Tryptic Peptide Mapping Research Articles

Characterization of polyphenol oxidase from aerial roots of an orchid, Aranda `Christine 130'

Four isoforms of polyphenol oxidase (PPO) were demonstrated in the aerial roots of a tropical orchid, Aranda `Christine 130'. They were extracted at neutral pH and purified to homogeneity as judged by SDS-gel electrophoresis. Purification was achieved by a combination of Triton X-114 treatment, temperature phase partitioning, gel filtration chromatography, ion-exchange separation and chromatofocusing. Two of the isoforms, designated PPO a and PPO d, differed in their N-terminal sequence, tryptic peptide map and substrate affinity for (+)-catechin, but exhibited similarity in their molecular mass under denaturing conditions, pH optimum and kinetic behaviour toward 4-methyl catechol. The other two isoforms, PPO b and PPO c, were identical to PPO a and PPO d, respectively, in terms of their N-terminal sequence, substrate preference and pH maximum, but were different with regard to their molecular mass under denaturing conditions. These four isoforms differed in their isoelectric point.

Identification of proteins from two-dimensional gel electrophoresis of human erythroleukemia cells using capillary high performance liquid chromatography/electrospray-ion trap-reflectron time-of-flight mass spectrometry with two-dimensional topographic map analysis of in-gel tryptic digest products.

Protein spots from two-dimensional (2-D) gel electrophoresis of a human erythroleukemia cell line have been identified by analysis of the in-gel tryptic digests using capillary high performance liquid chromatography (HPLC) separation with on-line detection using electrospray ionization mass spectrometry (ESI-MS). This is performed using an electrospray/ion trap storage/reflectron time-of-flight mass spectrometer system (ESI-IT-reTOFMS). A 2-D topographic mapping display developed to process the on-line data acquired with this TOF system has been used to obtain mass identification of each peptide, even though the capillary HPLC only provides limited separation capability of the tryptic peptide mixtures studied herein. Using this method, a substantial fraction of the protein sequence can be covered and identified using the tryptic map. It is demonstrated that by entering the cell species, the approximate MW and pI range as determined by 2-D gel electrophoresis, and the tryptic peptide map into the database a unique match for identification of the protein generally results. It is also demonstrated that a much improved coverage of the protein sequence is obtained by this method relative to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

P90RSK Is a Serum-stimulated Na+/H+ Exchanger Isoform-1 Kinase: REGULATORY PHOSPHORYLATION OF SERINE 703 OF Na+/H+ EXCHANGER ISOFORM-1

The Na+/H+ exchanger isoform-1 (NHE-1) is the key member of a family of exchangers that regulates intracellular pH and cell volume. Activation of NHE-1 by growth factors is rapid, correlates with increased NHE-1 phosphorylation and cell alkalinization, and plays a role in cell cycle progression. By two-dimensional tryptic peptide mapping of immunoprecipitated NHE-1, we identify serine 703 as the major serum-stimulated amino acid. Mutation of serine 703 to alanine had no effect on acid-stimulated Na+/H+ exchange but completely prevented the growth factor-mediated increase in NHE-1 affinity for H+. In addition, we show that p90 ribosomal S6 kinase (p90(RSK)) is a key NHE-1 kinase since p90(RSK) phosphorylates NHE-1 serine 703 stoichiometrically in vitro, and transfection with kinase-inactive p90(RSK) inhibits serum-induced phosphorylation of NHE-1 serine 703 in transfected 293 cells. These findings establish p90(RSK) as a serum-stimulated NHE-1 kinase and a mediator of increased Na+/H+ exchange in vivo.

Priming of Human Neutrophil Respiratory Burst by Granulocyte/Macrophage Colony-stimulating Factor (GM-CSF) Involves Partial Phosphorylation of p47 phox

