Purification and Properties of NADP-Dependent Maltose Dehydrogenase Produced by Alkalophilic Corynebacterium sp. No. 93–1

NADP-dependent maltose dehydrogenase (NADP-MalDH) was completely purified from the cell free extract of alkalophilic Corynebacterium sp. No. 93–1. The molecular weight of the enzyme was estimated as 45,000~48,000. The enzyme did not have a subunit structure. The isoelectric point of the enzyme was estimated as pH 4.48. The pH optimum of the enzyme activity was pH 10.2, and it was stable at pH 6 to 8. The temperature optimum was 40°C, and the enzyme was slightly protected from heat inactivation by 1 mm NADP. The enzyme oxidized d-xylose, maltose and maltotriose, and the Km values for these substrates were 150mm, 250 mm and 270 mm, respectively. Maltotetraose and maltopentaose were suitable substrates. The Km value for NADP was 1.5 mm with 100mm maltose as substrate. The primary product of this reaction from maltose was estimated as maltono-δ-lactone, and it was hydrolyzed non-enzymatically to maltobionic acid. The enzyme was inhibited completely by PCMB, Ag⁺ and Hg²⁺.     

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract

Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; A_dmf or A_Pac, G_dmf, C_ibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH₄OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N⁴ -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) as a control monomer and digestion by 3′-5′ selective P1 nuclease and alkaline phosphatase and HPLC analysis. Oligoribonucleotides were analyzed by Microgel capillary electrophoresis, anion-exchange HPLC, and the enzymatic digest/HPLC method.

     

Purification and characterization of a cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3

Purification of a cis-epoxysuccinic acid hydrolase was achieved by ammonium sulfate precipitation, ionic exchange chromatography, hydrophobic interaction chromatography followed by size-exclusion chromatography. The enzyme was purified 177-fold with a yield of 14.4%. The apparent molecular mass of the enzyme was determined to be 33 kDa under denaturing conditions. The optimum pH for enzyme activity was 7.0, and the enzyme exhibited maximum activity at about 45 °C in 50 mM sodium phosphate buffer (pH 7.5). EDTA and o-phenanthrolin inhibited the enzyme activity remarkably, suggesting that the enzyme needs some metal cation to maintain its activity. Results of inductively coupled plasma mass spectrometry analysis indicated that the cis-epoxysuccinic acid hydrolase needs Zn²⁺ as a cofactor. Eight amino acids sequenced from the N-terminal region of the cis-epoxysuccinic acid hydrolase showed the same sequence as the N-terminal region of the beta subunit of the cis-epoxysuccinic acid hydrolase obtained from Alcaligenes sp.     

Purification and Properties of Dimethylglycine Oxidase from Cylindrocarpon didymum M–1

Dimethylglycine oxidase was purified to homogeneity from the cell extract of Cylindrocarpon didymum M–1, aerobically grown in medium containing betaine as the carbon source. The molecular weight of the enzyme was estimated to be 170,000 by the gel filtration method and 180,000 by the sedimentation velocity method. The enzyme exhibited an absorption spectrum characteristic of a flavoprotein with absorption maxima at 277, 345 and 450 nm. The enzyme consisted of two identical subunits with a molecular weight of 82,000, and contained two mol of FAD per mol of enzyme. The flavin was shown to be covalently bound to the protein. The enzyme was inactivated by Ag⁺, Hg²⁺, Zn²⁺ and iodoacetate. The enzyme oxidized dimethylglycine but was inert toward choline, betaine, sarcosine and alkylamines. Km and Vmax values for dimethylglycine were 9.1 mm and 1.22 μmol/min/mg, respectively. The enzyme catalyzed the following reaction: Dimethylglycine+O₂+H₂O → sarcosine+formaldehyde+H₂O₂.     

Purification and Properties of Sarcosine Oxidase from Cylindrocarpon didymum M–1

Sarcosine oxidase was purified to homogeneity from the cell extract of Cylindrocarpon didymum M–1, aerobically grown in medium containing choline as the carbon source. The molecular weight of the enzyme was estimated to be 45,000 by gel filtration method and 48,000 by the sodium dodecylsulfate disc gel electrophoresis method. The enzyme exhibited an absorption spectrum with maxima at 277 and 450 run and shoulders at 370 and 470 nm. The anaerobic addition of sarcosine to the enzyme resulted in the disappearance of the peak at 450 nm. The enzyme contained one mol of covalently bound FAD per mol of enzyme. Enzyme activity was inhibited by Ag⁺, Cu²⁺, Hg²⁺, p-chloromercuribenzoate and iodoacetate. The enzyme oxidized sarcosine but was inert toward choline, betaine, dimethylglycine and N-methyl amino acids. Km and V_max values for sarcosine were 1.8 ihm and 26.2 μmol/min/mg, respectively. The enzyme catalyzed the following reaction: Sarcosine+O₂+H₂O→glycine +formaldehyde+H₂O₂.     

