Purification and Properties of Inorganic Pyrophosphatase from Porcine Brain

Abstract

Inorganic pyrophosphatase [EC 3.6.1.1] was purified from porcine brain to an electrophoretically homogeneous state. The molecular weight of the enzyme was estimated to be 62,000 by gel filtration and that of the subunit to be 33,000 by gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme consists of two identical subunits. The stability of the purified enzyme was dependent on its protein concentration. The enzyme was stable above 50 micrograms/ml at 20 degrees C, but it was gradually inactivated below this concentration, even at 0 degree C unless other proteins such as bovine serum albumin, calmodulin, etc. were present. Those added proteins not only protected the enzyme from inactivation, but also completely reactivated the enzyme after it had been once inactivated. The enzyme catalyzed the hydrolysis of inorganic pyrophosphate but not that of other phosphate esters. Only Mg2+ was required as an activating cation, and other divalent cations inhibited the activity to some degree. The addition of sulfhydryl reagents prevented the inhibition of activity by divalent cations.

Evidence for HTR1A and LHPP as interacting genetic risk factors in major depression

“…24 and subsequently purified from several additional mammalian organs. [25][26][27][28] A human LHPP cDNA has been cloned, and functional human Lhpp has been purified following heterologous expression in Escherichia coli. 29 Lhpp has been characterized in vitro as efficiently catalyzing the hydrolysis of P-N bonds in phosphohistidine and phospholysine, and less efficiently catalyzing the hydrolysis of P-N or P-O bonds in imidodiphosphate and pyrophosphate, respectively.…”

“…These transcripts are shown in Figure 4. Expression levels were categorized according to threshold cycle: very high ( < 20), high (20)(21)(22)(23)(24)(25), moderate (25)(26)(27)(28)(29)(30), low (30)(31)(32)(33)(34)(35), very low/questionable (35)(36)(37)(38)(39)(40) or not detected (no signal above background after 40 cycles). Because of the linkage and association of LHPP variants to MDD, there was particular focus on measuring expression of these transcripts in the brain (Figure 3b).…”

Bacillus subtilis ORF yybQ encodes a manganese-dependent inorganic pyrophosphatase with distinctive properties: the first of a new class of soluble pyrophosphatase?

“…These PP,ases are generally dimers, although the mitochondria1 members are heterodimers that generate a@, tetramers on association with the membrane. Although the PP,ases of S. cerevisiae, 1, Hachimori et al (1975), Schreier & Hohne (1978), Hachimori et al (1979), Schreier (1980), Ichiba et al (1990); 2, Shimizu et al (1997);3, Mitchell & Minnick (1997); 4, J o s e (1966), Wong et al (1970), Lahti et al (1988) ; 5, Fleischmann et al (1995) ; 6, Tomb et al (1997) ; 7, van Alebeek et al (1994) ; 8, Fraser et al (1995) ; 9, Himmelreich et al (1996) ; 10, Meyer et al (1995) ; 11, Richter & Schafer (1992a, b); 12, Kuranova et al (1987), Hohne et al (1988), Teplyakov et al (1994);13, Tominaga & Mori (1977); 14, Kieber & Signer (1991);15, Volk et al (1983), Lundin et al (1991);16, Hiraishi et al (1997), heterodimer;17, Yang & Wensel (1992a, b) ; 18, Syiem & Sharma (1996) ; 19, Thuillier (1978) ; 20, Stark & Milner (1989) ; 21, Hachimori et al (1983) ; 22, Felix & Fleisch (1975) ; 23, du Jardin et al (1995); 24, Morita & Yasui (1978); 25, Mansurova (1989);26, Mansurova (1989), heterodimer;27, Irie et al (1970), Yoshida et al (1982); 28, Irie et al (1970), Volk & Baykov (1984), heterodimer;29, Cooperman (1982), Kolakowski et al (1988);30, Lundin et al (1991), 30 am...…”

Characterization of the inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori

“…Cytoplasmic PPases are generally oligomeric proteins consisting of identical subunits. Eukaryotic PPases have a homodimeric structure with subunits of approximately 30 kDa (Cohen et al 1978;Hachimori et al 1983). Bacterial (Wong et al 1970), and archaeal (Verhoeven et al 1986;Richter and Shäfer 1992;Van Alebeek et al 1994) PPases form hexameric or tetrameric complexes with single subunits of about 20 kDa.…”