Neutrophil superoxide production can be potentiated by prior exposure to "priming" agents such as granulocyte/macrophage colony stimulating factor (GM-CSF). Because the mechanism underlying GM-CSF-dependent priming is not understood, we investigated the effects of GM-CSF on the phosphorylation of the cytosolic NADPH oxidase components p47(phox) and p67(phox). Preincubation of neutrophils with GM-CSF alone increased the phosphorylation of p47(phox) but not that of p67(phox). Addition of formyl-methionyl-leucyl-phenylalanine (fMLP) to GM-CSF-pretreated neutrophils resulted in more intense phosphorylation of p47(phox) than with GM-CSF alone and fMLP alone. GM-CSF-induced p47(phox) phosphorylation was time- and concentration-dependent and ran parallel to the priming effect of GM-CSF on superoxide production. Two-dimensional tryptic peptide mapping of p47(phox) showed that GM-CSF induced phosphorylation of one major peptide. fMLP alone induced phosphorylation of several peptides, an effect enhanced by GM-CSF pretreatment. In contrast to fMLP and phorbol 12-myristate 13-acetate, GM-CSF-induced phosphorylation of p47(phox) was not inhibited by the protein kinase C inhibitor GF109203X. The protein-tyrosine kinase inhibitor genistein and the phosphatidylinositol 3-kinase inhibitor wortmannin inhibited the phosphorylation of p47(phox) induced by GM-CSF and by fMLP but not that induced by phorbol 12-myristate 13-acetate. GM-CSF alone did not induce p47(phox) or p67(phox) translocation to the membrane, but neutrophils treated consecutively with GM-CSF and fMLP showed an increase (compared with fMLP alone) in membrane translocation of p47(phox) and p67(phox). Taken together, these results show that the priming action of GM-CSF on the neutrophil respiratory burst involves partial phosphorylation of p47(phox) on specific serines and suggest the involvement of a priming pathway regulated by protein-tyrosine kinase and phosphatidylinositol 3-kinase.

Identification and Mutation of Phosphorylation Sites in a Linker Histone: PHOSPHORYLATION OF MACRONUCLEAR H1 IS NOT ESSENTIAL FOR VIABILITY IN TETRAHYMENA

Linker histone phosphorylation has been suggested to play roles in both chromosome condensation and transcriptional regulation. In the ciliated protozoan Tetrahymena, in contrast to many eukaryotes, histone H1 of macronuclei is highly phosphorylated during interphase. Macronuclei divide amitotically without overt chromosome condensation in this organism, suggesting that requirements for phosphorylation of macronuclear H1 may be limited to transcriptional regulation. Here we report the major sites of phosphorylation of macronuclear H1 in Tetrahymena thermophila. Five phosphorylation sites, present in a single cluster, were identified by sequencing 32P-labeled peptides isolated from tryptic peptide maps. Phosphothreonine was detected within two TPVK motifs and one TPTK motif that resemble established p34(cdc2) kinase consensus sequences. Phosphoserine was detected at two non-proline-directed sites that do not resemble known kinase consensus sequences. Phosphorylation at the two noncanonical sites appears to be hierarchical because it was observed only when a nearby p34(cdc2) site was also phosphorylated. Cells expressing macronuclear H1 containing alanine substitutions at all five of these phosphorylation sites were viable even though macronuclear H1 phosphorylation was abolished. These data suggest that the five sites identified comprise the entire collection of sites utilized by Tetrahymena and demonstrate that phosphorylation of macronuclear H1, like the protein itself, is not essential for viability in Tetrahymena.

Purification of Tomato Sucrose Synthase Phosphorylated Isoforms by Fe(III)-Immobilized Metal Affinity Chromatography

The major phosphorylation site of maize sucrose synthase (SuSy) is well conserved among plant species but absent in the deduced peptide sequence of the tomato SuSy cDNA (TOMSSF). In this study, we report thein vitrophosphorylation of 25-day-old tomato fruits SuSy on seryl residue(s) by an endogenous Ca2+-dependent protein kinase activity. Two distinct32P-labeled peptides detected in the tryptic peptide map ofin vitro32P-radiolabeled tomato fruit SuSy were purified. Amino acid sequencing and phosphoamino acid analysis of the major32P-labeled peptide revealed the presence of a SuSy isozyme in young tomato fruit having the N-terminus phosphorylation site present in other plant species. By using Fe(III)-immobilized metal affinity chromatography [Fe(III)-IMAC] as a final purification step of tomato fruit SuSy, two32P-labeled tomato SuSy isoforms were separated from a nonradiolabeled SuSy fraction by using a pH gradient. The major32P-SuSy isoform was phosphorylated exclusively at the seryl residue related to the phosphorylation site of maize SuSy. The multiphosphorylated state of the second radiolabeled SuSy fraction was indicated by a higher retention during Fe(III)-IMAC and by tryptic peptide mapping analysis. Kinetic analyses of SuSy isoforms purified by Fe(III)-IMAC have revealed that phosphorylation of the major phosphorylation site of tomato fruit SuSy was not sufficient by itself to modulate tomato SuSy activity, whereas the affinity for UDP increased about threefold for the multiphosphorylated SuSy isoform.

