Subgrouping of facilitatory or inhibitory conditioned pain modulation responses in patients with chronic knee pain. Explorative analysis from a multicentre trial.

BACKGROUND: Facilitatory and inhibitory conditioned pain modulation (CPM) responses are observed in healthy volunteers and chronic pain patients, but the clinical implications for phenotyping are unknown. This study aimed to subgroup and compare chronic knee pain patients according to their CPM responses. METHODS: This explorative, cross-sectional study included 127 patients with chronic knee pain (osteoarthritis or following total knee arthroplasty). Individual CPM responses were categorized as facilitatory (test stimuli pain intensity increased when conditioning stimuli were applied), as inhibitory (test stimuli pain intensity decreased) or as no change (defined as less than 5.3% change in pain intensity). Outcomes were clinical pain intensities, temporal summation, widespread pain, self-reported physical function, PainDETECT questionnaire and Pain Quality Assessment Scale. Data were analysed as comparisons between the inhibitory and the facilitatory groups and using multivariate linear regression models. RESULTS: Fifty-four patients had facilitatory CPM responses, 49 had inhibitory CPM responses, and 24 showed no change in CPM response. A between-group difference was observed for self-reported physical function, with the facilitatory CPM group reporting better function (54.4 vs. 46.0, p = 0.028) and the facilitatory CPM group reported more deep pain sensations (3.2 vs. 2.0, p = 0.021). The remaining outcomes showed no between-group differences. Higher clinical pain intensity and facilitated temporal summation were associated in the facilitated CPM group but not in the inhibitory CPM group. CONCLUSION: These explorative findings indicated that quantitative clinical and experimental differences exist between facilitatory or inhibitory CPM responses in a chronic knee pain patient population. Differences in patients' CPM responses should be further investigated to unravel possible clinical importance. SIGNIFICANCE: Our findings confirm that conditioned pain modulation consist of inhibitory and facilitatory responders among a patient population with chronic knee pain. This explorative study indicates that patients with either facilitatory or inhibitory conditioned pain modulation could exhibit differences in pain outcomes. Subgrouping of chronic pain patients depending on individual conditioned pain modulation responses could be considered in phenotyping patients prior to inclusion in clinical trials or used for personalizing the management regime.

Slow Noncollinear Coulomb Scattering in the Vicinity of the Dirac Point in Graphene.

The Coulomb scattering dynamics in graphene in energetic proximity to the Dirac point is investigated by polarization resolved pump-probe spectroscopy and microscopic theory. Collinear Coulomb scattering rapidly thermalizes the carrier distribution in k directions pointing radially away from the Dirac point. Our study reveals, however, that, in almost intrinsic graphene, full thermalization in all directions relying on noncollinear scattering is much slower. For low photon energies, carrier-optical-phonon processes are strongly suppressed and Coulomb mediated noncollinear scattering is remarkably slow, namely on a ps time scale. This effect is very promising for infrared and THz devices based on hot carrier effects.

Isoprenylcysteine carboxyl methyltransferase deficiency in mice.

After isoprenylation, Ras and other CAAX proteins undergo endoproteolytic processing by Rce1 and methylation of the isoprenylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). We reported previously that Rce1-deficient mice died during late gestation or soon after birth. We hypothesized that Icmt deficiency might cause a milder phenotype, in part because of reports suggesting the existence of more than one activity for methylating isoprenylated proteins. To address this hypothesis and also to address the issue of other methyltransferase activities, we generated Icmt-deficient mice. Contrary to our expectation, Icmt deficiency caused a more severe phenotype than Rce1 deficiency, with virtually all of the knockout embryos (Icmt-/-) dying by mid-gestation. An analysis of chimeric mice produced from Icmt-/- embryonic stem cells showed that the Icmt-/- cells retained the capacity to contribute to some tissues (e.g. skeletal muscle) but not to others (e.g. brain). Lysates from Icmt-/- embryos lacked the ability to methylate either recombinant K-Ras or small molecule substrates (e.g. N-acetyl-S-geranylgeranyl-l-cysteine). In addition, Icmt-/- cells lacked the ability to methylate Rab proteins. Thus, Icmt appears to be the only enzyme participating in the carboxyl methylation of isoprenylated proteins.

