Subunits pyrophosphatase

Additional size estimates of the 5 M guanidine hydrochloride subunit can be made from the Svedberg equation (18), using the s°0 w and D 0.w values cited in Section II,F, and from the empirical equations of Tan-ford and associates (32, 33), which relate s and [17] values in concentrated guanidine hydrochloride solvents to polypeptide chain length. These calculations yield molecular weights of 21,300, 20,700, and 19,800, respectively, for the E. coli pyrophosphatase subunit (13). 

The hydrolysis of pyrophosphate to phosphate is catalyzed by yeast inorganic pyrophosphatase, which is a dimeric enzyme having two identical subunits.290 It now appears that three cations aTe required per active site, two bound to the enzyme and the third to the phosphate (equation 4). 

Hydrolytic driving force. The hydrolysis of pyrophosphate to orthophosphate is important in driving forward biosynthetic reactions such as the synthesis of DNA. This hydrolytic reaction is catalyzed in Escherichia coli hy a pyrophosphatase that has a mass of 120 kd and consists of six identical subunits. For this enzyme, a unit of activity is defined as the amount of enzyme that hydrolyzes 10 pmol of pyrophosphate in 15 minutes at 37°C under standard assay conditions. The purified enzyme has a of 2800 units per milligram of enzyme. 

Several membrane-bound ATPases occur in the genus Sulfolobus. There are two ATP-hydrolyzing activities in S. acidocaldarius strain 7. One has a pH optimum at 6.5 in the absence of sulfate, and the presence of that anion activates the enzyme and shifts the pH optimum to 5.0. ATP hydrolysis is unaffected by DCCD, azide, NEM, /7-hydroxymercuribenzoate, or vanadate [59]. The other ATPase is most active at pH 2.5, is inhibited by sulfate, and appears to be a pyrophosphatase [16]. The purified sulfate-activated ATPase (M, 360000) is composed of three subunits (Mr 69000, 54000, and 28000). It is most active at 85 C, stimulated some three-fold by sulfate, sulfite, and bicarbonate, but is unaffected by chloride. There are two pH optima. One is located at pH 5 and the other at pH 8.5 and neither is affected by sulfite. ATPase activity is inhibited by nitrate (63% at 20 mM) and NBD-Cl (90% at 1 mM) but is not significantly affected by azide (5mM), vanadate (100 pM), and NEM (100pM)[28]. 

In contrast to these results, Volk et al. in a very recent publication [77] using a detergent based solubilization described in [78], found very distinct differences between pyrophosphatases I and II. PPase I has a molecular weight of 60000 and consists of two different subunits, a and PPase II has a molecular weight of 185 000 and is composed of four subunits, a, j8, y and 8. Because of the similarity in mass between subunits a and in the two enzymes, Volk et al. propose that these two subunits are the same in both enzymes and form the catalytic part of PPase II. PPase I from mitochondria thus seems to differ from other soluble pyrophosphatases in that the two subunits are nonidentical. 

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. In Nature (London) 340 680-685 Laugwitz KL, Spicher K, Schultz G et al. (1994) Identification of receptor-activated G proteins selective immunoprecipitation of photolabeled G-protein a subunits. In Methods Enzymol. 237 283-294 Meyer T, Hilz H (1986) Production of anti-(ADP-ribose) antibodies with the aid of a dinucleotide-pyrophosphatase-resistent hapten and their application for the detection of mono(ADP-ribosyl)ated polypeptides. In Ear. J. Biochem. 155 157-165... 

Yeast inorganic pyrophosphatase (YIP) is an enzyme consisting of two identical subunits giving a molecular weight for the dimer of 64,000. Yeast inorganic pyrophosphatase catalyses the reversible hydrolysis of pyrophosphate. In the presence of Mg, YIP appears to be specific for pyrophosphate however, when other activating metal ions are employed, the enzyme becomes less specific and will hydrolyze a number of pyrophosphate esters (21). There is even a possibility that a mixture of enzymes is present in some preparations (55). 

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