Fraction IIA Research Articles

Reduction of nitrates in Cucumis sativus L. seedlings II. Influence of tungsten and vanadium on nitrate reductase and adenosine triphosphatase activities

ATPases isolated from the roots of cucumber seedlings activated by Mg<sup>+2</sup> ions in experiments in vitro, were fairly distinctly inhibited by Ca<sup>-2</sup> ions, very slightly inhibited by fluorides and molybdenum ions while NO<sub>3</sub><sup>-</sup> anions had no effect on the level of ATPase activity studied. Introduction into the nutrient of 10<sup>-4</sup> M Na<sub>2</sub>WO<sub>4</sub> or 10<sup>-3</sup> M Na VO<sub>3</sub> (inhibitors of nitrate reductase NR) distinctly inhibited activity of the ATPase under study especially of fractions IIa and III, and inhibited NR activity and lowered uptake of NO<sub>3</sub><sup>-</sup>. WO<sub>4</sub><sup>-2</sup> and VO<sub>3</sub><sup></sup> inhibited to the same extent absorption and reduction of NO<sub>3</sub><sup>-</sup> in the initial phase of NR induction, whereas at a later stage both inhibitors checked reduction to a greater degree than uptake of NO<sub>3</sub><sup>-</sup>. The results indicate the possibility of certain ATPase participation in assimilating nitrates, and suggest that in the initial stage of biosynthesis of the NR enzyme system, activity of the enzyme is distinctly dependent upon NO<sub>3</sub><sup>-</sup> transport and the level of NR activity limited by the amount of nitrate taken up. At a later an additional mechanism of NO<sub>3</sub><sup>-</sup> transport probably functions, not connected with simultaneous reduction of nitrates. On the basis of results the Butz and Jackson (1977) hypothesis concerning a model for the absorption and reduction of NO<sub>3</sub><sup>-</sup> by plant tissues is discussed.

Structural Characterization of Mesquite (Prosopis velutina) Gum and its Fractions

Structural and physicochemical characteristics of mesquite gum (from Prosopis velutina) were investigated using FT-IR spectroscopic, mass spectrometric and chromatographic methods. Four fractions (F-I, F-IIa, F-IIb and F-III) were isolated by hydrophobic interaction chromatography. The samples were characterized and analyzed for their monosaccharide and oligomers composition by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). L-Arabinose (L-Ara) and D-galactose (D-Gal) were found as the main carbohydrate constituent residues in the polysaccharides from mesquite gum and their ratio (L-Ara/D-Gal) varied within the range 2.54 to 3.06 among the various fractions. Small amounts of D-glucose (D-Glc), D-mannose (D-Man) and D-xylose (D-Xyl) were also detected, particularly in Fractions IIa, IIb and III. Infrared spectroscopy identified polysaccharides and protein in all the samples. Data from mass spectrometry (MALDI-TOF MS) was consistent with the idea that the structure corresponding to the periphereal chains of Fraction I is predominantly a chain of pentoses attached to uronic acid.

Assembly of Lysine 63-linked Ubiquitin Conjugates by Phosphorylated α-Synuclein Implies Lewy Body Biogenesis

alpha-Synuclein (alpha-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of alpha-syn in LBs is phosphorylated at serine 129 (Ser(129)). However, the role of Ser(129)-phosphorylated alpha-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)alpha-syn and Ub, coexpression of phospho-mimic mutant alpha-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D alpha-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT alpha-syn, S129D alpha-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H(2)O(2) and serum deprivation. These results suggest that the contribution of Ser(129)-phosphorylated alpha-syn to the Lys(63)-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies.

α-Synuclein and Parkin Contribute to the Assembly of Ubiquitin Lysine 63-linked Multiubiquitin Chains

Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.

Potential Chemopreventive Properties of Extract from Baked Sweet Potato (Ipomoea batatas Lam. Cv. Koganesengan)

The extract from baked sweet potato (Ipomoea batatas Lam. cv. Koganesengan) showed potential cancer-preventing effects. The extract was partially fractionated to four fractions (I, II-a II-b, and III) by Sephadex G-25 gel chromatography. The cytotoxicity against human myelocytic leukemia HL-60 cells, the suppression of TPA-induced transformation in mouse skin JB6 C141 cells, the apoptosis inducing activity in HL-60 cells, and the scavenging capacity against DPPH radical were tested on the four fractions. Fractions II-a and III showed markedly strong radical scavenging effects on the DPPH radical, coinciding with the high content of total phenolic compounds in the fractions. Both of these fractions suppressed strongly the proliferation of HL-60 cells with apoptosis induction in a dose-dependent manner. Moreover, the two fractions markedly blocked TPA-induced cell transformation in the JB6 cell line. Taken together, these data suggest that the water extract from baked sweet potato had potential chemopreventive properties.

