Edward Steers

A comparative study of spectrin: A protein isolated from red blood cell membranes

Abstract 1. 1. A membrane-bound protein, called spectrin, has been solubilized from human, guinea pig, horse, sheep, and rabbit erythrocyte ghosts by dialysis against EDTA and β-mercaptoethanol. 2. 2. Spectrin accounted for approx. 20% of the total membrane protein in all these species and was free of carbohydrate and lipid. 3. 3. Spectrin preparations from each species were homogeneous when studied by gel filtration, polyacrylamide gel electrophoresis, and immunoprecipitin reactions. 4. 4. Spectrin appeared to be a very similar, possibly homologous, protein in the red cell membranes of different species. The amino acid compositions and electrophoretic patterns were nearly identical. Antiserum to guinea pig and human spectrin cross-reacted with all the other species. 5. 5. Comparison by disc gel electrophoresis of peptides produced by CNBr cleavage of human, horse, and sheep spectrin showed similar numbers of peptides but different electrophoretic migrations, indicating some differences in amino acid sequence in the different molecules.

Phytochrome in etiolated annual rye: IV. Physical and chemical characterization of phytochrome

Abstract Isolated phytochrome was demonstrated to be free from all but trace impurities by high speed velocity and equilibrium ultracentrifugation, polyacrylamide electrophoresis, and gel filtration in Sephadex G-200. The results from these techniques and from electron microscopy, amino acid analyses, and tryptic peptide separations were consistent with the native phytochrome molecules existing as 14-S hexamers and 9-S tetramers of 2-S monomer units. The monomer unit appears to be chemically identical, existing as a single polypeptide chain of approx. 42 000 molecular weight. These monomer units appear to be associated through noncovalent type bonds. Fluorescence studies indicated that when the 660 nm-absorbing form of phytochrome was excited at 290 nm, it fluoresced intensely at 340 nm and slightly at 672 nm. When excited at 370 nm, it fluoresced at 672 nm.

Purification and characterization of the multiple forms of β-galactosidase of Escherichia coli

Abstract 1. 1. In constitutive or fully induced lactose fermenting strains of Escherichia coli , the β-galactosidase (EC 3.2.1.23) activity may be separated into at least ten well-defined zones of activity on polyacrylamide disc gels. A method is presented for the purification of these multiple forms of β-galactosidase exclusive of the major (95%) tetrameric species of this enzyme. 2. 2. Ultracentrifugation of a purified mixture of all isoenzymes yields six species with sedimentation values of approx. 16, 23, 27, 32, 36, and 41–45 S which are estimated to be aggregates of 4N, 6N, 8N, 10N, 12N, and 16N, respectively. 3. 3. The 16-S species has previously been shown to be a tetramer composed of identical subunits. The monomeric units of the 16-S and the 23–45-S species produced in urea appear to be identical by ultracentrifugation, electrophoresis in polyacrylamide disc gels, and amino acid composition. 4. 4. Mixtures of the 23–45-S species were found to be more sensitive than tetramer to thermal inactivation and denaturation. Dissociation of the tetramer in urea has previously been shown to proceed directly to monomer. Dissociation of the higher molecular weight forms proceeded through intermediate forms with sedimentation coefficients of approx. 16 and 10, presumably representing tetramer and dimer. 5. 5. Renaturation of urea-denatured heavy isoenzymes results in the formation of tetramers but not the heavier forms. This renatured tetramer is indistinguishable from the 16-S renatured tetramer in its sensitivity to urea and heat. 6. 6. The isoenzymes do not represent macromolecular complexes of β-galactosidase with other cell proteins or nucleic acids.

[34] β-Galactosidase

Publisher Summary The enzyme β-galactosidase is one of several oligosaccharide-splitting enzymes that have been purified and subsequently characterized to varying degrees both physically and mechanistically. It is found in a wide range of biological sources in both the plant and animal kingdoms, although the principal source of study has been in the bacterium Escherichia coli, where it gained prominence as a result of the studies of Jacob and Monod and their collaborators in enzyme regulation. The enzyme has been shown to catalyze hydrolytic and transfer reactions wherein a variety of substances may act as acceptor for the glycone moiety of the substrate. β-Galactosidase from E. coli has been purified by various standard procedures. The chapter discusses the use of affinity chromatography with agarose derivatives of the substrate analog—p-aminophenyl-β-D-galactoside—for the purification of the enzyme from E. coli and Bacillus megaterium . The chapter concludes with a discussion of various physical and chemical properties of the enzyme.

The effect of urea on subunit interactions of ß-galactosidase from Escherichia coli K12

Abstract The effect of increasing concentrations of urea on sedimentation velocity, enzymatic activity, antigenicity, and protein fluorescence of s-galactosidase (s- d -galactoside galactohydrolase, EC 3.2.1.23) indicates that the enzymatically active tetramer is completely dissociated into inactive monomer in 6 M urea. Removal of urea by dialysis results in reaggregation to the active tetramer together with a return to normal values of those properties which have been studied. Protein which is immunologically related to s-galactosidase but incapable of forming an enzymatically active tetramer was added to wild-type enzyme in the presence of 8 M urea. After renaturation, the enzymatically active tetramer was found to be a hybrid consisting of mutant and wild-type subunits containing full enzymatic activity.