The Xenopus laevis Aurora-related Protein Kinase pEg2 Associates with and Phosphorylates the Kinesin-related Protein XlEg5

We have previously reported on the cloning of XlEg5, a Xenopus laevis kinesin-related protein from the bimC family (Le Guellec, R., Paris, J., Couturier, A., Roghi, C., and Philippe, M. (1991) Mol. Cell. Biol. 11, 3395-3408) as well as pEg2, an Aurora-related serine/threonine kinase (Roghi, C., Giet, R., Uzbekov, R., Morin, N., Chartrain, I., Le Guellec, R., Couturier, A., Dorée, M., Philippe, M., and Prigent, C. (1998) J. Cell Sci. 111, 557-572). Inhibition of either XlEg5 or pEg2 activity during mitosis in Xenopus egg extract led to monopolar spindle formation. Here, we report that in Xenopus XL2 cells, pEg2 and XlEg5 are both confined to separated centrosomes in prophase, and then to the microtubule spindle poles. We also show that pEg2 co-immunoprecipitates with XlEg5 from egg extracts and XL2 cell lysates. Both proteins can directly interact in vitro, but also through the two-hybrid system. Furthermore immunoprecipitated pEg2 were found to remain active when bound to the beads and phosphorylate XlEg5 present in the precipitate. Two-dimensional mapping of XlEg5 tryptic peptides phosphorylated in vivo first confirmed that XlEg5 was phosphorylated by p34(cdc2) and next revealed that in vitro pEg2 kinase phosphorylated XlEg5 on the same stalk domain serine residue that was phosphorylated in metabolically labeled XL2 cells. The kinesin-related XlEg5 is to our knowledge the first in vivo substrate ever reported for an Aurora-related kinase.

CAMP-dependent phosphorylation of the tetrodotoxin-resistant voltage-dependent sodium channel SNS.

1. Protein kinase A (PKA) modulation of tetrodotoxin-resistant (TTX-r) voltage-gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned alpha-subunit of the rat sensory neurone-specific TTX-r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intracellular I-II loop had been eliminated by site-directed mutagenesis (serine to alanine). 2. In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I-II loops expressed as glutathione-S-transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I-II loop. 3. SNS and SNS(SA) channels were transiently expressed in COS-7 cells and their electrophysiological properties compared. In wild-type SNS channels, forskolin and 8-bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I-II loop PKA consensus sites caused a marked reduction in PKA modulation of wild-type channels. 4. Under control conditions, the voltage dependence of activation of SNS(SA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(SA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild-type channels under basal conditions.5. We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I-II loop.

An ether-linked tetrafunctional acylating reagent and its cross-linking reactions with hemoglobin

A new type of tetrafunctional reagent for cross-linking proteins has been prepared and used to modify human hemoglobin A. DPEE (1,2-bis{2-[3,5-bis(3,5-dibromosalicyloxycarbonyl) phenoxy]ethoxy}ethane)) has two separate pairs of reacting sites connected by a flexible tetraether chain. DPEE is capable of connecting a cross-link within a protein to another cross-link, either within the same protein molecule or between molecules. DPEE was readily prepared by esterification of a tetraether-linked bisphthalate (prepared by coupling of 1,2-bis(2-iodoethoxy)ethane and 5-hydroxyisophthalic acid). DPEE reacts with deoxy hemoglobin to produce a mixture of modified proteins. Ion-exchange HPLC was used to separate the modified proteins in the mixture. The most abundant products were selected for structural analysis, which used data from reverse-phase chromatography and tryptic peptide mapping. To prevent dissociation of the modified proteins during analysis, the products were further reacted with the bifunctional reagent, bis(3,5-dibromosalicyl) fumarate, which produces fumaryl cross-links between α-subunits. From peptide analysis of the separated products, the major modified protein from DPEE was identified as a novel species with four links within the same α2β2 tetramer. In addition, a minor product that involves cross-links in two different proteins was observed. These results imply that the reagent reacts primarily in a folded state within the protein.Key words: acylation: cross-linking, multifunctional, hemoglobin, reaction pattern.