The C-terminal polylysine region and methylation of K-Ras are critical for the interaction between K-Ras and microtubules.

After synthesis in the cytosol, Ras proteins must be targeted to the inner leaflet of the plasma membrane for biological activity. This targeting requires a series of C-terminal posttranslational modifications initiated by the addition of an isoprenoid lipid in a process termed prenylation. A search for factors involved in the intracellular trafficking of Ras has identified a specific and prenylation-dependent interaction between tubulin/microtubules and K-Ras. In this study, we examined the structural requirements for this interaction between K-Ras and microtubules. By using a series of chimeras in which regions of the C terminus of K-Ras were replaced with those of Ha-Ras and vice versa, we found that the polylysine region of K-Ras located immediately upstream of the prenylation site is required for binding of K-Ras to microtubules. Studies in intact cells confirmed the importance of the K-Ras polylysine region for microtubule binding, as deletion or replacement of this region resulted in loss of paclitaxel-induced mislocalization of a fluorescent K-Ras fusion protein. The additional modifications in the prenyl protein processing pathway also affected the interaction of K-Ras with microtubules. Removal of the three C-terminal amino acids of farnesylated K-Ras with the specific endoprotease Rce1p abolished its binding to microtubules. Interestingly, however, methylation of the C-terminal prenylcysteine restored binding. Consistent with these results, localization of the fluorescent K-Ras fusion protein remained paclitaxel-sensitive in cells lacking Rce1, whereas no paclitaxel effect was observed in cells lacking the methyltransferase. These studies show that the polylysine region of K-Ras is critical for its interaction with microtubules and provide the first evidence for a functional consequence of Ras C-terminal proteolysis and methylation.

Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.

After isoprenylation and endoproteolytic processing, the Ras proteins are methylated at the carboxyl-terminal isoprenylcysteine. The importance of isoprenylation for targeting of Ras proteins to the plasma membrane is well established, but the importance of carboxyl methylation, which is carried out by isoprenylcysteine carboxyl methyltransferase (Icmt), is less certain. We used gene targeting to produce homozygous Icmt knockout embryonic stem cells (Icmt-/-). Lysates from Icmt-/- cells lacked the ability to methylate farnesyl-K-Ras4B or small-molecule Icmt substrates such as N-acetyl-S-geranylgeranyl-L-cysteine. To assess the impact of absent Icmt activity on the localization of K-Ras within cells, wild-type and Icmt-/- cells were transfected with a green fluorescent protein (GFP)-K-Ras fusion construct. As expected, virtually all of the GFP-K-Ras fusion in wild-type cells was localized along the plasma membrane. In contrast, a large fraction of the fusion in Icmt-/- cells was trapped within the cytoplasm, and fluorescence at the plasma membrane was reduced. Also, cell fractionation/Western blot studies revealed that a smaller fraction of the K-Ras in Icmt-/- cells was associated with the membranes. We conclude that carboxyl methylation of the isoprenylcysteine is important for proper K-Ras localization in mammalian cells.

The membrane binding domains of prostaglandin endoperoxide H synthases 1 and 2. Peptide mapping and mutational analysis.