Isoenzymes of bacterial β-galactosidases: Purification and characterization of a nonisoenzyme forming β-galactosidase of Aerobacter cloacae☆

Abstract The β-galactosidase (β- d -galactoside galactohydrolase, EC 3.2.1.23) of an inducible strain of Aerobacter cloacae does not form isoenzymes unlike several strains examined to date. The single band of enzymatic activity on polyacrylamide disc gels had a lower mobility than the tetramer of E. coli and could be detected only if divalent cations had been added to the running gel during their preparation. The enzyme was purified by several gel filtration steps, with a final purification on a column of anti-β-galactosidase antibody which had been linked to Sepharose. Its amino acid composition, sedimentation coefficient, and K m were not detectably different than that of the E. coli tetramer. The A. cloacae β-galaetosidase was weakly cross-reactive immunologically with that of E. coli , had a slightly shifted pH optimum, was more heat labile, and was more sensitive to urea denaturation. Sedimentation velocity analyses in the presence of various concentrations of urea showed that the urea-induced loss of activity correlated precisely with dissociation of the tetramer into subunits. Exposure to ion-exchange resins or extended storage at 4 ° also dissociated the A. cloacae β-galactosidase into its subunits. Therefore, the increased sensitivity to urea (and presumably to heat) of this nonisoenzyme-forming β-galactosidase reflects lower bond strengths between monomers in the tetramer.

Characterization of the ß-galactosidase from a lactose-negative, complementing mutant of Escherichia coli K12

Abstract The enzyme s-galactosidase (s- d -galactoside galactohydrolase, EC 3.2.1.23) synthesized by a lactose-negative, cross-reacting material (CRM) positive mutant of Escherichia coli K12, was purified and characterized by several physicochemical procedures. The basal level of enzymatic activity associated with extracts from this mutant are the result of low levels (5%) of naturally occurring tetramer which has a specific activity on the order of 10 −3 that of the wild-type 3300 strain and is more labile to thermal inactivation. The major CRM-positive, complementing protein has been shown, however, to be a dimer with a molecular weight of approx. 250 000. The complementing dimer shows complete immunological identity with the wild-type protein demonstrating that the dissociation of tetramer to dimer does not qualitatively effect the immunochemical properties of the enzyme. Finally, the mutational effect on the enzyme which prohibits the formation of the normal tetramer from dimers also prohibits the normal polymerization of monomers into more highly aggregated isozymes such as those which occur in the wild-type strain.

Purification of the i-antigen 51A fromParamecium tetraurelia by immunoaffinity chromatography

Abstract Purification of the surface antigen of Paramecium tetraurelia (termed i-antigen) by previously published procedures has been shown to result in preparations which frequently contain contaminating proteins including a thiol-activated protease. Alternate procedures for the purification of i-antigen were developed using ion-exchange chromatography. While certain of these procedures result in a homogeneous preparation of i-antigen, the poor yields obtained make these procedures impractical. An alternate method for the purification of i-antigen was developed using immunoaffinity chromatography. Purification by this technique proved to be superior to the various standard procedures described previously. The immunoaffinity column is rapid and results in a recovery, on the average, of over 95% of the applied material. The columns are readily regenerated and may be used numerous times without loss of capacity or purification ability. The i-antigen purified by this method appears homogeneous immunologically and electrophoretically on polyacrylamide gel electrophoresis and isoelectric focusing.

Bacillus megaterium, KM β-galactosidase: Purification by affinity chromatography and characterization of the active species

Abstract The enzyme β-galactosidase from Bacillus megaterium, strain KM has been purified by affinity chromatography. The enzyme was found to have a dimeric subunit structure, with the monomer having a molecular weight of 120,000. The Keq of the monomer-dimer equilibrium was strongly shifted towards dissociation in the isolated state. Inclusion of 5% sucrose in the buffer (and maintenance of the temperature at 5 °) minimized this dissociation. Molecularly homogeneous monomer and dimer could be prepared on sucrose gradients. The dimer was determined to have an S20,w of 8, while the monomer had an S20,w of 3. The amino acid composition was found to be similar to that of the E. coli β-galactosidase although significant differences occur. The activity of the monomer was studied by both urea-denaturation experiments and by immobilization of the monomer on Sepharose-4B. The monomer, bound to Sepharose-4B, was found to be inactive but still capable of binding the inhibitor thio-methyl galactoside. Activity was reconstituted by adding free monomer, in 8 M urea, to the Sepharose-bound monomer, followed by removal of the urea by dialysis. In addition, free monomers from E. coli β-galactosidase were found to form active hybrids with Sepharose-bound B. megaterium β-galactosidase monomers. We conclude on the basis of these studies that the free monomer is inactive, and that the dimer is the active species, in marked contrast to E. coli β-galactosidase where only the tetrameric form is active.

Evidence for pyrrolidone carboxylic acid in β-galactosidase from E. coli

Abstract Acidic peptides isolated from pronase digests of β-galactosidase by chromatography on Dowex 50-X2 contain N-terminal pyrrolidone carboxylic acid. This was not due to cyclization of glutamine or glutamic acid to pyrrolidone carboxylic acid during the procedure as addition of excess fluorodinitrobenzene immediately following digestion and prior to the chromatographic step did not alter the results. Acetylation of β-galactosidase prior to the procedure resulted in recovery of additional amino acids which were compatible with the previously established N-terminal threonine. These data indicate the presence of multiple polypeptide chains in the β-galactosidase monomers of 135,000 molecular weight, with pyrrolidone carboxylic acid and threonine as N-terminal residues.