Macromolecular dimensions and mechanical properties of monolayer films of Sonorean mesquite gum.

Mesquite gum sourced from Prosopis velutina trees and gum arabic (Acacia spp.) were characterized using light scattering and Langmuir isotherms. Both gum materials were fractionated by hydrophobic interaction chromatography, yielding four fractions for both gums: FI, FIIa, FIIb and FIII in mesquite gum and FI, FII, FIIIa and FIIIb in gum arabic. In mesquite gum, the obtained fractions had different protein content (7.18-38.60 wt.-%) and macromolecular dimensions (M approximately 3.89 x 10(5)-8.06 x 10(5) g.mol(-1), RG approximately 48.83-71.11 nm, RH approximately 9.61-24.06 nm) and architecture given by the structure factor (RG/RH ratio approximately 2.96-5.27). The mechanical properties of Langmuir monolayers at the air-water interface were very different on each gum and their fractions. For mesquite gum, the most active species at the interface were those comprised in Fractions IIa and IIb and III, while Fraction I the pi/A isotherm lied below that of the whole gum. In gum arabic only Fraction III developed greater surface pressure at the same surface per milligram of material than whole gum. This is rationalized in terms of structural differences in both materials. Mesquite gum tertiary structure seems to fit best with an elongated polydisperse macrocoil in agreement with the "twisted hairy rope" proposal for arabinogalactan proteoglycans.

Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test.

It has been shown recently that a flavonoid fraction (fraction II) obtained from a crude extract of Hypericum perforatum (St. John's Wort) was remarkably active in the forced swimming test (FST). Fraction II was further separated using MLCCC to give fractions IIa and IIb. Both fractions proved to be active in the FST at different dosages. Further separation of fraction IIa by preparative HPLC yielded fraction IIa1 which mainly was composed of hyperoside, isoquercitrin, miquelianin and quercitrin, and fraction IIa2 which contained small amounts of hyperoside and astilbin, while most compounds were not known. Both fractions were active after acute treatment in the FST. Isolates obtained from these fractions including hyperoside, isoquercitrin, quercitrin, miquelianin, the aglycone quercetin and astilbin, were tested for activity in the FST. Except for quercetin, quercitrin and astilbin all compounds were active. To exclude false positive results in the FST the validity was checked in open field experiments and in the FST after 12 days of daily treatment.

Cellular proteins required for adeno-associated virus DNA replication in the absence of adenovirus coinfection.

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3' primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase alpha-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase delta and epsilon, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase delta or polymerase epsilon for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.

Characterization of synaptosomal membrane Na +, K +-ATPase inhibitors

Previous work carried out in this laboratory has led to the isolation from rat brain of an aqueous soluble fraction (peak II) inhibiting synaptosomal membrane Na +, K +-ATPase and possessing other ouabain-like properties. Brain peak II was subjected to several treatments or fractionation by reversed-phase or anionic exchange HPLC and the effect of resultant fractions tested on synaptosomal membrane ATPase activity. The inhibitory components proved highly hydrophilic since they were neither extracted by hexane nor retained by a C-18 HPLC column, ruling out a lipidic nature. By anionic exchange chromatography, peak II was separated into eight fractions, two of which, named II-A and II-E, presented inhibitory activity, had low molecular weight, reacted with ninhydrin and were sensitive to acid hydrolysis. Fraction II-A was further chromatographed through a C-18 column, rendering five fractions, II-A, to II-A 5 inhibitory activity being confined to the most hydrophilic one (II-A 1). Fraction II-E seems non-peptidic in nature, and its inhibitory activity was completely lost by alkalinization. II-E differs from authentic ouabain in u.v. spectrum, chromatographic behaviour and alkali sensitivity. It is suggested that two small hydrophilic compounds, probably one peptidic (II-A 1) and another non-peptidic (II-E) in nature are involved in the regulation of Na +, K +-ATPase activity at the synaptic membranes.

Resolution and some properties of enzymes involved in enantioselective transformation of 1,3-dichloro-2-propanol to (R)-3-chloro-1,2-propanediol by Corynebacterium sp. strain N-1074.

