Contributions of the C-terminal domain to the control of P2X receptor desensitization

The P2X purinergic receptor channels (P2XRs) differ among themselves with respect to the rates of desensitization during prolonged agonist stimulation. Here we studied the desensitization of recombinant channels by monitoring the changes in intracellular free Ca(2+) concentration in cells stimulated with ATP, the native and common agonist for all P2XRs. The focus in our investigations was on the relevance of the P2XR C terminus in controlling receptor desensitization. When expressed in GT1 cells, the P2XRs desensitized with rates characteristic to each receptor subtype: P2X(1)R = P2X(3)R > P2X(2b)R > P2X(4)R > P2X(2a)R > P2X(7)R. A slow desensitizing pattern of P2X(2a)R was mimicked partially by P2X(3)R and fully by P2X(4)R when the six-amino acid sequences of these channels located in the cytoplasmic C terminus were substituted with the corresponding arginine 371 to proline 376 sequence of P2X(2a)R. Changing the total net charge in the six amino acids of P2X(4)R to a more positive direction also slowed the receptor desensitization. On the other hand, substitution of arginine 371-proline 376 sequence of P2X(2a)R with the corresponding sequences of P2X(1)R, P2X(3)R, and P2X(4)R increased the rate of receptor desensitization. Furthermore, heterologous polymerization of wild-type P2X(2a)R and mutant P2X(3)R having the C-terminal six amino acids of P2X(2a)R at its analogous position resulted in a functional channel whose desensitization was significantly delayed. These results suggest that composition of the C-terminal six-amino acid sequence and its electrostatic force influence the rate of receptor desensitization.     

Involvement of inorganic polyphosphate in expression of SOS genes

Inorganic polyphosphate (poly(P)) is a linear polymer that has been found in every organism so far examined. To elucidate the functions of poly(P) in the regulation of gene expression, the level of cellular poly(P) in Escherichia coli was reduced to a barely detectable concentration by overproduction of exopolyphosphatase (exopoly(P)ase) with a plasmid encoding yeast exopoly(P)ase (Shiba et al., Proc. Natl. Acad. Sci. USA 94 (1997) 11210-11215). It was found that exopoly(P)ase-overproducing cells were more sensitive to UV or mitomycin C (MMC) than were control cells. Poly(P) accumulation was observed after treatment with MMC, whereas the poly(P) level was below the detectable level in cells that overproduced exopoly(P)ase. When exopoly(P)ase-overproducing cells were transformed again by a multiple copy number plasmid that carries the polyphosphate kinase gene (ppk), the cells accumulated a great amount of poly(P) and restored the UV and MMC sensitivities to the level of control cells. In exopoly(P)ase-overproducing cells, the expression of recA and umuDC were not induced by MMC. In addition, a strain containing multiple copies of ppk accumulated not only a large amount of poly(P) but also recA mRNA. Since recA expression was induced in a recA-deletion strain harboring a plasmid with the ppk gene, poly(P) could be necessary for regulating the expression of SOS genes without depending on the RecA-LexA regulatory network.     

Mutation of the putative hydrogen-bond donor to P700 of photosystem I

The primary electron donor of photosystem I (PS1), called P(700), is a heterodimer of chlorophyll (Chl) a and a'. The crystal structure of photosystem I reveals that the chlorophyll a' (P(A)) could be hydrogen-bonded to the protein via a threonine residue, while the chlorophyll a (P(B)) does not have such a hydrogen bond. To investigate the influence of this hydrogen bond on P(700), PsaA-Thr739 was converted to alanine to remove the H-bond to the 13(1)-keto group of the chlorophyll a' in Chlamydomonas reinhardtii. The PsaA-T739A mutant was capable of assembling active PS1. Furthermore the mutant PS1 contained approximately one chlorophyll a' molecule per reaction center, indicating that P(700) was still a Chl a/a' heterodimer in the mutant. However, the mutation induced several band shifts in the visible P(700)(+) - P(700) absorbance difference spectrum. Redox titration of P(700) revealed a 60 mV decrease in the P(700)/P(700)(+) midpoint potential of the mutant, consistent with loss of a H-bond. Fourier transform infrared (FTIR) spectroscopy indicates that the ground state of P(700) is somewhat modified by mutation of ThrA739 to alanine. Comparison of FTIR difference band shifts upon P(700)(+) formation in WT and mutant PS1 suggests that the mutation modifies the charge distribution over the pigments in the P(700)(+) state, with approximately 14-18% of the positive charge on P(B) in WT being relocated onto P(A) in the mutant. (1)H-electron-nuclear double resonance (ENDOR) analysis of the P(700)(+) cation radical was also consistent with a slight redistribution of spin from the P(B) chlorophyll to P(A), as well as some redistribution of spin within the P(B) chlorophyll. High-field electron paramagnetic resonance (EPR) spectroscopy at 330-GHz was used to resolve the g-tensor of P(700)(+), but no significant differences from wild-type were observed, except for a slight decrease of anisotropy. The mutation did, however, provoke changes in the zero-field splitting parameters of the triplet state of P(700) ((3)P(700)), as determined by EPR. Interestingly, the mutation-induced change in asymmetry of P(700) did not cause an observable change in the directionality of electron transfer within PS1.     