Prostaglandin endoperoxide H synthases 1 and 2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs. Both isozymes are integral membrane proteins but lack transmembrane domains. X-ray crystallographic studies have led to the hypothesis that PGHS-1 and -2 associate with only one face of the membrane bilayer through a novel, monotopic membrane binding domain (MBD) that is comprised of four short, consecutive, amphipathic alpha-helices (helices A-D) that include residues 74-122 in ovine PGHS-1 (oPGHS-1) and residues 59-108 in human PGHS-2 (hPGHS-2). Previous biochemical studies from our laboratory showed that the MBD of oPGHS-1 lies somewhere between amino acids 25 and 166. In studies reported here, membrane-associated forms of oPGHS-1 and hPGHS-2 were labeled using the hydrophobic, photoactivable reagent 3-trifluoro-3-(m-[(125)I]iodophenyl)diazirine, isolated, and cleaved with AspN and/or GluC, and the photolabeled peptides were sequenced. The results establish that the MBDs of oPGHS-1 and hPGHS-2 reside within residues 74-140 and 59-111, respectively, and thus provide direct provide biochemical support for the hypothesis that PGHS-1 and -2 do associate with membranes through a monotopic MBD. We also prepared HelA, HelB, and HelC mutants of oPGHS-1, in which, for each helix, three or four hydrophobic residues expected to protrude into the membrane were replaced with small, neutral residues. When expressed in COS-1 cells, HelA and HelC mutants exhibited little or no catalytic activity and were present, at least in part, as misfolded aggregates. The HelB mutant retained about 20% of the cyclooxygenase activity of native oPGHS-1 and partitioned in subcellular fractions like native oPGHS-1; however, the HelB mutant exhibited an extra site of N-glycosylation at Asn(104). When this glycosylation site was eliminated (HelB/N104Q mutation), the mutant lacked cyclooxygenase activity. Thus, our mutational analyses indicate that the amphipathic character of each helix is important for the assembly and folding of oPGHS-1 to a cyclooxygenase active form.

Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.

Little is known about the enzyme(s) required for the endoproteolytic processing of mammalian Ras proteins. We identified a mouse gene (designated Rce1) that shares sequence homology with a yeast gene (RCE1) implicated in the proteolytic processing of Ras2p. To define the role of Rce1 in mammalian Ras processing, we generated and analyzed Rce1-deficient mice. Rce1 deficiency was lethal late in embryonic development (after embryonic day 15.5). Multiple lines of evidence revealed that Rce1-deficient embryos and cells lacked the ability to endoproteolytically process Ras proteins. First, Ras proteins from Rce1-deficient cells migrated more slowly on SDS-polyacrylamide gels than Ras proteins from wild-type embryos and fibroblasts. Second, metabolic labeling of Rce1-deficient cells revealed that the Ras proteins were not carboxymethylated. Finally, membranes from Rce1-deficient fibroblasts lacked the capacity to proteolytically process farnesylated Ha-Ras, N-Ras, and Ki-Ras or geranylgeranylated Ki-Ras. The processing of two other prenylated proteins, the farnesylated Ggamma1 subunit of transducin and geranylgeranylated Rap1B, was also blocked. The absence of endoproteolytic processing and carboxymethylation caused Ras proteins to be mislocalized within cells. These studies indicate that Rce1 is responsible for the endoproteolytic processing of the Ras proteins in mammals and suggest a broad role for this gene in processing other prenylated CAAX proteins.

Cloning and characterization of a mammalian prenyl protein-specific protease.

Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Ggamma1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.

Photolabeling of prostaglandin endoperoxide H synthase-1 with 3-trifluoro-3-(m-[125I]iodophenyl)diazirine as a probe of membrane association and the cyclooxygenase active site.

Previous studies of the crystal structure of the ovine prostaglandin endoperoxide H synthase-1 (PGHS-1)/S-flurbiprofen complex (Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249) suggest that the enzyme is associated with membranes through a series of four amphipathic helices located between residues 70 and 117. We have used the photoactivatable, hydrophobic reagent 3-trifluoro-3-(m-[125I]iodophenyl)diazirine ([125I]TID) which partitions into membranes and other hydrophobic domains to determine which domains of microsomal PGHS-1 are subject to photolabeling. After incubation of ovine vesicular gland microsomes with [125I]TID, ovine PGHS-1 was one of the major photolabeled proteins. Proteolytic cleavage of labeled PGHS-1 at Arg277 with trypsin established that [125I]TID was incorporated into both the 33-kDa tryptic peptide containing the amino terminus and the 38-kDa tryptic peptide containing the carboxyl terminus. This pattern of photolabeling was not affected by the presence of 20 mM glutathione, indicating that the photolabeling observed for PGHS-1 was not due to the presence of [125I]TID in the aqueous phase. However, nonradioactive TID as well as two inhibitors, ibuprofen and sulindac sulfide, which bind the cyclooxygenase active site of PGHS-1, prevented the labeling of the 38-kDa carboxyl-terminal tryptic peptide. These results suggest that [125I]TID can label both the cyclooxygenase active site in the tryptic 38-kDa fragment and a membrane binding domain located in the 33-kDa fragment. Cleavage of photolabeled PGHS-1 with endoproteinase Lys-C yielded a peptide containing residues 25-166 which was labeled with [125I]TID. This peptide contains the putative membrane binding domain of ovine PGHS-1. Our results provide biochemical support for the concept developed from the crystal structure that PGHS-1 binds to membranes via four amphipathic helices located near the NH2 terminus of the protein.

