Purification, properties, and sequence specificity of SslI, a new type II restriction endonuclease from Streptococcus salivarius subsp. thermophilus.

SslI, a type II restriction endonuclease, was purified from Streptococcus salivarius subsp. thermophilus strain BSN 45. SslI is an isoschizomer of BstNI. SslI activity was maximum at pH 8.8, 0 to 50 mM NaCl, 2 to 8 mM Mg2+, and 42 degrees C. Activity against phage DNA in vitro was demonstrated.     

Purification, properties, and sequence specificity of SslI, a new type II restriction endonuclease from Streptococcus salivarius subsp. thermophilus.

SslI, a type II restriction endonuclease, was purified from Streptococcus salivarius subsp. thermophilus strain BSN 45. SslI is an isoschizomer of BstNI. SslI activity was maximum at pH 8.8, 0 to 50 mM NaCl, 2 to 8 mM Mg2+, and 42 degrees C. Activity against phage DNA in vitro was demonstrated.     

Phosphoenolpyruvate-insensitive phosphofructokinase isozyme from Thermus thermophilus HB8.

In the previous paper [Xu, J., Oshima, T., & Yoshida, M. (1990) J. Mol. Biol. 215, 597-606], we reported that phosphofructokinase from Thermus thermophilus is allosterically inhibited by phosphoenolpyruvate, which induces dissociation of the active four-subunit enzyme into an inactive two-subunit form. When T. thermophilus was cultured in a glucose-containing medium, another phosphofructokinase (PFK2) appeared in addition to the reported one (PFK1). The molecular weights of the native PFK2 molecule (132,000) and its subunit (34,500), which are slightly smaller than those of PFK1, suggest that PFK2 is also composed of four identical subunits. However, the hyperbolic kinetics and molecular form of PFK2 are not affected at all by phosphoenolpyruvate. The NH2-terminal amino acid sequences of subunits of PFK1 and PFK2 revealed that they are composed of very similar but different polypeptides.     

Phosphofructokinases from Escherichia coli. Purification and characterization of the nonallosteric isozyme.

The main phosphofructokinase of Escherichia coli (PFK I) is an extensively studied allosteric enzyme specified by the pfkA gene. A nonallosteric phosphofructokinase was reported (Fraenkel, D.G., Kotlarz, D., and Bluc, H. (1973) J. Biol. Chem. 248, 4865-4866) in strains carrying the pfkB1 mutation, a suppressor of pfkA mutants, and very low levels of this enzyme have also been detected in strains not carrying the suppressor (i.e. pfkB+). The nonallosteric protein has now been prepared pure from three strains, one carrying pfkB1 and pfkA+, one carrying pfkB1 and completely deleted for pfkA, and one carrying pfkB+ and also deleted for pfkA. It is apparently the same enzyme (PFK II) in all three strains, which shows that pfkB1 is a mutation affecting the amount of a normally minor isozyme. PFK II is a tetramer of slightly larger subunit molecular weight than PFK I (36,000 and 34,000, respectively). No immunological cross-reactivity was detected between PFK II and PFK I. Unlike PFK I, PFK II does not show cooperative interactions with fructose-6-P, inhibition by P-enolpyruvate, or activation by ADP. Also unlike PFK I, PFK II is somewhat sensitive to inhibition by fructose-1,6-P2 and can use tagatose-6-P as substrate. Both enzymes can perform the reverse reaction, fructose-6-P + ATP from fructose-1,6-P2 + ADP in vitro, but not in vivo. The normal function of PFK II is not known.     

Purification and properties of sheep liver phosphofructokinase

1. The activity of phosphofructokinase in sheep liver was found to be dependent on the composition and molarity of the buffer used in extraction. Under optimum conditions a value of 4-7mumoles/min./g. wet wt. of tissue was obtained. 2. The enzyme was purified 480-fold by a combination of ammonium sulphate fractionation, heat treatment in the presence of ethanol, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. The final specific activity was 18.5mumoles/min./mg. of protein. 3. The purified enzyme was inhibited by ATP and citrate, the degree of inhibition depending on the concentration of fructose 6-phosphate, magnesium chloride and ammonium sulphate, as well as on the pH. ATP and citrate inhibition was overcome by AMP and fructose 1,6-diphosphate. 4. The enzyme was also inhibited by NADH and NADPH in a manner largely independent of other components of the assay medium. AMP and fructose 1,6-diphosphate were not able to overcome this type of inhibition. 5. Octanoate was not an inhibitor of phosphofructokinase. 6. Differences between these results and those of other workers are discussed.     