Regulation of the (pro)renin-renin receptor in cardiac remodelling

The (pro)renin-renin receptor [(P)RR] was discovered as an important novel component of the renin-angiotensin system (RAS). The functional significance of (P)RR is widely studied in renal and vascular pathologies and has sparked interest for a potential role in cardiovascular disease. To investigate the role of (P)RR in cardiac pathophysiology, we aimed to assess (P)RR regulation in adverse cardiac remodelling of the failing heart. In particular, we evaluated the expression of (P)RR in different models of heart failure and across different species. Significantly increased levels of (P)RR mRNA were found in post-myocardial infarcted (MI) hearts of rats (1.6-fold, P < 0.05) and mice (5-fold, P < 0.01), as well as in transgenic rats with overexpression of the mouse renin gene (Ren2) (2.2-fold, P < 0.01). Moreover, we observed a strong increase of (P)RR expression in hearts of dilated cardiomyopathy (DCM) patients (5.3-fold, P < 0.001). Because none of the tested commercially available antibodies appeared to detect endogenous (P)RR, a (P)RR-specific polyclonal antibody was generated to study (P)RR protein levels. (P)RR protein levels were significantly increased in the post-MI rat heart (1.4-fold, P < 0.05) as compared to controls. Most interestingly in DCM patients, a significant 8.7-fold (P < 0.05) increase was observed. Thus, protein expression paralleled gene expression. These results demonstrate that (P)RR expression is strongly up-regulated both in rodent models of heart failure and in the failing human heart, hinting to a potential role for (P)RR in cardiac pathophysiology.     

Osmotic stress-induced increase of phosphatidylinositol 3,5-bisphosphate requires Vac14p, an activator of the lipid kinase Fab1p

Phosphatidylinositol 3,5-bisphosphate (PtdIns[3,5]P(2)) was first identified as a non-abundant phospholipid whose levels increase in response to osmotic stress. In yeast, Fab1p catalyzes formation of PtdIns(3,5)P(2) via phosphorylation of PtdIns(3)P. We have identified Vac14p, a novel vacuolar protein that regulates PtdIns(3,5)P(2) synthesis by modulating Fab1p activity in both the absence and presence of osmotic stress. We find that PtdIns(3)P levels are also elevated in response to osmotic stress, yet, only the elevation of PtdIns(3,5)P(2) levels are regulated by Vac14p. Under basal conditions the levels of PtdIns(3,5)P(2) are 18-28-fold lower than the levels of PtdIns(3)P, PtdIns(4)P, and PtdIns(4,5)P(2). After a 10 min exposure to hyperosmotic stress the levels of PtdIns(3,5)P(2) rise 20-fold, bringing it to a cellular concentration that is similar to the other phosphoinositides. This suggests that PtdIns(3,5)P(2) plays a major role in osmotic stress, perhaps via regulation of vacuolar volume. In fact, during hyperosmotic stress the vacuole morphology of wild-type cells changes dramatically, to smaller, more highly fragmented vacuoles, whereas mutants unable to synthesize PtdIns(3,5)P(2) continue to maintain a single large vacuole. These findings demonstrate that Vac14p regulates the levels of PtdIns(3,5)P(2) and provide insight into why PtdIns(3,5)P(2) levels rise in response to osmotic stress.     

FTIR study of the primary electron donor of photosystem I (P700) revealing delocalization of the charge in P700(+) and localization of the triplet character in (3)P700.