The hepatitis delta virus large antigen is farnesylated both in vitro and in animal cells.

The hepatitis delta virus large antigen (lHDAg) is a virally encoded protein that contains a prenylation signal sequence at its carboxyl terminus consisting of the tetrapeptide Cys-Arg-Pro-Gln. Although the presence of the Gln as the COOH-terminal residue generally specifies addition of the 15-carbon farnesyl isoprenoid, earlier reports had suggested that the protein is modified by the 20-carbon geranylgeranyl. The prenylation of lHDAg was examined in vitro using a fusion protein between glutathione S-transferase and the COOH-terminal 117 amino acids of lHDAg (GST-lHDAg). When recombinant GST-lHDAg was incubated with bovine brain cytosol in the presence of either farnesyl diphosphate or geranylgeranyl diphosphate, GST-lHDAg was preferentially farnesylated. Geranylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified recombinant protein prenyltransferases, GST-lHDAg was found to be an excellent substrate (apparent Km = 0.8 microM) for protein farnesyltransferase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranylgeranylation of GST-lHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-lHDAg observed in cytosolic preparations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-lHDAg fusion protein, isoprenoid analysis of authentic lHDAg expressed in COS cells demonstrated that the protein was farnesylated. Geranylgeranylation of lHDAg expressed in COS cells was not observed. As prenylation of lHDAg is required for the assembly of the hepatitis delta viral particle, these results suggest that inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection.

Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2.

The subcellular locations of prostaglandin endoperoxide synthase-1 and -2 (PGHS-1 and -2) were determined by quantitative confocal fluorescence imaging microscopy in murine 3T3 cells and human and bovine endothelial cells using immunocytofluorescence with isozyme-specific antibodies. In all of the cell types examined, PGHS-1 immunoreactivity was found equally distributed in the endoplasmic reticulum (ER) and nuclear envelope (NE). PGHS-2 immunoreactivity was also present in the ER and NE. However, PGHS-2 staining was twice as concentrated in the NE as in the ER. A histofluorescence staining method was developed to localize cyclooxygenase/peroxidase activity. In quiescent 3T3 cells, which express only PGHS-1, histofluorescent staining was most concentrated in the perinuclear cytoplasmic region. In contrast, histochemical staining for PGHS-2 activity was about equally intense in the nucleus and in the cytoplasm, a pattern of activity staining distinct from that observed with PGHS-1. Our results indicate that there are significant differences in the subcellular locations of PGHS-1 and PGHS-2. It appears that PGHS-1 functions predominantly in the ER whereas PGHS-2 may function in the ER and the NE. We speculate that PGHS-1 and PGHS-2 acting in the ER and PGHS-2 functioning in the NE represent independent prostanoid biosynthetic systems.

Localization of prostaglandin endoperoxide synthase-1 to the endoplasmic reticulum and nuclear envelope is independent of its C-terminal tetrapeptide-PTEL.

