Isolation and characterization of the inositol cyclic phosphate products of polyphosphoinositide cleavage by phospholipase C. Physiological effects in permeabilized platelets and Limulus photoreceptor cells

Cleavage of the polyphosphoinositides, catalyzed by phospholipase C purified from ram seminal vesicles, produces phosphorylated inositols containing cyclic phosphate esters (Wilson, D. B., Bross, T. E., Sherman, W. R., Berger, R. A., and Majerus, P. W. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4013-4017). In the present study we describe the isolation and characterization of inositol 1:2-cyclic 4-bisphosphate and inositol 1:2-cyclic 4,5-trisphosphate, the two cyclic phosphate products of phospholipase C catalyzed cleavage of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, respectively. We established the structures of these two cyclic compounds through 18O labeling of phosphate moieties, phosphomonoesterase digestion, and fast atom bombardment-mass spectrometry. We examined the physiological effects of these compounds in two systems: saponin-permeabilized platelets loaded with 45Ca2+ and intact Limulus photoreceptors. Both inositol 1:2-cyclic 4,5-trisphosphate and the noncyclic inositol 1,4,5-trisphosphate, but not inositol 1:2-cyclic 4-bisphosphate, release 45Ca2+ from permeabilized platelets in a concentration-dependent manner. Injection of inositol 1:2-cyclic 4,5-trisphosphate into Limulus ventral photoreceptor cells induces both a change in membrane conductance and a transient increase in intracellular calcium ion concentration similar to those induced by light. We injected inositol 1,4,5-trisphosphate and inositol 1:2-cyclic 4,5-trisphosphate into the same photoreceptor cell and found that the cyclic compound is approximately five times more potent than the noncyclic compound in stimulating a conductance change. We speculate that inositol 1:2-cyclic 4,5-trisphosphate may function as a second messenger in stimulated cells.     

Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain

Two different forms of inositol phospholipid-specific phospholipase C (PLC) have been purified 2810- and 4010-fold, respectively, from a crude extract of rat brain. The purification procedures consisted of chromatography of both enzymes on Affi-Gel blue and cellulose phosphate, followed by three sequential high performance liquid chromatography steps, which were different for the two enzymes. The resultant preparations each contained homogeneous enzyme with a Mr of 85,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One of these enzymes (PLC-II) was found to hydrolyze phosphatidyl-inositol 4,5-bisphosphate (PIP2) at a rate of 15.3 mumol/min/mg of protein and also phosphatidylinositol 4-monophosphate and phosphatidylinositol (PI) at slower rates. For hydrolysis of PI, this enzyme was activated by an acidic pH and a high concentration of Ca2+ and showed a Vmax value of 19.2 mumol/min/mg of protein. The other enzyme (PLC-III) catalyzed hydrolysis of PIP2 preferentially at a Vmax rate of 12.9 mumol/min/mg of protein and catalyzed that of phosphatidylinositol 4-monophosphate slightly. The rate of PIP2 hydrolysis by this enzyme exceeded that of PI under all conditions tested. Neither of these enzymes had any activity on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, or phosphatidic acid. These two enzymes showed not only biochemical but also structural differences. Western blotting showed that antibodies directed against PLC-II did not react with PLC-III. Furthermore, the two enzymes gave different peptide maps after digestion with alpha-chymotrypsin or Staphylococcus aureus V8 protease. These results suggest that these two forms of PLC belong to different families of PLC.     

Activation of cytosolic phosphoinositide phospholipase C by G-protein beta gamma subunits.

Bovine liver cytosol contains a phosphoinositide phospholipase C (PLCcyt) that is activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S)-activated G-proteins from liver plasma membranes. Heparin-Sepharose chromatography indicated that PLCcyt was immunologically distinct from PLC-beta 1, PLC-gamma 1, or PLC-delta 1 from brain. Initial purification of the GTP gamma S-activated G-proteins that stimulated PLCcyt indicated that the beta gamma complex was responsible. G-proteins were subsequently extracted from liver membranes as heterotrimers and purified in the presence of AlCl3, MgCl2, and NaF to allow reversible activation. Immunoblot analysis with an antiserum selective for the beta subunit showed that the stimulatory activity corresponded with the presence of this protein at every chromatographic step. When liver beta gamma complex was purified and separated from all detectable alpha subunits, as shown by immunoblotting and silver staining, it strongly stimulated PLCcyt after removal of the activating ligand [AlF4]- by gel filtration. beta gamma prepared from brain was approximately equipotent with that from liver. beta gamma was half-maximally effective at 33 nM and produced a maximal 50-fold activation of the PLC. Under identical conditions, beta gamma had no effect on brain PLC-gamma 1 or PLC-delta 1 and produced a 2-fold stimulation of PLC-beta 1 activity. Addition of purified GDP-bound alpha o, which had no effect by itself, completely reversed the beta gamma activation of PLCcyt, confirming that beta gamma was the active species. These data provide evidence for a novel mechanism by which beta gamma subunits of pertussis toxin-sensitive or -insensitive G-proteins activate phospholipase C.     