The effect of global (15)N or (2)H labeling on the light-induced P700(+)/P700 FTIR difference spectra has been investigated in photosystem I samples from Synechocystis at 90 K. The small isotope-induced frequency shifts of the carbonyl modes observed in the P700(+)/P700 spectra are compared to those of isolated chlorophyll a. This comparison shows that bands at 1749 and 1733 cm(-)(1) and at 1697 and 1637 cm(-)(1), which upshift upon formation of P700(+), are candidates for the 10a-ester and 9-keto C=O groups of P700, respectively. A broad and relatively weak band peaking at 3300 cm(-)(1), which does not shift upon global labeling or (1)H-(2)H exchange, is ascribed to an electronic transition of P700(+), indicating that at least two chlorophyll a molecules (denoted P(1) and P(2)) participate in P700(+). Comparisons of the (3)P700/P700 FTIR difference spectrum at 90 K with spectra of triplet formation in isolated chlorophyll a or in RCs from photosystem II or purple bacteria identify the bands at 1733 and 1637 cm(-)(1), which downshift upon formation of (3)P700, as the 10a-ester and 9-keto C=O modes, respectively, of the half of P700 that bears the triplet (P(1)). Thus, while the P(2) carbonyls are free from interaction, both the 10a-ester and the 9-keto C=O of P(1) are hydrogen bonded and the latter group is drastically perturbed compared to chlorophyll a in solution. The Mg atoms of P(1) and P(2) appear to be five-coordinated. No localization of the triplet on the P(2) half of P700 is observed in the temperature range of 90-200 K. Upon P700 photooxidation, the 9-keto C=O bands of P(1) and P(2) upshift by almost the same amount, giving rise to the 1656(+)/1637(-) and 1717(+)/1697(-) cm(-)(1) differential signals, respectively. The relative amplitudes of these differential signals, as well as of those of the 10a-ester C=O modes, appear to be slightly dependent on sample orientation and temperature and on the organism used to generate the P700(+)/P700 spectrum. If it is assumed that the charge density on ring V of chlorophyll a, as measured by the perturbation of the 10a-ester or 9-keto C=O IR vibrations, mainly reflects the spin density on the two halves of the oxidized P700 special pair, a charge distribution ranging from 1:1 to 2:1 (in favor of P(2)) is deduced from the measurements presented here. The extreme downshift of the 9-keto C=O group of P(1), indicative of an unusually strong hydrogen bond, is discussed in relation with the models previously proposed for the PSI special pair.     

P52rIPK regulates the molecular cochaperone P58IPK to mediate control of the RNA-dependent protein kinase in response to cytoplasmic stress

The 52 kDa protein referred to as P52(rIPK) was first identified as a regulator of P58(IPK), a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52(rIPK) and P58(IPK) each possess structural domains implicated in stress signaling, including the charged domain of P52(rIPK) and the tetratricopeptide repeat (TPR) and DnaJ domains of P58(IPK). The P52(rIPK) charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52(rIPK) function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58(IPK). This domain bound specifically to P58(IPK) TPR domain 7, the domain adjacent to the TPR motif required for P58(IPK) interaction with PKR, thus providing a mechanism for P52(rIPK) inhibition of P58(IPK) function. Both the charged domain of P52(rIPK) and the TPR 7 domain of P58(IPK) were required for P52(rIPK) to mediate downstream control of PKR activity, eIF2alpha phosphorylation, and cell growth. Furthermore, we found that P52(rIPK) and P58(IPK) formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52(rIPK) charged domain functions as a TPR-specific signaling motif to directly regulate P58(IPK) within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.     

Absence of clustering of phosphatidylinositol-(4,5)-bisphosphate in fluid phosphatidylcholine

Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] plays a key role in the modulation of actin polymerization and vesicle trafficking. These processes seem to depend on the enrichment of PI(4,5)P(2) in plasma membrane domains. Here, we show that PI(4,5)P(2) does not form domains when in a fluid phosphatidylcholine matrix in the pH range of 4.8-8.4. This finding is at variance with the spontaneous segregation of PI(4,5)P(2) to domains as a mechanism for the compartmentalization of PI(4,5)P(2) in the plasma membrane. Water/bilayer partition of PI(4,5)P(2) is also shown to be dependent on the protonation state of the lipid.     