During the course of the transformation of 1,3-dichloro-2-propanol (DCP) into (R)-3-chloro-1,2-propanediol [(R)-MCP] with the cell extract of Corynebacterium sp. strain N-1074, epichlorohydrin (ECH) was transiently formed. The cell extract was fractionated into two DCP-dechlorinating activities (fractions Ia and Ib) and two ECH-hydrolyzing activities (fractions IIa and IIb) by TSKgel DEAE-5PW column chromatography. Fractions Ia and Ib catalyzed the interconversion of DCP to ECH, and fractions IIa and IIb catalyzed the transformation of ECH into MCP. Fractions Ia and IIa showed only low enantioselectivity for each reaction, whereas fractions Ib and IIb exhibited considerable enantioselectivity, yielding R-rich ECH and MCP, respectively. Enzymes Ia and Ib were isolated from fractions Ia and Ib, respectively. Enzyme Ia had a molecular mass of about 108 kDa and consisted of four subunits identical in molecular mass (about 28 kDa). Enzyme Ib was a protein of 115 kDa, composed of two different polypeptides (about 35 and 32 kDa). The specific activity of enzyme Ib for DCP was about 30-fold higher than that of enzyme Ia. Both enzymes catalyzed the transformation of several halohydrins into the corresponding epoxides with liberation of halides and its reverse reaction. Their substrate specificities and immunological properties differed from each other. Enzyme Ia seemed to be halohydrin hydrogen-halide-lyase which was already purified from Escherichia coli carrying a gene from Corynebacterium sp. strain N-1074.

Purification of DNA polymerase delta as an essential simian virus 40 DNA replication factor

DNA replication from the SV40 origin can be reconstituted in vitro using purified SV40 large T antigen, cellular topoisomerases I and II, replication factor A (RF-A), proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and a phosphocellulose fraction (IIA) made from human cell extracts (S100). Fraction IIA contains all DNA polymerase activity required for replication in vitro in addition to other factors. A newly identified factor has been purified from fraction IIA. This factor is required for complete reconstitution of SV40 DNA replication and co-purifies with a PCNA-stimulated DNA polymerase activity. This DNA polymerase activity is sensitive to aphidicolin, but is not inhibited by butylanilinodeoxyadenosine triphosphate or by monoclonal antibodies which block synthesis by DNA polymerase alpha. The polymerase activity is synergistically stimulated by the combination of RF-A, PCNA, and RF-C in an ATP-dependent manner. Purified calf thymus polymerase delta can fully replace the purified factor in DNA replication assays. We conclude that this factor, required for reconstitution of SV40 DNA replication in vitro, corresponds to human DNA polymerase delta.

Studies on Wakan-Yakus (traditional herbal drugs) : Especially on the effects of Gaiyoh (Artemisiae folium) on blood coagulation

Wakan-Yakus (traditional herbal drugs) such as Akyoh (Glutinum), Gaiyoh (Artemisiae folium), Sanshishi (Gardeniae fructus), Kizutsu (Aurantii fructus), and Taisoh (Zizyphi fructus) were studied in relation to their effects on blood coagulation-fibrinolysis. (1) All of the water extracts of the Wakan-Yakus prolonged aPTT and PT. The potency of the effectiveness on aPTT was in the order of Gaiyoh (Artemisiae folium)> Kizutsu (Aurantii fructus)> Sanshishi (gardeniae fructus)> Taisoh (Zizyphi fructus )> Akyoh (Glutium). (2) Gaiyoh (Aremisiae folium)> Kizutsu (Aurantii fructus) Akyoh (glutinum) Taisoh (Zizyphi fructus) showed the antifibrinolytic effects in this order. On the other hand, Sanshishi showed the accelerating effect on fibrinolysis. (3) The inhibition modes of both thrombin and plasmin by Gaiyoh (Artemisiae folium) were shown to be competitive on Lineweaver-Burk plot. (4) Gayoh (Artemisiae folium) was gel-filtrated on Sephadex G-25 column (1.5×90cm) equilibrated with distilled water at room temperature. Five fractions were obtained, and in the first to fourth fraction, strong anticoagulant effects on aPTT and PT were observed. We pooled first and second to make fraction I, and make fraction II from peak 3. The recovery rate was 4.2 % by weight, and 36.7 % by inhibition activity, and specific activity on the basis of inhibition to aPTT was 34.8 % U/mg in the case with fraction II. Fraction I was found to be the same characteristically on blood coagulation. Fraction II was further purified by Sephadex LH-20 column (1.5 × 80 cm) at room temperature. Three fractions (Fraction IIa, IIb, IIc) were obtained, and the strong inhibitory effects was observed on both aPTT and PT in each fraction. The first fraction (fraction IIa) showed the strong inhibitory effect on aPTT, and the heightened specific activity with 17.6 % as the recovery rate. Fraction IIb showed strong inhibitory effect on fibrinolysis. Fraction IIb and IIc were found to be similar with fraction IIa characteristically on blood coagulation-fibrinolysis, but could separate the substance for coagulation and fibrinolysis each other on the basis of weight/volume. (5) by ex vivo study using rabbits, aPTTs were prolonged gradually after infusion (60 mg of fraction IIa). White blood cells and platelets were decreased gradually.