Metabolism of thyrotropin releasing hormone in brain extracts. Isolation and characterization of an imidopeptidase for histidylprolineamide.

An extract of porcine brain acetone powder incubated with thyrotropin-releasing hormone (TRH; pGlu-His-ProNH2) produces acid TRH (pGlu-His-Pro), histidine, and prolineamide. Fractionation of the brain extract by DEAE-cellulose chromatography produces three protein fractions which metabolize TRH. The activity of these fractions was characterized using TRH with a 3H-label on the histidine or proline as well as [His-3H]His-ProNH2. Fraction I contains pyroglutamate aminopeptidase and Fraction II contains TRH deamidase. Fraction III was found to contain a previously unrecognized enzyme which cleaves His-ProNH2 to histidine and proline. The histidylprolineamide imidopeptidase has been characterized. A competition study using a variety of compounds containing histidine or proline suggests that the best substrates for the imidopeptidase contain a free alpha-amino group on histidine and a blocked carboxyl group on proline, as is found in His-ProNH2. A survey of a variety of polypeptide hormones indicates that many of them inhibit the imidopeptidase activity. A kinetic study of the inhibition of the enzyme by adrenocorticotropic hormone (1-24) shows that the inhibition by polypeptide hormones is noncompetitive. We hypothesize that pituitary hormones may stimulate the production of (cyclo)-His-Pro by inhibiting alternate routes of TRH metabolism.     

The formation of inositol 1,2-cyclic phosphate on agonist stimulation of phosphoinositide breakdown in mouse pancreatic minilobules. Evidence for direct phosphodiesteratic cleavage of phosphatidylinositol

It is generally thought that formation of inositol 1,2-cyclic phosphate (IcP) on agonist-stimulated "breakdown" of endogenous phosphatidylinositol in intact cells would provide strong evidence for the direct phosphodiesteratic cleavage of phosphatidylinositol. We report here that on ionophoresis of extracts of pancreatic minilobules incubated with the cholecystokinin/pancreozymin congener, caerulein, the usual inositol phosphates, i.e. inositol 1-phosphate (IP), inositol 4,5-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3) were seen. In addition, an [3H]inositol-labeled unknown was present with the correct electrophoretic mobility of IcP. There was only a trace of "IcP" in the unstimulated pancreatic minilobules. Several lines of evidence indicate that the unknown peak was IcP. 1) It ran on ionophoresis with standard [14C]IcP, and the ratio of 3H to 14C for each point on the peak was a constant within experimental error. 2) The putative IcP peak which had been eluted from the electropherogram also coincided with standard [14C]IcP on paper chromatography. 3) On mild acid hydrolysis in the presence of standard 14C-labeled IP, the putative [3H] IcP peak disappeared and appeared in the exact position of the standard [14C]IP peak, as to be predicted of IcP. The formation of IcP on agonist stimulation supports direct phosphodiesteratic cleavage of phosphatidylinositol on stimulation of phosphoinositide breakdown in pancreatic minilobules.     

Structure of the membrane protein of influenza virus. I. Isolation and characterization of cyanogen bromide cleavage products.

After cleavage of the membrane (M) protein of influenza A/WSN virus by using cyanogen bromide (CNBr), six peptide peaks representing approximate molecular weights of 6,000, 4,000, 2,200, 1,600, 1,200, and 1,000 were resolved by gel filtration on BioGel P6. Analysis by thin-layer chromatography indicates that the first, second, fourth, and fifth peaks contain single-peptide components, whereas the third and sixth peaks contain more than one peptide. By using Whatman CM52 ion-exchange chromatography in 5 M urea, four peptides were resolved from the third BioGel P6 peak. The amino acid composition of each of the purified peptides has been determined, and partial sequences were obtained for several peptides. Based on finding a blocked amino terminal residue, the 6,000-dalton fragment appears to contain the amino terminus of the M protein, whereas the carboxy terminal peptide was identified as a 2,000-dalton peptide.     