Differential stereoselectivity of D- and L-myo-inositol 1,2,4, 5-tetrakisphosphate binding to the inositol 1,4,5-trisphosphate receptor and 3-kinase

D- and L-myo-inositol 1,2,4,5-tetrakisphosphate (Ins(1,2,4,5)P(4)) were investigated for their ability to bind to the D-myo-inositol 1, 4,5-trisphosphate (Ins(1,4,5)P(3)) receptor in a bovine adrenal cortical membrane fraction, to mobilize intracellular Ca(2+) stores in Xenopus oocytes, and to bind to the rat brain Ins(1,4,5)P(3) 3-kinase overexpressed and purified in E. coli. In competitive binding experiments with the Ins(1,4,5)P(3) receptor, D-Ins(1,2,4, 5)P(4) effectively displaced [(3)H]Ins(1,4,5)P(3) in a concentration-dependent manner with a potency comparable to that of D-Ins(1,4,5)P(3), while L-Ins(1,2,4,5)P(4) was approximately 50-fold less effective than D-Ins(1,4,5)P(3) and D-Ins(1,2,4,5)P(4). The DL-Ins(1,2,4,5)P(4) racemate bound to the Ins(1,4,5)P(3) receptor with an apparent intermediate efficiency. Injection of D-Ins(1,2,4, 5)P(4) into oocytes evoked a chloride current dependent on intracellular Ca(2+) mobilization in which the agonists ranked in a similar order of potency as in the Ins(1,4,5)P(3) receptor binding. On the other hand, D-Ins(1,2,4,5)P(4) only inhibited the binding of [(3)H]Ins(1,4,5)P(3) to 3-kinase very weakly with a markedly reduced potency compared to D-Ins(1,4,5)P(3), indicating that D-Ins(1,2,4, 5)P(4) is not an effective competitor in the phosphorylation of [(3)H]-Ins(1,4,5)P(3) by 3-kinase. The results, therefore, clearly indicate that D-Ins(1,2,4,5)P(4) is as effective as D-Ins(1,4,5)P(3) in the binding to the receptor but not 3-kinase, and access of Ins(1, 2,4,5)P(4) over the Ins(1,4,5)P(3) receptor calls for stringent stereospecificity with D-Ins(1,2,4,5)P(4) being the active form in DL-Ins(1,2,4,5)P(4)-mediated Ca(2+) mobilization.     

Rules for optimization of biocatalysis in organic solvents

General rules for the optimization of different biocatalytic systems in various types of media containing organic solvents are derived by combining data from the literature, and the logarithm of the partition coefficient, log P, as a quantitative measure of solvent polarity. (1) Biocatalysis in organic solvents is low in polar solvents having a log P < 2, is moderate in solvents having a log P between 2 and 4, and is high in a polar solvents having a log P > 4. It was found that this correlation between polarity and activity parallels the ability of organic solvents to distort the essential water layer that stabilizes the biocatalysts. (2) Further optimization of biocatalysis in organic solvents is achieved when the polarity of the microenvironment of the biocatalyst (log P(i)) and the continuous organic phase (log P(cph)) is tuned to the polarities of both the substrate (log P(s)) and the product (log P(p)) according to the following rules: |log P(i) - log P(s)| and |log P(cph) - log P(p)| should be minimal and |log P(cph) - log P(s)| and |log P(i) - log P(p)| should be maximal, with the exception that in the case of substrate inhibition log P(i), should be optimized with respect to log P(s) In addition to these simple optimization rules, the future developments of biocatalysis in organic solvents are discussed.     

Evidence that inositol 1-phosphate in brain of lithium-treated rats results mainly from phosphatidylinositol metabolism.