Role of inositol-containing sphingolipids in Saccharomyces cerevisiae during inositol starvation.

The in vitro lipid requirements of UDP-N-acetylglucosamine-dolichol phosphate N-acetylglucosamine-1-phosphotransferase for the inositol-containing sphingolipids from Saccharomyces cerevisiae were characterized in terms of concentration and specificity. The effects of combinations of lipids, especially phosphatidylinositol and the inositol-containing sphingolipids, were also tested on the transferase. Phosphatidylinositol and phosphatidylglycerol stimulated the enzyme 3.3- and 2.8-fold, respectively. The inositol-containing sphingolipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine did not stimulate the activity of the transferase. Phosphatidylcholine and phosphatidylethanolamine in combination with phosphatidylinositol had no effect on the transferase activity; however, the inositol-containing sphingolipids markedly inhibited the stimulation of the transferase by phosphatidylinositol. This inhibition by the sphingolipids was prevented if phosphatidylcholine, in addition to the other lipids, was present in the assay mixture. In addition, changes due to inositol starvation in the in vivo membrane lipid environment, i.e., phosphatidylinositol and the inositol-containing sphingolipids, were analyzed to determine whether they corresponded to the observed in vitro effects. Three hours after the beginning of inositol starvation, there were 9- and 14-fold reductions in the accumulation of phosphatidylinositol in membrane fractions IIA (vesicles) and IV (endoplasmic reticulum), respectively, although there was only a 6-fold reduction in membrane fraction I (plasma membrane). The accumulation of [14C]inositol into inositol-containing sphingolipids also reflected the differences in the cellular location of membranes.

Dephosphorylation of skeletal muscle phosphorylase, glycogen synthase, and phosphorylase kinase β-subunit by a Mn 2+-activated protein phosphatase

An Mn 2+-activated phosphoprotein phosphatase of M r = 80,000 from rabbit muscle catalyzes the dephosphorylation of skeletal muscle proteins that are phosphorylated by either phosphorylase kinase or cAMP-dependent protein kinase. Phosphorylase or glycogen synthase labeled by phosphorylase kinase at seryl residues 14 or 7, respectively, are both dephosphorylated by the phosphatase. Phosphorylase a and glycogen synthase compete with one another for the phosphatase. The phosphatase discriminates between different sites labeled by the cAMP-dependent protein kinase: glycogen synthase phosphorylated either to 1.0 or 1.8 mol phosphate/mol, or phosphorylase kinase phosphorylated on its β-subunit serve as substrates for the phosphatase, but the phosphorylase kinase α-subunit, the phosphorylated phosphatase inhibitor 1, or casein do not. Histone fraction IIA, phosphorylated by the catalytic subunit, was a poor substrate even at a concentration of 100 μ m. Phosphorylation of the α-subunit of phosphorylase kinase had no influence on the kinetics of dephosphorylation of the β-subunit. Thus, the M r = 80,000 phosphatase meets the functional definition of a protein phosphatase 1 [Cohen, P. (1978) Curr. Top. Cell. Regul. 14, 117–196]. Furthermore, from a comparison of the known phosphorylated sites of these proteins, it appears that the phosphatase discriminates between different sites present in the phosphoproteins tested on the basis of the K m values for the reactions. It displays a preferential activity toward proteins with a primary structure wherein basic residues are two positions amino-terminal from the phosphoserine, AgrLysX-YSer(P) or LysArgX-YSer(P), rather and one residue away, ArgArgX-Ser(P).