Purification and properties of citrate lyase from Streptococcus diacetilactis.

Citrate lyase from Streptococcus diacetilactis has been purified to yield a protein that was homogeneous as judged by sedimentation velocity and sedimentation equilibrium experiments. The enzyme's sedimentation coefficient is 16.8 S and its molecular weight is around 585,000. It contains three nonidentical subunits of about 53,000, 34,000, and 10,000 daltons. The enzyme in its active form contains an acetyl group which turns over during the citrate cleavage reaction. Removal of the acetyl group inactivates the enzyme. The deacetyl enzyme can be partially reactivated by acetylation with acetic anhydride. The enzyme undergoes slow "reaction-inactivation." The rate of inactivation is first order and the rate constant of inactivation is much lower than that for a similar inactivation process of the citrate lyase from Klebsiella aerogenes. Like the latter enzyme it contains stoichiometric amounts of phosphopantothenate. The enzyme is inactivated at pH greater than 8.1 and the presence of citrate provides protection against this inactivation. Sedimentation studies of the enzyme at pH 8.7 indicate that the enzyme is dissociated, which may account for the inactivation. The enzyme is immunologically different from citrate lyases of K. aerogenes and Escherichia coli.     

Purification and properties of pyruvate kinase from Streptococcus mutans.

Pyruvate kinase (EC 2.7.1.40) from Streptococcus mutans strain JC2 was purified, giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was 180,000 to 190,000, and the enzyme was considered to consist of four identical subunits. This enzyme was completely dependent on glucose 6-phosphate for activity, and the saturation curve for activation by glucose 6-phosphate was sigmoidal. In the presence of 0.5 mM glucose 6-phosphate, the saturation curves for the substrates phosphoenolpyruvate and ADP were hyperbolic, and the Km values were 0.22 and 0.39 mM, respectively. GDP, IDP, and UDP could replace ADP, and the Km for GDP (0.026 mM) was 0.067 of that for ADP. The enzyme required not only divalent cations, Mg2+ or Mn2+, but also monovalent cations, K+ or NH4+, for activity, and it was strongly inhibited by Pi. When the concentration of Pi was increased, the half-saturating concentration and Hill coefficient for glucose 6-phosphate increased. However, the enzyme was immediately inactivated in a solution without Pi. The intracellular concentration of glucose 6-phosphate, in cooperation with that of Pi, may regulate pyruvate kinase activity in S. mutans.     

Purification and properties of phosphoglycerate kinase from Thermus thermophilus strain HB8.

(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.     

Purification and regulatory properties of phosphofructokinase from Trypanosoma (Trypanozoon) brucei brucei.

Phosphofructokinase (EC 2.7.1.11) from Trypanosoma (Trypanozoon) brucei brucei was purified to homogeneity by using a three-step procedure that may be performed within 1 day. Proteolysis, which removes a fragment of Mr approx. 2000, may occur during the purification, but this can be prevented by including antipain, an inhibitor of cysteine proteinases, in the buffers during the purification. The subunits of the enzyme appear to be identical in size, with an Mr of 49 000. The Mr of the native enzyme was estimated to be approx. 220 000, suggesting a tetrameric structure. Kinetic studies showed the activity to depend hyperbolically on the concentration of ATP but sigmoidally on the concentration of fructose 6-phosphate. Although cyclic AMP, AMP and ADP stimulated the enzyme activity at low concentrations of fructose 6-phosphate, the last two nucleotides were inhibitory at high concentrations of this substrate. Phosphoenolpyruvate behaved as an allosteric inhibitor of the phosphofructokinase. Citrate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and Pi did not influence significantly the activity of the enzyme.