In cerebral cortex of rats treated with increasing doses of LiCl, the relative concentrations of Ins(1)P, Ins(4)P and Ins(5)P (when InsP is a myo-inositol phosphate) are approx. 10:1:0.2 at all doses. In rats treated with LiCl followed by increasing doses of pilocarpine a similar relationship occurs. myo-Inositol-1-phosphatase (InsP1ase) from bovine brain hydrolyses Ins(1)P, Ins(4)P and Ins(5)P at comparable rates, and these substrates have similar Km values. The hydrolysis of Ins(4)P is inhibited by Li+ to a greater degree than is hydrolysis of Ins(1)P and Ins(5)P. D-Ins(1,4,5)P3 and D-Ins(1,4)P2 are neither substrates nor inhibitors of InsP1ase. A dialysed high-speed supernatant of rat brain showed a greater rate of hydrolysis of Ins(1)P than of D-Ins(1,4)P2 and a lower sensitivity of the bisphosphate hydrolysis to LiCl, as compared with the monophosphate. That enzyme preparation produced Ins(4)P at a greater rate than Ins(1)P when D-Ins(1,4)P2 was the substrate. The amount of D-Ins(3)P [i.e. L-Ins(1)P, possibly from D-Ins(1,3,4)P3] is only 11% of that of D-Ins(1)P on stimulation with pilocarpine in the presence of Li+. DL-Ins(1,4)P2 was hydrolysed by InsP1ase to the extent of about 50%; both Ins(4)P and Ins(1)P are products, the former being produced more rapidly than the latter; apparently L-Ins(1,4)P2 is a substrate for InsP1ase. Li+, but not Ins(2)P, inhibited the hydrolysis of L-Ins(1,4)P2. The following were neither substrates nor inhibitors of InsP1ase; Ins(1,6)P2, Ins(1,2)P2, Ins(1,2,5,6)P4, Ins(1,2,4,5,6)P5, Ins(1,3,4,5,6)P5 and phytic acid. myo-Inositol 1,2-cyclic phosphate was neither substrate nor inhibitor of InsP1ase. We conclude that the 10-fold greater tissue contents of Ins(1)P relative to Ins(4)P in both stimulated and non-stimulated rat brain in vivo are the consequence of a much larger amount of PtdIns metabolism than polyphosphoinositide metabolism under these conditions.     

Dependence of purinergic P2X receptor activity on ectodomain structure

Purinergic receptors (P2XRs) activate and desensitize in response to the binding of extracellular nucleotides in a receptor- and ligand-specific manner, but the structural bases of their ligand preferences and channel kinetics have been incompletely characterized. Here we tested the hypothesis that affinity of agonists for binding domain accounts for a ligand-specific desensitization pattern. We generated chimeras using receptors with variable sensitivity to ATP in order: P2X(4)R > P2X(2a)R = P2X(2b)R P2X(7)R. Chimeras having the ectodomain Ile(66)-Tyr(310) sequence of P2X(2)R and Val(61)-Phe(313) sequence of P2X(7)R in the backbone of P2X(4)R were expressed but were non-functioning channels. P2X(2a) + X(4)R and P2X(2b) + X(4)R chimeras having the Val(66)-Tyr(315) ectodomain sequence of P2X(4)R in the backbones of P2X(2a)R and P2X(2b)R were functional and exhibited increased sensitivity to ligands as compared with both parental receptors. These chimeras also desensitized faster than parental receptors and in a ligand-nonspecific manner. However, like parental P2X(2b)R and P2X(2a)R, chimeric P2X(2b) + X(4)R desensitized more rapidly than P2X(2a) + X(4)R, and the rate of desensitization of P2X(2a)+X(4)R increased by substituting its Arg(371)-Pro(376) intracellular C-terminal sequence with the Glu(376)-Gly(381) sequence of P2X(4)R. These results indicate the relevance of interaction between the ectodomain and flanking regions around the transmembrane domains on ligand potency and receptor activation. Furthermore, the ligand potency positively correlates with the rate of receptor desensitization but does not affect the C-terminal-specific pattern of desensitization.     

Equivalent radius of paracellular "pores" of the mesothelium.

Diffusional permeability (P) to water (P(w)), Cl(-) (P(Cl(-))), and mannitol (P(man)) was determined in specimens of rabbit parietal pericardium without and with phospholipids added on the luminal side, as previously done with sucrose and Na(+). P to the above-mentioned molecules and to Na(+) (P(Na(+))) was also determined after mesothelium was scraped away from specimens. P(w), P(Cl(-)), P(Na(+)), and P(man) of connective tissue were the following (x10(-5) cm/s): 73.1 +/- 7.3 (SE), 59.5 +/- 4.5, 41.7 +/- 3.4, and 23.4 +/- 2.4, respectively. From these and corresponding data on integer pericardium, P(w), P(Cl(-)), P(Na(+)), and P(man) of mesothelium were computed. They were the following: 206, 17.9, 9.52, and 3.93, and 90.2, 14.4, 4.34, and 1.75 x 10(-5) cm/s without and with phospholipids, respectively. As previously found for P to sucrose, P to solutes is smaller in mesothelium than in connective tissue, although the latter is approximately 35-fold thicker; instead, P(w) is higher in mesothelium, suggesting marked water diffusion through cell membrane. Equivalent radius of paracellular "pores" of mesothelium was computed with two approaches, disregarding P(w). The former, a graphical analysis on a P-molecular radius diagram, yielded 6.0 and 1.7 nm without and with phospholipids, respectively. The latter, on the basis of P(man), P to sucrose, and function for restricted diffusion, yielded 7.8 and 1. 1 nm, respectively.     