An improved method of preparing rat brain synaptic membranes. Eliminatio of a contaminating membrane containing 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity

Synaptosomes were prepared from rat cortex by subjecting a washed crude mitochondrial pellet to centrifugation first on discontinuous Ficoll-isotonic sucrose gradients and then on discontinuous sucrose gradients. The synaptosome fraction, collected from the 7.5–14% Ficoll band (II), was further separated into two additional fractions, designated IIA and IIB, which band at the 0.32–1.05 M and at the 1.05–1.6 M sucrose interfaces, respectively. Electron microscopic analysis showed that fraction IIB contained synaptosomes and extra terminal mitochondria and was essentially free of membrane fragments. Further characterization showed that IIB contained 69% of the protein and 83% of the lactic dehydrogenase activity of fraction II and had a specific activity of a 2′,3′-cyclic nucleotide 3′-phosphohydrolase approximately 1% of that obtained with myelin. Fraction IIA had approximately 50% the specific activity of the 2′,3′-cyclic nucleotide 3′-phosphohydrolase found in myelin. Synaptic plasma membranes were prepared by lysing fraction IIB in 1 mM sodium phosphate, 0.1 mM EDTA at pH 8.5 and subjecting this preparation to centrifugation on a discontinuous sucrose density gradient. Enzymatic analysis indicated that membranes banding at the 0.6–0.8 M sucrose interface had highgh specific activities of plasma membrane enzymes (e.g. acetylcholinesterase, ATPase, 5′-nucleotidase). The specific activity of the (Na + + K +)-ATPase in the purified membrane preparation was 8-fold higher than that in the original homogenate. Specific activities of various marker enzymes indicated that the composition of these membrane preparations for the most part was synaptic plasma membranes, approximately 7% mitochondrial outer membranes and 3% a membrane containing 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity. The polypeptide compositions of three possible contaminating membranes and of synaptic membranes were compared by electrophoresis in 6–20% gradient polyacrylamide gels in the presence of sodium dodecyl sulfate. Whereas mitochondrial and myelin membranes had distinct compositions, the compositions of the microsomal and synaptosomal plasma membranes were similar. Synaptic plasma membranes contained at least 27 polypeptides; the three major polypeptdes had molecular weights of 103,000; 54,000; and 50,000. The major polypeptides of soluble synaptosomal proteins had molecular weights of 54,000 and 42,000.

Characterization of cell wall polymers secreted into the growth medium of lysis-defective pneumococci during treatment with penicillin and other inhibitors of cell wall synthesis.

Autolysin-defective pneumococci secrete large quantities of choline-containing cell wall polymers into the growth medium during treatment with inhibitors of peptidoglycan synthesis. The secreted polymers were separated into three fractions by a combination of gel filtration on agarose and sodium dodecyl sulfate-gel electrophoresis. Fraction I had a high apparent molecular size and contained the Forssman antigen in complex with material exhibiting properties of cell wall teichoic acid. Choline-containing polymers of as yet uncharacterized structure were present in both fractions IIA and IIB, and fraction IIA also contained peptidoglycan components.

Enzymic hydrolysis of the potassium chloride soluble fraction of carrageenan: properties of "lambda carrageenases" from Pseudomonas carrageenovora.

An enzyme complex which hydrolyzed the KCl soluble carrageenan extracted from the red alga Chondrus crispus has been isolated from the cell-free medium of a culture of Pseudomonas carrageenovora grown on this polysaccharide. Three hydrolases could be separated. Fraction I, which caused a rapid decrease in the specific viscosity of the polysaccharide preparation with only minimal release of reducing sugar, could be distinguished from fraction II chromatographically on Sephadex G-100 and electrophoretically on agarose gel. Fraction IIa caused release of reducing sugar at pH 6.2, which activity was depressed at pH 7.5. Fraction IIb exhibited viscometric activity only at both pH 6.2 and pH 7.5. Fraction IIa had a sharp pH optimum at pH 6.2 and a temperature optimum at 28°. All hydrolases were inactivated by freezing, by dialysis against distilled water, by heating at 35° for 30 min, and by Hg2+ and 0.0001 mM EDTA. When fraction II (a and b) isolated after chromatographic resolution on Sephadex G-100 was incubated at pH 6.2 with KCl soluble carrageenan from C. crispus, products which had Rgal values of 0.74 and 0.17 were detected, were sulfated, and contained no 3,6-anhydrogalactose.