An ether-linked tetrafunctional acylating reagent and its cross-linking reactions with hemoglobin

A new type of tetrafunctional reagent for cross-linking proteins has been prepared and used to modify human hemoglobin A. DPEE (1,2-bis{2-[3,5-bis(3,5-dibromosalicyloxycarbonyl) phenoxy]ethoxy}ethane)) has two separate pairs of reacting sites connected by a flexible tetraether chain. DPEE is capable of connecting a cross-link within a protein to another cross-link, either within the same protein molecule or between molecules. DPEE was readily prepared by esterification of a tetraether-linked bisphthalate (prepared by coupling of 1,2-bis(2-iodoethoxy)ethane and 5-hydroxyisophthalic acid). DPEE reacts with deoxy hemoglobin to produce a mixture of modified proteins. Ion-exchange HPLC was used to separate the modified proteins in the mixture. The most abundant products were selected for structural analysis, which used data from reverse-phase chromatography and tryptic peptide mapping. To prevent dissociation of the modified proteins during analysis, the products were further reacted with the bifunctional reagent, bis(3,5-dibromosalicyl) fumarate, which produces fumaryl cross-links between α-subunits. From peptide analysis of the separated products, the major modified protein from DPEE was identified as a novel species with four links within the same α2β2 tetramer. In addition, a minor product that involves cross-links in two different proteins was observed. These results imply that the reagent reacts primarily in a folded state within the protein.Key words: acylation: cross-linking, multifunctional, hemoglobin, reaction pattern.

Characterization and hormonal modulation of immunoreactive thiamin carrier protein in immature rat Sertoli cells in culture

Immature rat Sertoli cells synthesize and secrete a protein species which has immunological similarity with chicken egg thiamin carrier protein (TCP) as assessed by immunocytochemical localization, liquid phase radioimmunoassay (RIA), immunoprecipitation of [ 35S]-methionine incorporated newly synthesized proteins by polyclonal antibodies (pAbs) to chicken TCP and tryptic peptide mapping of iodinated immunoprecipitated proteins. FSH and testosterone together bring about 4-fold induction of Sertoli cell TCP over the control levels which is inhibitable upto 75% by an aromatase inhibitor. Addition of optimal concentrations of exogenous estradiol-17β to the cultures causes 2-fold enhancement of secretion of TCP which can significantly be inhibited by tamoxifen, when added along with estradiol-17β. These results show that Sertoli cells produce estrogen-inducible TCP, presumably to transport the vitamin to the developing germ cells.

Regulatory light chain phosphorylation and the assembly of myosin II into the cytoskeleton of microcapillary endothelial cells.

During the crawling movements of non-muscle cells, myosin II-containing structures assemble and disassemble with a high degree of spatial and temporal heterogeneity. In order to understand how this is controlled, we examined factors that influence the association of myosin II with detergent-resistant cytoskeletons of cultured endothelial cells. Treatment of cells with 0.05% Triton X-100 in an actin-stabilizing buffer released approximately 42% of the myosin II from the cytoplasm. Most remaining myosin II was dissociated from the cytoskeleton by treatment with ATP or AMPPNP, but not ADP, suggesting that myosin II is retained as ATP-sensitive filaments or via rigor-like binding to F-actin. Disruption of actin filaments with cytochalasin or latrunculin prior to detergent permeabilization sharply decreased the amount of myosin II retained, suggesting the latter type of association. Because phosphorylation of myosin II affects filament assembly and actin binding in vitro, phosphorylation levels in soluble and cytoskeletal myosin II were measured. Phosphorylation of myosin heavy chains was not significantly different between the two fractions, but regulatory light chains of cytoskeletal myosin II were 5 times more phosphorylated than in soluble myosin II. Tryptic-peptide mapping showed that cytoskeletal light chains were phosphorylated predominantly at serine 19/threonine 18, which regulates myosin II assembly in vitro, whereas soluble light chains were not phosphorylated or were phosphorylated at threonine 9. Treating cells with the kinase inhibitor, staurosporine, prior to permeabilization decreased light-chain phosphorylation with concomitant reduction in myosin retention. These observations suggest that assembly of myosin II into cytoskeletal structures, where it can generate and resist forces, is regulated in vivo by phosphorylation of myosin light chains at serine 19/threonine 18.