Rapid accumulation of inositol trisphosphate reveals that agonists hydrolyse polyphosphoinositides instead of phosphatidylinositol.

The agonist-dependent hydrolysis of inositol phospholipids was investigated by studying the breakdown of prelabelled lipid or by measuring the accumulation of inositol phosphates. Stimulation of insect salivary glands with 5-hydroxytryptamine for 6 min provoked a rapid disappearance of [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and [3H]phosphatidylinositol 4-phosphate (PtdIns4P) but had no effect on the level of [3H]phosphatidylinositol (PtdIns). The breakdown of PtdIns(4,5)P2 was associated with a very rapid release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reached a peak 5 1/2 times that of the resting level after 5 s of stimulation. This high level was not maintained but declined to a lower level, perhaps reflecting the disappearance of PtdIns(4,5)P2. 5-Hydroxytryptamine also induced a rapid and massive accumulation of inositol 1,4-bisphosphate [Ins(1,4)P2]. The fact that these increases in Ins(1,4,5)P3 and Ins(1,4)P2 precede in time any increase in the level of inositol 1-phosphate or inositol provides a clear indication that the primary action of 5-hydroxytryptamine is to stimulate the hydrolysis of PtdIns(4,5)P2 to yield diacylglycerol and Ins(1,4,5)P3. The latter is then hydrolysed by a series of phosphomonoesterases to produce Ins(1,4)P2, Ins1P and finally inositol. The very rapid agonist-dependent increases in Ins(1,4,5)P3 and Ins(1,4)P2 suggests that they could function as second messengers, perhaps to control the release of calcium from internal pools. The PtdIns(4,5)P2 that is used by the receptor mechanism represents a small hormone-sensitive pool that must be constantly replenished by phosphorylation of PtdIns. Small changes in the size of this small energy-dependent pool of polyphosphoinositide will alter the effectiveness of the receptor mechanism and could account for phenomena such as desensitization and super-sensitivity.     

Partial purification of inositol polyphosphate 1-phosphomonoesterase with characterization of its substrates and products by nuclear magnetic resonance spectroscopy

A study of the enzyme activities that degrade Ins(1,3,4)P3 in rat brain showed that it was dephosphorylated primarily by a Mg2+-dependent inositol polyphosphate 1-phosphomonoesterase to Ins(3,4)P2 and then to Ins(3)P by a 4-phosphomonoesterase. A less active enzyme activity with the properties of a 4-phosphomonoesterase that converted Ins(1,3,4)P3 to Ins(1,3)P2 was also detected. The inositol polyphosphate 1-phosphomonoesterase was separated from the 4-phosphomonoesterase and the inositol monophosphate phosphomonoesterase by chromatography on phosphocellulose, DE-52 anion exchange and hydroxylapatite columns. Kinetic characterization of the partially purified inositol polyphosphate 1-phosphomonoesterase indicated that both Ins(1,3,4)P3 and Ins(1,4)P2 were substrates with apparent Km values of 0.9 microM and 0.7 microM, respectively. Either substrate was a competitive inhibitor of the other substrate and dephosphorylation of both substrates was directly inhibited by Li+ in an uncompetitive manner. These data strongly suggest that a single enzyme dephosphorylates both Ins(1,3,4)P3 and Ins(1,4)P2. The 4-phosphomonoesterase that dephosphorylated Ins(3,4)P2 to Ins(3)P was insensitive to Mg2+ and Li+ and was probably the same enzyme that degraded Ins(1,3,4)P3 to Ins(1,3)P2. The isomeric configurations of the major inositol polyphosphates formed from the degradation of Ins(1,3,4,5)P4 were determined using 1H- and 31P-NMR spectroscopy, and confirmation of the structures assigned to Ins(1,3,4,5)P4, Ins(1,3,4)P3 and Ins(3,4)P2 was obtained.     