Purification and characterization of HIV–human protein complexes

To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen–host protein–protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10–100 proteins), generation of comprehensive host–virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral–host protein–protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host–pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome.     

Purification,Crystallization and Some Enzymatic Properties of Acid Protease of Cladosporium sp. No. 45–2

An acid protease of Cladosporium sp. No. 45–2 was purified and crystallized by precipitation with ammonium sulfate, fractional precipitation with acetone, and pH adjustment. About 600 mg of third crystallized preparation was obtained from one liter of culture broth. The purified enzyme was chromatographically homogeneous and confirmed to be monodispersive by physicochemical criteria such as uhracentrifugal and electrophoretical analysis. The enzyme was most active at pH values between 2.5 and 2.7 toward both casein and hemoglobin and was stable at pH values from 2.5 to 7.0 on twenty hour incubation at 30°C.

Millimolar concentration of sodium lauryl sulfate markedly inhibited the enzyme, wheares diisopropyl phosphorofluoridate, sulfhydryl reagents, ethylenediaminetetra acetic acid, and divalent metal ion relatively little affected the activity. The enzyme was most resistant toward S-PI among the acid proteases tested.     

Purification and Properties of L-Methionine γ-Lyase from Aeromonassp.

Four types of β-xylosidases from a concentrated culture filtrate of Pénicillium wortmanni IFO 7237, designated as xylosidase-1, -2, -3, and -4 were purified to homogeneity on SDS polyacrylamide gel electrophoresis by an alcohol precipitation, DEAE-Sephadex A-25 ion exchange chromatography, and isoelectric focusing. The molecular weights of xylosidase-1, -2, -3, and -4 were estimated to be 110,000, 195,000, 210,000, and 180,000 respectively and their isoelectric points to be 3.7, 4.28, 4.6, and 4.8. The pH optima of β-xylosidase activities were from 3 to 4.5. The optimum temperature for enzyme activities was from 55°C to 65°C. On the enzymic hydrolysis of phenyl ß-d- xyloside, the reaction product of each enzyme was found to be β-d-xylose with retention of configuration. All the four ß-xylosidases were free of α-xylosidase and ß-glucosidase activities. All the enzyme activities of four β-xylosidases were strongly inhibited by Hg²⁺ and N- bromosuccinimide. With respect to the hydrolysis patterns and HPLC analysis of hydrolyzates from xylooligosaccharides, xylosidase-2 was totally different from other three as a distinct enzyme. Xylosidase-1 was also in a separate group although xylosidase-3 and -4 showed closely related action patterns as a different group.     

Lipid and phase specificity of α-toxin from S. aureus

The pore forming toxin Hla (α-toxin) from Staphylococcus aureus is an important pathogenic factor of the bacterium S. aureus and also a model system for the process of membrane-induced protein oligomerisation and pore formation. It has been shown that binding to lipid membranes at neutral or basic pH requires the presence of a phosphocholine-headgroup. Thus, sphingomyelin and phosphatidylcholine may serve as interaction partners in cellular membranes. Based on earlier studies it has been suggested that rafts of sphingomyelin are particularly efficient in toxin binding. In this study we compared the oligomerisation of Hla on liposomes of various lipid compositions in order to identify the preferred interaction partners and conditions. Hla seems to have an intrinsic preference for sphingomyelin compared to phosphatidylcholine due to a higher probability of oligomerisation of membrane bound monomer. We also can show that increasing the surface density of Hla-binding sites enhances the oligomerisation efficiency. Thus, preferential binding to lipid rafts can be expected in the cellular context. On the other hand, sphingomyelin in the liquid disordered phase is a more favourable binding partner for Hla than sphingomyelin in the liquid ordered phase, which makes the membrane outside of lipid rafts the more preferred region of interaction. Thus, the partitioning of Hla is expected to strongly depend on the exact composition of raft and non-raft domains in the membrane.     

