Human lysosomal beta-glucosidase: purification by affinity chromatography

Two Sepharose-bound substrate analogs, 6'-aminohexanoyl-(2-N-sphingosyl-O-beta-D-glucoside) and 6'-aminohexyl-dodecanedioyl-1-(2-N-sphingosyl-1-O-beta-D-glu coside), were synthesized and used sequentially for the affinity purification of lysosomal beta-glucosidase (N-acyl-sphingosyl-1-O-beta-D-glucoside:glucohydrolase, EC 3.2.1.45). The capacities of these nondegradable affinity supports were 0.1 and 0.15 mg enzyme/ml settled gel, respectively. The purified enzyme had a specific activity of 75 mumol min-1 mg-1. The preparation had a single protein band with a molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, evidencing its apparent homogeneity. Isoelectric focusing on granular gels revealed four molecular forms of the enzyme with pI values of 4.0, 4.5, 4.7, and 5.8 to 6.2. The purified enzyme hydrolyzed glucosyl ceramide and 4-methylumbelliferyl-beta-D-glucoside with Km and Vmax values of 0.6 and 2.5 mM, and 101 and 26.1 mumol min-1 mg-1, respectively. The enzyme also hydrolyzed octyl beta-glucoside, a linear mixed-type inhibitor of the enzyme. Binding constants (Ki) were determined for the inhibitors, sphingosyl-1-O-beta-D-glucoside (Ki = 20 microM) and its N-hexyl derivative (Ki = 0.3 microM). The enzyme had a half-life of 65 and 30 min at 50 degrees C and pH 5.0 or 6.0, respectively. In addition, two other classes of ligands were used for the purification of lysosomal beta-glucosidase, and their capacities and specificities were compared to those of the substrate analog affinity supports. These included (i) the alkyl amine inhibitors octylamine, decylamine, and tetradecylamine; and (ii) the inhibitors, 6-aminohexanoyl-beta-glucosylamine and aminododecanoyl-1-(2-N-sphingosyl-1-O-beta-D-glucoside). Compared to these other ligand columns, the substrate analog affinity supports had about 100- to 1000-fold greater capacities or afforded 8- to 40-fold greater purification of human lysosomal beta-glucosidase.     

The binding of human milk lactoferrin to immunoglobulin A

To define the step at which translational initiation factor IF1 exercises its stimulation, initial rate kinetic analyses of 30 S initiation complex formation were carried out in the presence and absence of this factor. It was shown that, without affecting the affinity of the ribosomes either for the initiator tRNA or for the poly(AUG) used as template, IF1 increases approximately 2.5-fold the limiting Vmax of the 'pre-ternary complex'----ternary complex transition which represents the rate-limiting step in 30 S initiation complex formation. This kinetic effect titrates with the 30 S ribosomal subunit which must therefore represent the target of IF1 action.     

Mass spectrometric measurements of methane and oxygen utilization by methanotrophic bacteria

Abstract A mass spectrometer with membrane inlet was used to measure methane and oxygen utilization rates at various methane concentrations in Methylosinus trichosporium and a locally isolated strain of a methane-oxidizing coccus (OU-4-1). The apparent K _m for methane was found to be 2 μM for M. trichosporium and 0.8 μM for strain OU-4-1. These K _m-values are 10–30 times lower than most previously reported values. The ratio of oxygen to methane utilization rates was 1.7 for M. trichosporium and 1.5 for strain OU-4-1 corresponding to a growth yield of 0.38 and 0.63 g dry weight/g methane, respectively.     

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaeamaderae

The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [³H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaeamaderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.     

Synthesis of charged amphipathic nitroxide lipid spin labels and an example of their application in membrane studies

The synthesis of a series of amphipathic nitroxide lipid spin labels is reported. Thus, 12-proxylhexadecanol has been converted into the versatile fatty acid spin label 14-proxylstearic acid. This substance was used to prepare 14-proxylstearyltrimethylammonium methanesulfonate, a positively charged label, and 14-proxylstearylmethyl phosphate sodium salt, a negatively charged label. Also prepared in the doxyl series were quaternary ammonium salts derived from 16-doxyl- and 7-doxylstearic acid. The positively charged and negatively charged proxyl labels were used in a preliminary experiment to investigate the role of charge in their interaction with reconstituted cytochrome oxidase. The average binding affinity of the negatively charged label is approximately 2-fold higher than that of the positively charged label at pH 7.4. At pH 5.5 the average relative affinity for negatively charged label is about 3.5-fold higher than that of positively charged label, suggesting that the ionizable group(s) on the protein can interact with the lipid headgroup.     