Hydrogen bonding to P700: site-directed mutagenesis of threonine A739 of photosystem I in Chlamydomonas reinhardtii

The primary electron donor P700 of photosystem I is a dimer comprised of chlorophyll a (P(B)) and chlorophyll a' (P(A)). P(A) is involved in a hydrogen bond network with several surrounding amino acid residues and a nearby water molecule. To investigate the influence of hydrogen bond interactions on the properties of P700, the threonine at position A739, which donates a putative hydrogen bond to the 13(1)-keto group of P(A), was replaced with valine, histidine, and tyrosine in Chlamydomonas reinhardtii using site-directed mutagenesis. Growth of the mutants was not impaired. (i) The (P700(+)* - P700) FTIR difference spectra of the mutants lack a negative band at 1634 cm(-1) observed in the wild-type spectrum and instead exhibit a new negative band between 1658 and 1672 cm(-1) depending on the mutation. This band can therefore be assigned to the 13(1)-keto group of P(A) which is upshifted to higher frequencies upon removal of the hydrogen bond. (ii) The main bleaching band in the Q(y)() region of the (P700(+)* - P700) and ((3)P700 - P700) absorption difference spectra is blue shifted for the mutants by approximately 6 nm compared to that of the wild type. A blue shift is also observed for the main bleaching in the Soret region. (iii) The (P700(+)* - P700) CD difference spectrum of the wild type reveals two bands at 694 nm (positive CD) and 680 nm (negative CD) of approximately equal area. For each mutant, these two components are blue-shifted to the same extent. The results strongly suggest that a blue shift of the Q(y) absorption band of P(A) is responsible for a blue shift of the exciton bands. (iv) Redox titrations yielded a decrease in the midpoint potential for the oxidation of P700 by 32 mV for the exchange of Thr against Val. (v) ENDOR spectroscopy shows that the hfc of the methyl protons at position 12 of the spin-carrying Chl P(B) is decreased due to the removal of the hydrogen bond to P(A). This indicates a redistribution of spin density in P700(+)* compared to that in the wild type. This gives evidence for an electronic coupling between the two halves of the dimer in the wild type and mutants.     

Turnover of the phosphomonoester groups of polyphosphoinositol lipids in unstimulated human platelets

The metabolic activity of the polyphosphoinositol lipids in unstimulated human platelets was studied by short-term labelling with [32P]Pi, by replacement of [32P]Pi from pre-labelled platelets with unlabelled phosphate, and by depriving the cells of metabolic ATP. Under short-term labelling conditions, the 4- and 5-phosphate groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] had the same specific 32P radioactivity as the gamma-phosphate of metabolic ATP. The specific 32P radioactivity of the 1-phosphates of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2 was similar, but only 4-13% compared to that of the ATP-gamma-phosphate. When [32P]Pi pre-labelled platelets were incubated with up to 25 mM of unlabelled phosphate, the displacement of the 32P label from PtdIns4P, PtdIns(4,5)P2 and metabolic ATP followed similar kinetics. Inhibition of ATP regeneration in platelets pre-labelled with [32P]Pi resulted in a rapid fall in metabolic ATP with a much slower fall in [32P]PtdIns(4,5)P2, whereas [32P]PtdIns4P increased initially. However, ATP turnover was not abolished, as indicated by the marked (25% of the control) incorporation of extracellular [32P]Pi into PtdIns4P and PtdIns(4,5)P2 in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PtdIns4P and PtdIns(4,5)P2 to metabolic inhibition. We conclude that PtdIns4P and PtdIns(4,5)P2 are present as a single metabolic pool in human platelets. Turnover of the 4- and 5-phosphates of PtdIns4P and PtdIns(4,5)P2 in unstimulated platelets is as rapid as that of the gamma-phosphate of metabolic ATP, and accounts for about 7% of basal ATP consumption.     

The effects of N-dodecane pretreatment on the metabolism and distribution of benzo(A) pyrene in the isolated perfused rabbit lung