Prostaglandin endoperoxide H (PGH) synthases 1 and 2 are both membrane-associated proteins localized to the endoplasmic reticulum (ER) and nuclear envelope. The carboxyl terminal tetrapeptides of PGH synthases 1 and 2 are of the form -P/STEL. These sequences are similar to the -KDEL retention signal sequence characteristic of many proteins localized to the ER. To determine if the -PTEL sequence (residues 597-600) functions as an ER retention signal for ovine PGH synthase-1, we prepared and analyzed five mutants (L600N, L600R, L600V, E599Q, and delta 597), all having modifications that would be expected to alter the subcellular location of PGH synthase-1 if the -PTEL sequence were involved in ER targeting. Native ovine PGH synthase-1 and each of the five mutants were subcloned into the pSVT7 expression vector and were expressed transiently in cos-1 cells. The L600N, L600R, E599Q, and delta 597 mutants retained both cyclooxygenase and peroxidase activities. Moreover, when subjected to immunocytofluorescent staining, cos-1 cells expressing native and mutant enzymes showed similar patterns of fluorescence corresponding to ER and nuclear envelope localization. Finally, culture media bathing cos-1 cells transfected with native or mutant PGH synthases were tested for secreted PGH synthase-1 protein by Western blotting, but no PGH synthase-1 was detected in any of the culture media. Our results demonstrate that mutations in the C-terminal sequence-PTEL do not change the subcellular location of ovine PGH synthase-1. Thus, targeting of PGH synthase-1 to the ER can occur independent of its -PTEL sequence.

The orientation of prostaglandin endoperoxide synthases-1 and -2 in the endoplasmic reticulum.

Prostaglandin endoperoxide H synthases (PGHS)-1 and -2 are integral membrane proteins of the endoplasmic reticulum (ER). The luminal versus cytoplasmic orientations of several epitopes of PGHS-1 and PGHS-2 were determined by immunocytofluorescent staining of cells following treatment with membrane-selective permeants. With serum-stimulated, murine NIH/3T3 cells expressing PGHS-2, an anti-peptide antibody directed against a domain near the COOH terminus of this isozyme caused staining only after all membranes were permeabilized with 0.2% saponin; no staining occurred with 3T3 cells treated with digitonin to permeabilize only the plasma membrane. Similarly, cos-1 cells expressing ovine PGHS-1 were stained with anti-peptide antibodies directed against (a) the amino terminus (residues 25-35), (b) a domain containing the tryptic cleavage site at Arg277 (residues 272-284), or (c) a region near the carboxyl terminus (residues 583-594) following permeabilization with saponin but not with digitonin or streptolysin O. The results obtained with the antibodies against the Arg277-containing domain of PGHS-1 were surprising because the enzyme is susceptible to tryptic cleavage at Arg277 in microsomal preparations. However, enzymatic and immunochemical analyses of microsomes prepared from ovine vesicular glands and cos-1 cells indicated that these microsomes are not intact. Accordingly, our results indicate that the trypsin cleavage site (Arg277) as well as the NH2 and COOH termini of ovine PGHS-1 are on the luminal side of the ER. The NH2 terminus, the Arg277 domain, and the N-glycosylation sites of ovine PGHS-1 are part of a large soluble, globular structure in crystalline ovine PGHS-1 (Picot, D., Loll, P. J., and Garavito, M. (1994) Nature, 367, 243-249). We conclude that PGHS-1 and, by analogy, the highly homologous PGHS-2 are luminal ER proteins. Assuming that the PGHS-1 and PGHS-2 present in the ER are functional in intact cells, our results indicate that PGH2 synthesis from arachidonate occurs in the lumen of the ER.

N-glycosylation of prostaglandin endoperoxide synthases-1 and -2 and their orientations in the endoplasmic reticulum.