Sugar Transport

Abstract The Enzymes II of a bacterial phosphotransferase system are sugar-specific membrane components, most of which are inducible. The present studies are concerned with the constitutive Enzymes II of Escherichia coli. In the presence of purified Enzyme I and HPr of the phosphotransferase system, an E. coli membrane fraction catalyzed the transfer of phosphate from phosphoenolpyruvate to d-glucose, d-mannose, and d-fructose. Extraction of the membrane fraction with a mixture of urea and 1-butanol gave a protein fraction (II-A), which catalyzed the reaction only in the presence of the residual pellet. Fraction II-A was purified free of lipid, and subfractionated by isoelectric focusing to three proteins, each being required for phosphorylation of one of the sugars. The pellet was treated with deoxycholate and fractionated, giving another protein fraction (II-B) which was purified free of lipid. The combination of II-A and II-B was inactive in the phosphorylation reaction unless supplemented with divalent cation and with crude E. coli lipid. A minor component of crude E. coli lipid, phosphatidylglycerol, was found to be the active lipid factor. Thus, reconstitution of Enzyme II activity, specific for one of the three sugars, was effected by combining the corresponding specific II-A with II-B, phosphatidylglycerol, and either Ca++ or Mg++. However, activity was dependent upon the sequence of mixing of the four components. The activity of reconstituted Enzyme II was found in a sedimentable pellet. Further study of this phenomenon showed that II-B, phosphatidylglycerol, and divalent cation interacted to give a sedimentable pellet which catalyzed the phosphorylation of the sugars in the presence of II-A and the phospho-HPr generating system. Activity of the pellet was studied as a function of the concentration of phosphatidylglycerol added to the mixture, and the optimum was reached when all of the lipid sedimented with the protein; additional quantities of lipid gave less active pellets. At the optimum, the pellet contained II-B and lipid in a ratio of 1:1 (w/w). Each of the II-A proteins appeared homogeneous when examined by disc gel electrophoresis. Protein II-B, which showed a marked tendency to aggregate in the absence of detergent, was studied by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate, and only one protein band was detected, its mobility corresponding to a molecular weight of about 36,000. If II-B is homogeneous it represents at least 10% of the protein in the membrane preparation.

The identification of metabolites of retinal and retinoic acid in rat bile

1. 1. The present work was conducted to determine whether retinoic acid is normally formed from retinal in the rat and what the metabolic products of retinoic acid are. Within 6 h after the intraportal injection of 3 mg of [6,7- 14C 2]retinal to rats with cannulated bile ducts, a minimum of 6% of the injected dose appeared in the bile as free or conjugated acid. 2. 2. In similar experiments with 6,7- 14C 2]retinoic acid (Fraction IIA) was demonstrated both in the liver and the intestine at 4.5 h, and in the bile which was collected for 17 h. Non-acidic esters of retinoic acid accounted for at least 60% of the radioactivity in Fraction I of bile, whereas retinoyl-β-glucuronide accounted for at least 45% of the radioactivity in Fraction III of bile. Furthermore, another acidic metabolite of retinoic acid (Fraction IIB) accounted for about 10% of the biliary radioactivity. 3. 3. We concluded that retinoic acid is normal, but not necessarily an obligatory, metabolite of retinal in the rat, and that retinoic acid is rapidly converted in the liver to its β-glucuronide and to other conjugates which are excreted in the bile. Presumably little or no cleavage of the carbon chain of retinoic acid occurs during these processes.

The acid extraction of histones from calf thymus deoxyribonucleoprotein

Histones extracted from calf thymus deoxyribonucleoprotein at several low pH values have been fractionated by chromatography on Amberlite IRC50. Chromatographic fractions of the histones were characterized further by their amino acid composition, behavior on zone electrophoresis, and fingerprints of tryptic digests. Histone was not removed from the nucleoprotein at or above pH 2·65. Only histone fractions la and lb (arginine/lysine ratio, about 0·1) were removed in the pH interval 2·6 to 1·75. Below pH 1·75, histone fractions lib, III and IV (arginine/lysine ratios, 0·6, 1·4 and 1·4) were removed simultaneously, but histone fraction IIa (arginine/lysine ratio, 1·1) was extracted only below pH 1·2. Removal of histone was complete at pH 0·7, but the residual material contained much protein. From the size and composition of the histone fractions, it appears that the ease of removal of the fractions from nucleic acid is inversely proportional to the number of ionic bonds to be broken. When removal (partial or complete) of histones by acid was effected at temperatures below 0°C, the remaining nucleoprotein was of high molecular weight; it was not depurinated and its DNA was not denatured. The progressive removal of histones led to an enhanced efficiency in priming RNA synthesis.