synthesis of new glycoproteins such as mucins.     

Desaturation of oleate-[14C] in leaves of barley

Etiolated barley leaves when exposed to light desaturate oleate-[¹⁴C] to linoleate. The production of substantial amounts of radioactive linolenate was found only in very young, tightly rolled leaves. In oleate-[¹⁴C] pulse experiments, radioactive linolenate first appeared in phosphatidylcholine (PC) and only after a lag period did it begin to accumulate in monogalactosyldiacylglycerol (MGDG). The results indicate that in young, immature barley leaves linolenate is synthesized from oleate on the parent lipid, PC, and is then transferred to MGDG.     

Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase

Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 ± 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum raised against the β-subunit of mitochondrial ATPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subcellular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial F₁F₀-ATPase and the lysosomal V-type ATPase.     

Direct photoaffinity labeling of the primary region of the ouabain binding site of (Na+ + K+)-ATPase with [3H]ouabain, [3H]digitoxin and [3H]digitoxigenin

The tritiated cardiotonic steroids, ouabain, digitoxin, and digitoxigenin are shown to photolabel the large polypeptide but not the glycoprotein or proteolipid component of the (Na⁺ + K⁺)-ATPase when they are bound to the inhibitory site and exposed to light of 220 or 254 nm. The extent of photolabeling is low, less than 1%, and is limited by photocross-linking of the enzyme. The mechanism of photoincorporation does not appear to be either photolysis of the lactone ring in ouabain or photolysis of tryptophan or tyrosine residues in the polypeptide.     

Expression,purification and preliminary biochemical studies of the N-terminal domain of leucine-rich repeat kinase 2

Leucine-rich repeat kinase 2 gene is a key factor for Parkinson's disease and encodes for a large protein kinase LRRK2 (280 kDa) with multiple domains, including the different repeat sequences at the N-terminus such as ankyrin domain. Here, we successfully expressed and purified two kinds of LRRK2's N-terminal fragments N1 (aa12–320) and N2 (aa12–860). The purified N2 protein was identified by mass spectrometry and N1's molecular weight was determined to be 33.23 kDa. Gel filtration revealed that N1 exhibits as monomer, dimer and tetramer and N2 as oligomer in solution. N1's multiple oligomeric states were further proved by native-page and cross-linking gel experiments. Circular dichroism spectrum indicated that N1 and N2 contain both α helixes and β sheets. The polymerization character of LRRK2 N-terminal region would be speculated to relate with its biological function.     

Indole-3-carbinol protects against covalent binding of benzo[a]pyrene and N-nitrosodimethylamine metabolites to mouse liver macromolecules

Benzo[a]pyrene (BaP) and N-nitrosodimethylamine (NDMA) are carcinogens and indirect acting mutagens. A naturally occurring dietary indole, indole-3-carbinol (I-3-C), has been shown to decrease the incidence of aryl hydrocarbon induced neoplasia in experimental animals. We examined the relationship between the ability of I-3-C to alter the rate of carcinogen oxidation and its ability to decrease the rate of covalent binding of carcinogen metabolites to DNA and protein. We found that I-3-C inhibited the covalent binding of NDMA oxidation products to DNA in vitro in proportion to its ability to inhibit carcinogen metabolism. Pretreatment of mice by gavage with I-3-C resulted in no change in the rate of aryl hydrocarbon hydroxylase or NDMA demethylase in hepatic post-mitochondrial supernatant. However, this pretreatment resulted in a 60-90% decrease in the ability of carcinogen oxidative metabolites to bind covalently to DNA or protein in vitro. Similarly, in in vivo experiments, gavage with I-3-C, followed by gavage with BaP or NDMA, resulted in a 63-85% decrease in covalent binding to macromolecules, with no concomitant change in carcinogen metabolism. The results suggest that the in vivo administration of I-3-C may confer protection for hepatic macromolecules against covalent binding of the metabolites of these two indirect acting mutagens.     

Mutation of the small acidic tract A1 drastically reduces nucleoplasmin activity

Xenopus laevis nucleoplasmin is a molecular chaperone that mediates sperm decondensation and nucleosome assembly. Nucleoplasmin has three acidic tracts (A1, A2 and A3) and until recent years the long polyglutamic tract A2 was thought to be the binding site for basic proteins. However, the latest publications in this field show that neither A2 nor A3 is indispensable for histone and sperm-specific protein binding. In this work, we show that the mutation of only four acidic amino acid residues of the small A1 tract drastically reduces nucleoplasmin decondensing activity, pointing out this region as the potential binding site for sperm proteins.     

Polymorphism of 14C vitamin D3 binding protein in cattle and water buffalo serum

Cattle and water buffalo sera labelled with vitamin D₃[¹⁴C] (300 and 480 individual samples respectively) were subjected to starch gel electrophoresis followed by autoradiography in an attempt to identify a possible polymorphism of the proteins capable of binding this vitamin.
Three phenotypes controlled by two codominant autosomal alleles were identified in cattle while in water buffalo six phenotypes controlled by three codominant autosomal alleles were observed.     

Structural and functional abnormalities in the adipocyte plasma membrane from db/db mouse, and the effect on the abnormalities of oral treatment with AS-6

The protein bands of adipocyte plasma membranes from the genetically obese diabetic mice C57BL/KsJ db/db (db/db mice) showed slight but significant changes compared with their lean littermates. The treatment for 1 week with a new antidiabetic agent, AS-6, caused the changes to revert toward the condition in the lean littermates. In the absence of insulin, the plasma membrane and mitochondria mixture (P3 fraction) of the lean littermates densely labeled 55000 and 57000 dalton protein bands by phosphorylating with (a-32P)-ATP, whereas the labeling was less in the P3 from AS-6 treated and untreated db/db mice. Insulin inhibited phosphorylation of these bands in P3 from the lean littermates and untreated db/db mice, while the hormone enhanced the labeling in AS-6 treated db/db mice compared with the basal condition without insulin. Ca2+ greatly enhanced the labeling in all three groups, whereas Mg2+ mimicked the insulin action diminishing the labeling of these bands in the lean and untreated db/db groups. However, Mg2+ enhanced the phosphorylation in the P3 from AS-6 treated db/db mice compared with the basal condition.     

The incorporation of [3H]glucosamine, [3H]fucose and [14C]mannose into protein in the rat anterior pituitary incubated in vitro

Rat anterior hemipituitaries incubated in vitro rapidly take up and incorporate into protein D-[6-³H]-glucosamine · HCl, D-[1-¹⁴C]mannose and L-[G-³H]fucose. The newly labeled protein was only slowly released into a Krebs-Ringer bicarbonate incubation medium. Glucosamine- or mannose-labeled protein was barely detectable in the medium after a 30–60 min incubation whereas about 4% of all fucose-labeled protein had already been released into the incubation medium by 30 min. Puromycin · 2HCl (1 mM) inhibited incorporation of glucosamine or mannose into protein to 40% or less of control values within 30 min; fucose incorporation was not significantly inhibited before 45 min. Acid hydrolysis followed by amino acid analysis of glucosamine-labeled protein yielded significant amounts of label in glucosamine, galactosamine and apparent glucosamine-degradation products but no significant amount of label in any amino acid.