Re-examination of the Glycosylation of High Mr Subunits of Wheat Glutenin

SDS−PAGE-separated high Mr wheat glutenin subunits stained positively in the DIG-glycan procedure, suggesting that they were glycosylated, but control experiments indicated that these were probably false positives. Lack of reaction with concanavalin A indicated the absence of terminal mannose, glucose, or N-acetylglucosamine. GC/MS analysis indicated that A-PAGE-prepared subunits contained glucose, mannose, xylose, and galactose. Because gel regions containing no protein contained similar amounts of those sugars, they were most likely unbound contaminants. RP-HPLC-purified subunits from cv. Apollo wheat contained variable amounts of glucose only, probably representing starch contamination. RP-HPLC-purified subunits 1Ax1 and 1Bx7 contained no GC/MS-detectable sugars even though the procedure was sensitive enough to detect 1 sugar residue per 40 subunit molecules. Mapping of tryptic or chymotryptic peptides, before and after deglycosylation, and GC/MS analysis of isolated tryptic peptides provided no evidence for glycosylation of high Mr subunits. It appears unlikely that high Mr wheat glutenin subunits are glycosylated. Keywords: Wheat; glutenin; glycosylation; subunits

Characterization of a Non-electrophoretic Genetic Variant of β-Casein by Peptide Mapping and Mass Spectrometric Analysis

Abstract From a total of 634 Holstein milk samples, 158 were identified as homozygous β -casein A 1 according to PAGE. All the β -casein A 1 samples were isolated in pure form by anion exchange chromatography followed by tryptic hydrolysis and peptide mapping of the hydrolysates by reversed-phase HPLC. A comparison of the chromatogram revealed that the β -casein A 1 samples could be classified into two groups based on differences in elution times of fragment 114–169 due to a change in hydrophobicity caused by amino acid substitution. Mass spectrometric analysis showed that 7 β -casein samples with more hydrophobic 114–169 fragment had a higher molecular mass (difference of 16 Da) than the remaining 151 samples with less hydrophobic fragment. Amino acid composition and sequence analysis confirmed the difference in mass of peptide 114–169 by a Pro→Leu substitution at position 137. It is proposed to identify the newly found variant as β -casein G.

P40phox Is Phosphorylated on Threonine 154 and Serine 315 during Activation of the Phagocyte NADPH Oxidase: IMPLICATION OF A PROTEIN KINASE C-TYPE KINASE IN THE PHOSPHORYLATION PROCESS

The superoxide-generating NADPH oxidase complex of phagocytic cells is a multicomponent system containing a membrane-bound flavocytochrome b and a small G protein Rac as well as cytosolic factors p67(phox) (phagocyte oxidase), p47(phox), and p40(phox), which translocate to the membrane upon activation. In a previous paper, we reported that p40(phox) undergoes phosphorylation on multiple sites upon stimulation of the NADPH oxidase by either phorbol 12-myristate 13-acetate or by formyl peptide with a time course that is strongly correlated with that of superoxide production (Fuchs, A., Bouin, A. P., Rabilloud, T., and Vignais, P. V. (1997) Eur. J. Biochem. 249, 531-539). In this study, through phosphoamino acid and tryptic peptide maps of in vivo and in vitro phosphorylated p40(phox), we show that p40(phox) is phosphorylated on serine and threonine residues during activation of the NADPH oxidase in dimethyl sulfoxide-differentiated HL60 promyelocytes as well as in isolated human neutrophils. In vitro phosphorylation studies using casein kinase II and protein kinase C (PKC) as well as the effect of various protein kinase inhibitors on the isoelectric focusing pattern of p40(phox) in whole cell lysates point to a role of a PKC type kinase in the phosphorylation of p40(phox). Directed mutagenesis of all PKC consensus sites enable us to conclude that Thr154 and Ser315 in p40(phox) are phosphorylated during activation of the NADPH oxidase.

Covalent modification of cyclooxygenase-2 (COX-2) by 2-acetoxyphenyl alkyl sulfides, a new class of selective COX-2 inactivators.