Using site-directed mutagenesis, we have determined that Asn68, Asn144, and Asn410 of ovine prostaglandin endoperoxide (PGH) synthase-1 are N-glycosylated. A fourth consensus N-glycosylation sequence at Asn104 is not glycosylated. Glycosylation of PGH synthase-1 at Asn410 and at either Asn68 or Asn144 was required for expression of both the cyclooxygenase and the peroxidase activities of the enzyme. Inactive PGH synthase-1 glycosylation site mutant proteins do not appear to achieve their native conformations. However, the native enzyme, once in an active conformation, does not appear to require attached carbohydrate for cyclooxygenase or peroxidase activities. N-Glycosylation consensus sequences corresponding to the three glycosylation sites of ovine PGH synthase-1 are conserved in the deduced amino acid sequences of PGH synthases-2. Using site-directed mutagenesis, we determined that there is an additional site of N-glycosylation in murine PGH synthase-2 located at Asn580. This site is N-glycosylated in about 50% of PGH synthase-2 molecules, resulting in two peptide bands on SDS-polyacrylamide gel electrophoresis (72 and 74 kDa). Glycosylation of PGH synthase-2 is necessary for expression of enzyme activity, but glycosylation of PGH synthase-2 at Asn580 per se does not affect activity. Assuming that the N-glycosylation sites of PGH synthase-1 are on the luminal side of the endoplasmic reticulum (ER), and that the site of tryptic cleavage of ovine PGH synthase-1 (Arg277) is on the cytoplasmic side of the ER, we propose that both the NH2 and COOH termini of PGH synthase-1 are located in the lumen of the ER and that there are two transmembrane domains located between Asn144 and Arg277 and between Arg277 and Asn410, respectively. A similar orientation is predicted for PGH synthase-2.

Trp387 and the putative leucine zippers of PGH synthases-1 and -2.

The active site sequence 385-YHWH-388 of ovine prostaglandin endoperoxide synthase-1 (PGHS-1) has residues critical for cyclooxygenase and peroxidase catalysis. Tyr385 is essential for cyclooxygenase activity, His386, for peroxidase activity, and His388, for both activities. To determine the importance of Trp387, we used site-directed mutagenesis to replace Trp387 of PGHS-1 with arginine, phenylalanine, and serine. W387R and W387S lacked significant activity. W387F retained both cyclooxygenase and peroxidase activities. Thus, we conclude that Trp387 is not essential for catalysis by PGHS-1. Purified PGHS-1 is a homodimer. There are two putative leucine zipper regions in ovine PGHS-1 involving residues 345-366 and 487-508. We tested for a role of these leucine zippers as determinants of dimer formation. Helix-breaking proline mutations were introduced at Leu359 or Leu501. Neither of these residues proved to be essential for peroxidase activity; but, mutations at each residue greatly reduced or eliminated cyclooxygenase activity. Both mutant proteins chromatographed as dimers on Sephacryl G-200. Thus, neither of these putative leucine zipper regions alone is responsible for PGHS-1 dimer formation.

Effect of divalent ions on protein precipitation with polyethylene glycol: mechanism of action and applications.

Polyethylene glycol (PEG) is extensively employed for protein purification by fractional precipitation. Efficiency of precipitation is highest when the solution pH is near the isoelectric point of the target protein. At pH values far from the isoelectric point of the target protein, proteins develop a net positive or negative charge and are not more resistant to precipitation. We have found that divalent cations (Ba2+, Sr2+, and Ca2+) or divalent anions (SO4(2-)) significantly change the pattern of PEG precipitation when the ion is chosen so as to counteract the expected net charge on the target protein. At moderate (5-50 mM) concentrations of Ba2+, negatively charged proteins can be precipitated from solution at pH values as high as 10 with efficiency unchanged from precipitation at pH values near their isoelectric point values. The mechanism of PEG precipitation of protein at these high pH values appears to be unchanged from the mechanism operative at the protein isoelectric point. Precipitation is rapid and the capacity for protein precipitation is high. There is no detectable coprecipitation of small molecules (AMP, ATP, and NADH) or soluble proteins (carbonic anhydrase) induced when large quantities of protein are precipitated by this method. The purification of bovine carbonic anhydrase from erythrocyte lysate is more efficient at pH 10 in the presence of Ba2+ than is conventional PEG precipitation carried out at the isoelectric point of carbonic anhydrase. Application of these observations should broaden the utility of protein purification by fractional precipitation with PEG.