我国各类基物上的酵母菌分布及其尿素酶活性

本文讨论了2300株由我国各地分离到的酵母菌和类酵母菌与各类基物的关系。共述及23属,占Lodder(1970)系统的半数以上。在十几类基物中酵母菌的分布各有不同,叶子上有大量掷孢酵母,果实上比其它基物更能获得克勒克酵母属(Kloeckera),在咸腌食品中相对地有较多耐高渗透压的酵母如球拟酵母属(Torulopsis)与德巴利酵母属(Debaryomyces)。对部分属的定种工作也已进行,说明某些属的种在我国均有发现而且为数不少。此外还研究了600余株代表各类酵母的尿素酶活性,结果说明担子菌酵母或系统上接近担子菌的酵母都有尿素酶活性,而子囊菌酵母则无。根据作者的观察子囊菌酵母中裂殖酵母属(Schizosaccharomyces)则常常例外地有尿素酶反应,且常有辅酶Q_(10),从而可见裂殖酵母属有其特有的生物学特性,在系统上很可能与其它子囊菌酵母不同。对属于不完全菌的无孢子酵母也进行了尿素酶活性测定,结果表明它们部分与子囊菌有关,部分与担子菌有关。     

Affinity purification of insoluble recombinant fusion proteins containing glutathione-S-transferase

Prokaryotic expression of polypeptides as fusion proteins with glutathione-S-transferase has recently been reported as a one-step means of purifying recombinant protein. The usefulness of the glutathione-S-transferase/glutathioneagarose system, however, is significantly limited by the frequent synthesis of recombinant proteins in insuluble form by Escherichia coli. We have found that for 5 separate fusion proteins containing glutathione-S-transferase and different domains of the large cystic fibrosis transmembrane conductance regulator, all were packaged in insoluble form by E. coli. Insolubility of these products made them inaccessible to one-step purification utilizing this scheme requires proper folding of recombinant glutathione-S-transferase to allow recognition on glutathione affinity agarose, we investigated the suitability of several alternative approaches for converting insoluble recombinant fusion proteins to a soluble form amenable to glutathione-agarose affinity purification. Low-temperature induction of fusion protein synthesis, but not incubation with anion-exchange resins, led to improved one-step purification of glutathione-S-transferase fusion proteins from E. coli cell lysate using mild, nondenaturing conditions. Solubilization in 8 mol/L urea, but not with other chaotropic agents or detergents, also allowed preparative yields of affinity-purified fusion protein. These techniques increase the usefulness of this recombinant protein purification scheme, and should be broadly applicable to diverse polypeptides synthesized as fusions with glutathione-S-transferase.     

Studies on the isolation of penicillin acylase from Escherichia coli by aqueous two-phase partitioning

A method of enzyme release and aqueous two-phase extraction is described for the separation of penicillin acylase from Escherichia coli cells. Butyl acetate, 12% (v/v), treatment combined with freeze-thawing gives up to 70% enzyme release. For polyethylene glycol (PEG) + phosphate two-phase extraction systems the enzyme purity and yield were rather low. Modified PEG, including PEG-ampicillin, PEG-aniline, PEG-phosphate, and PEG-trimethylamine, were synthesized and used in aqueous two-phase systems; PEG-trimethylamine is the most satisfactory. A system containing 12% (w/w) PEG4000, 8% (w/w) of which is PEG-trimethylamine, with 0.7M potasium phosphate at pH 7.2, resulted in the enzyme selective partition being greatly enhanced by charge directed effects. Possible mechanisms for the separation process are discussed. (c) 1992 John Wiley & Sons, Inc.     

Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation

Aqueous two-phase extraction incorporated affinity precipitation was examined as a technique for protein purification. An enteric coating polymer, Eudragit S100, was employed as a ligand carrier. Eudragit was specifically partitioned to the top phase in the aqueous two-phase systems. For application of this method to purification of recombinant protein A using human IgG coupled to Eudragit in an aqueous two-phase system, 80% of protein A added was recovered with 81% purity. The purity was enhanced 26-fold by thid method. The IgG-Eudragit could be used repeatedly for the purification process. This seperation method should be applicable to industrial-scale purification as a new purification procedure combining the advantages and compensating for the disadvantages of the aqueous two-phase method and affinity precipitation method. (c) 1992 John Wiley & Sons, Inc.     

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.