Identification and characterization of a phosphoinositide phosphate kinase homolog

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) plays a central role in regulating the actin cytoskeleton as a substrate for phosphoinositide 3-kinase and phospholipase C as well as by binding directly to proteins that control the processes of actin monomer sequestration, filament severing, capping, nucleation, cross-linking, and bundling (Ma, L., Cantley, L. C., Janmey, P. A., and Kirschner, M. W. (1998) J. Cell Biol. 140, 1125-1136; Hinchliffe, K. (2000) Curr. Biol. 10, R104-R1051). Three related phosphatidylinositol 4-phosphate 5-kinases (PI(4)P 5-kinases) have been identified in mammalian cells (types Ialpha, Ibeta, and Igamma) and appear to play distinct roles in actin remodeling. Here we have identified a fourth member of this family by searching the human genome and EST data bases. This new protein, which we have designated phosphatidylinositol phosphate kinase homolog (PIPKH), is expressed at relatively high levels in brain and testis. Immunoprecipitates of PIPKH expressed in mammalian cells contain PI(4)P 5-kinase activity, but this activity is not affected by mutations in residues that inactivate other type I PI(4)P 5-kinases. We show that the PI(4)P 5-kinase activity in PIPKH immunoprecipitates can be explained by the ability of PIPKH to heterodimerize with other type I PI(4)P 5-kinases. Transfection of 293t cells with PIPKH resulted in >8-fold increase in total phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) without a significant net increase in total PI(4,5)P(2). When coexpressed with PIPKH, green fluorescent protein (GFP) fusion construct of the pleckstrin homology domain from Bruton's tyrosine kinase (GFP-BTK-PH) localized in intracellular vesicular structures, suggesting an unusual intracellular site of PI(3,4,5)P(3) production. Finally, expression of PIPKH induced the reorganization of actin from predominantly stress fibers to predominantly foci and comets similar to those observed previously in cells infected with the intracellular pathogen Listeria or transfected with recombinant PIPKIalpha. These results suggest that PIPKH acts as a scaffold to localize and regulate type I PI(4)P 5-kinases and the synthesis of PI(3,4,5)P(3).     

Mammalian cells that express Bacillus cereus phosphatidylinositol-specific phospholipase C have increased levels of inositol cyclic 1:2-phosphate, inositol 1-phosphate, and inositol 2-phosphate.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.     

Identification in bovine liver plasma membranes of a Gq-activatable phosphoinositide phospholipase C.

Phosphoinositide phospholipase C (PLC) activity extracted from bovine liver plasma membranes with sodium cholate was stimulated by GTP gamma S-activated G alpha q/G alpha 11, whereas the enzyme from liver cytosol was not. The membrane-associated PLC was subjected to chromatography on heparin-Sepharose, Q Sepharose, and S300HR, enabling the isolation of the G-protein stimulated activity and its resolution from PLC-gamma and PLC-delta. Following gel filtration, two proteins of 150 and 140 kDa were found to correspond to the activatable enzyme. These proteins were identified immunologically as members of the PLC-beta family and were completely resolved by chromatography on TSK Phenyl 5PW. The 150-kDa enzyme was markedly responsive to GTP gamma S-activated alpha-subunits of G alpha q/G alpha 11 or to purified Gq/G11 in the presence of GTP gamma S. The response of this PLC was of much greater magnitude than that of the 140-kDa enzyme. The partially purified 150-kDa enzyme showed specificity for PtdIns(4,5)P2 and PtdIns4P as compared to PtdIns and had an absolute dependence upon Ca2+. These characteristics were similar to those of the brain PLC-beta 1. The immunological and biochemical properties of the 150-kDa membrane-associated enzyme are consistent with its being the PLC-beta isozyme that is involved in receptor-G-protein-mediated generation of inositol 1,4,5-triphosphate in liver.     

Carbon-carbon bond cleavage of fucosterol-24, 28-oxide by cell-free extracts of silkworm Bombyx mori.

The 1500 X g supernatant of the silkworm $\text{Bombyx mori}$ gut homogenate catalyzed the conversion of 24, 28-epoxystigmast-5-en-3β-ol(III) to cholesta-5, 24-dien-3β-ol(IV) which is a key step of stigmast-5-en-3β-ol(I) dealkylation in the insects. A structural analog 24, 28-imino-stigmast-5-en-3β-ol(VI) was a potent inhibitor of this conversion.