Purification and Properties of a New l-Fucose-specific Lectin Produced by Streptomyces No. 100–2

The inhibitory effects of 3-nitro-2,4,6-trihydroxybenzamide derivatives on human 5-lipoxygenase (5-LO), a key enzyme in arachidonic acid cascades, were examined using 5-LO produced by Escherichia coli. Some of the tested compounds inhibited the conversion of arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), and in particular the N-phenylbutyl derivative was about 30 times more active (IC₅₀ = 35 μm) than caffeic acid (IC₅₀ = 1000 μm), a known selective 5-LO inhibitor.     

Purification and properties ofβ-fructofuranosidase fromAureobasidium sp. ATCC 20524

Summary Purification and properties of two -fructofuranosidases, which produce 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, fromAureobasidium sp. ATCC 20524 are reported. The enzymes were purified to homogeneity by fractionations involving ethanol, calcium acetate and ammonium sulfate and DEAE-Cellulofine and Sephadex G-200 chromatography. Molecular weights of the enzymes were estimated to be about 318000 (P-1) and 346000 (P-2) daltons by gel filtration. The enzymes were glycoproteins that contained about 30% (w/v) (P-1) and 53% (w/v) (P-2) carbohydrate. The optimum pH for the enzymatic reactions were 4.5–5.5 (P-1) and 4.5–6 (P-2). The enzymes were stable over a wide pH range (4–9). The optimum reaction temperatures for both enzymes were 50–55°C and they retained more than 94% (P-1) and 98% (P-2) activities at 50°C after 15 min. TheK _m values for sucrose were 0.47 M (P-1) and 0.65 M (P-2). The enzymes were inhibited by mercury, copper and lead ions as well asp-chloromercuribenzoate.     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Purification and Characterization of α-Hydroxyglutarate Dehydrogenase from Alcaligenes sp.

NADH-dependent soluble l-α-hydroxyglutarate dehydrogenase (l-2-hydroxyglutarate: NAD⁺ 2-oxidoreductase) was found in a bacterium belonging to the genus Alcaligenes obtained from soil by citrate enrichment culture. A mutant with about 2.5-fold higher activity of the enzyme was derived from the bacterium and used as the enzyme source. High level of the enzyme was produced at the late stage of cultivation in the presence of citrate and with limited aeration. The enzyme was purified from the cells to homogeneity to give crystals, and its enzymatic properties were studied. The enzyme strongly reduced α-ketoglutarate to stereochemically pure l-α-hydroxyglutarate with NADH as a coenzyme, but it oxidized d-α-hydroxyglutarate with about 1/10 of the rate for l-form oxidation.     

Purification and Chemistry of Bacteriophage ?

From a stock of varkappa phage grown on Salmonella, a host-range mutant which attacks Escherichia coli was isolated. As in the case of Salmonella, only motile strains of E. coli are sensitive to varkappa. The phage shows an eclipse period of 35 min and a minimal latent period of 52 min. The adsorption rate constant is 3 x 10(-9) ml/min. Adsorption shows a marked dependence on temperature. Bacteriophage varkappa was purified by differential centrifugation and CsCl density gradient centrifugation. It contains deoxyribonucleic acid (DNA) which is double-stranded. The DNA has a molecular weight of 42 million and a guanine plus cytosine content of 57%. Of 68 molecules of DNA inspected, 7 were circular. The phage particle weight is about 90 million.     

Causal specificity and the instructive–permissive distinction

I use some recent formal work on measuring causation to explore a suggestion by James Woodward: that the notion of causal specificity can clarify the distinction in biology between permissive and instructive causes. This distinction arises when a complex developmental process, such as the formation of an entire body part, can be triggered by a simple switch, such as the presence of particular protein. In such cases, the protein is said to merely induce or "permit" the developmental process, whilst the causal "instructions" for guiding that process are already prefigured within the cells. I construct a novel model that expresses in a simple and tractable way the relevant causal structure of biological development and then use a measure of causal specificity to analyse the model. I show that the permissive-instructive distinction cannot be captured by simply contrasting the specificity of two causes as Woodward proposes, and instead introduce an alternative, hierarchical approach to analysing the interaction between two causes. The resulting analysis highlights the importance of focusing on gene regulation, rather than just the coding regions, when analysing the distinctive causal power of genes.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.