B(a)P is an ubiquitous potent carcinogen that has been associated with the increased incidence of human bronchiogenic carcinoma in occupational and urban populations. An isolated perfused rabbit lung preparation (IPL) was used to study the influence of pretreatment with a cocarcinogen, n-dodecane, on the metabolism of benzo (a)-pyrene (B(a)P). ¹⁴C-B(a)P was administered intratracheally to the IPL following biweekly inhalation exposure of the rabbits to n-dodecane. Results from n-dodecane pretreatment group were compared to those from the no-pretreatment group and an intratracheally administered B(a)P pretreatment group (B(a)P IT). Metabolites were isolated from serial blood samples up to 3 hours after administration of the ¹⁴C-B(a)P to the IPL. Patterns of B(a)P metabolites were determined using thin layer and high performance liquid chromatography, and liquid scintillation counting. The rates of appearance of B(a)P metabolites in the blood and the B(a)P metabolic pattern for n-dodecane groups were compared to B(a)P IT and no-pretreatment groups. The rates of appearance of metabolites in the blood (ng/hr/g of lung) were similar for B(a)P IT and n-dodecane pretreatment groups, but were five and four times greater, respectively, when compared to rates of appearance of metabolites for the no-pretreatment group. The B(a)P metabolic pattern of n-dodecane pretreatment showed an increase in the dihydrodiol and nonextractable formation and a decrease in the monohydroxy and diones compared to the no-pretreatment group. The metabolite pattern for B(a)P IT pretreatment group was similar to the metabolite pattern for the n-dodecane pretreatment group. The significance of the findings is that a known cocarcinogen, n-dodecane, appears to be as good an enzyme inducer as B(a)P in stimulating metabolism of B(a)P in lung. Therefore, man's risk in developing cancer may not be solely a result of exposure to a carcinogen, but may be dependent upon other constituents in his environment.     

Metabolism of inositol phosphates in alpha 1-adrenoceptor-stimulated and homogenized cardiac myocytes of adult rats.

Previous studies demonstrated the accumulation of inositol mono- and poly-phosphates in carbamoylcholine-stimulated cultured cardiac ventricular myocytes of adult rats [Berg, Guse & Gercken (1989) Biochim. Biophys. Acta 1010, 100-107]. Stimulation with noradrenaline (50 microM) in the presence of propranolol (10 microM) led to a time-dependent pattern of inositol mono- and poly-phosphates in cultured cardiac-ventricular myocytes. Ins(1,4,5)P3 and Ins(1,3,4,5)P4 increased in a rapid initial phase. The degradation products of Ins(1,4,5)P3, namely Ins(1,4)P2 and Ins(4)P, accumulated between 1 and 15 min. Ins(1,3,4,5)P4 was rapidly dephosphorylated to Ins(1,3,4)P3, which was then metabolized to Ins(1,3)P2 and Ins(3,4)P2. The last two InsP2 isomers and their degradation products, Ins(1)P and Ins(3)P (determined as an enantiomeric mixture), increased at extended stimulation periods. To confirm the pathway of Ins(1,3,4,5)P4 degradation, homogenates of isolated ventricular myocytes were incubated with [3H]INs(1,3,4,5)P4. The subsequent products were Ins(1,3,4)P3, Ins(3,4)P2, Ins(1,3)P2 and InsP. The effect of noradrenaline was antagonized by prazosin (0.1 microM), but not by yohimbine (0.1 microM), indicating phosphoinositidase activation via the alpha 1-adrenoceptor.     

Allosteric activation of PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor that is lost in many human tumors and encodes a phosphatidylinositol phosphate phosphatase specific for the 3-position of the inositol ring. Here we report a novel mechanism of PTEN regulation. Binding of di-C8-phosphatidylinositol 4,5-P2 (PI(4,5)P2) to PTEN enhances phosphatase activity for monodispersed substrates, PI(3,4,5)P3 and PI(3,4)P2. PI(5)P also is an activator, but PI(4)P, PI(3,4)P2, and PI(3,5)P2 do not activate PTEN. Activation by exogenous PI(4,5)P2 is more apparent with PI(3,4)P2 as a substrate than with PI(3,4,5)P3, probably because hydrolysis of PI(3,4)P2 yields PI(4)P, which is not an activator. In contrast, hydrolysis of PI(3,4,5)P3 yields a potent activator, PI(4,5)P2, creating a positive feedback loop. In addition, neither di-C4-PI(4,5)P2 nor inositol trisphosphate-activated PTEN. Hence, the interaction between PI(4,5)P2 and PTEN requires specific, ionic interactions with the phosphate groups on the inositol ring as well as hydrophobic interactions with the fatty acid chains, likely mimicking the physiological interactions that PTEN has with the polar surface head groups and the hydrophobic core of phospholipid membranes. Mutations of the apparent PI(4,5)P2-binding motif in the PTEN N terminus severely reduced PTEN activity. In contrast, mutation of the C2 phospholipid-binding domain had little effect on PTEN activation. These results suggest a model in which a PI(4,5)P2 monomer binds to PTEN, initiates an allosteric conformational change and, thereby, activates PTEN independent of membrane binding.