All of the selective COX-2 inhibitors described to date inhibit the isoform by binding tightly but noncovalently at the substrate binding site. Recently, we reported the first account of selective covalent modification of COX-2 by a novel inactivator, 2-acetoxyphenyl hept-2-ynyl sulfide (70) (Science 1998, 280, 1268-1270). Compound 70 selectively inactivates COX-2 by acetylating the same serine residue that aspirin acetylates. This paper describes the extensive structure-activity relationship (SAR) studies on the initial lead compound 2-acetoxyphenyl methyl sulfide (36) that led to the discovery of 70. Extension of the S-alkyl chain in 36 with higher alkyl homologues led to significant increases in inhibitory potency. The heptyl chain in 2-acetoxyphenyl heptyl sulfide (46) was optimum for COX-2 inhibitory potency, and introduction of a triple bond in the heptyl chain (compound 70) led to further increments in potency and selectivity. The alkynyl analogues were more potent and selective COX-2 inhibitors than the corresponding alkyl homologues. Sulfides were more potent and selective COX-2 inhibitors than the corresponding sulfoxides or sulfones or other heteroatom-containing compounds. In addition to inhibiting purified COX-2, 36, 46, and 70 also inhibited COX-2 activity in murine macrophages. Analogue 36 which displayed moderate potency and selectivity against purified human COX-2 was a potent inhibitor of COX-2 activity in the mouse macrophages. Tryptic digestion and peptide mapping of COX-2 reacted with [1-14C-acetyl]-36 indicated that selective COX-2 inhibition by 36 also resulted in the acetylation of Ser516. That COX-2 inhibition by aspirin resulted from the acetylation of Ser516 was confirmed by tryptic digestion and peptide mapping of COX-2 labeled with [1-14C-acetyl]salicyclic acid. The efficacy of the sulfides in inhibiting COX-2 activity in inflammatory cells, our recent results on the selectivity of 70 in attenuating growth of COX-2-expressing colon cancer cells, and its selectivity for inhibition of COX-2 over COX-1 in vivo indicate that this novel class of covalent modifiers may serve as potential therapeutic agents in inflammatory and proliferative disorders.

Identification of Protein-ArginineN-Methyltransferase as 10-Formyltetrahydrofolate Dehydrogenase

S-Adenosylmethionine:protein-arginine N-methyltransferase (EC 2.1.1. 23; protein methylase I) transfers the methyl group of S-adenosyl-L-methionine to an arginine residue of a protein substrate. The homogeneous liver protein methylase I was subjected to tryptic digestion followed by reverse phase high performance liquid chromatography (HPLC) separation and either "on-line" mass spectrometric fragmentation or "off-line" Edman sequencing of selected fractions. Data base searching of both the mass spectrometric and Edman sequencing data from several peptides identified the protein methylase as 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6; Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Chem. 266, 4965-4973; Swiss accession number). This identification was confirmed by comparative HPLC tryptic peptide mapping and affinity chromatography of the methylase on the 5-formyltetrahydrofolate-Sepharose affinity gel used to purify the dehydrogenase. The purified rat liver methylase had approximately 33% of the 10-formyltetrahydrofolate dehydrogenase and 36% of the aldehyde dehydrogenase activity as compared with the recombinant dehydrogenase, which also had protein methylase I activity. Polyclonal antibodies against recombinant dehydrogenase reacted with protein methylase I purified either by polyacrylamide gel electrophoresis or 5-formyltetrahydrofolate affinity chromatography. In each instance there was only a single immunoreactive band at a molecular weight of approximately 106,000. Together, these results confirm the co-identity of protein-arginine methyltransferase and 10-formyltetrahydrofolate dehydrogenase.

Regulation of the p21-activated Kinase-relatedDictyostelium Myosin I Heavy Chain Kinase by Autophosphorylation, Acidic Phospholipids, and Ca2+-Calmodulin

The Dictyostelium myosin I heavy chain kinase (MIHCK) is a member of the p21-activated kinase family (Lee, S.-F., Egelhoff, T. T., Mahasneh, A., and Côté, G. P. (1996) J. Biol. Chem. 271, 27044-27048). MIHCK incubated with MgATP in the absence of effectors incorporates 1 mol of phosphate/mol, resulting in an approximately 40-fold increase in kinase activity. Sequence analysis of tryptic peptides has identified the major site of phosphorylation as Ser-8. A peptide and a glutathione S-transferase fusion protein containing the Ser-8 phosphorylation site were good substrates for MIHCK, indicating that MIHCK can catalyze its own activation. Guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)-Rac1 stimulates MIHCK autophosphorylation and kinase activity 10-fold. Phosphatidylserine, phosphatidylinositol, and phosphatidylinositol 4,5-bisphosphate, but not phosphatidylcholine or sphingosine, were as effective as GTPgammaS-Rac1 in enhancing MIHCK autophosphorylation and activity. Acidic lipids and GTPgammaS-Rac1 induced the autophosphorylation of a similar set of sites as judged by two-dimensional tryptic peptide maps. It is proposed that GTP-Rac and acidic phospholipids function cooperatively to associate MIHCK with membranes. Ca2+-calmodulin bound MIHCK and inhibited activation by acidic phospholipids but not by GTPgammaS-Rac1. These studies reveal a number of similarities between the regulatory properties of the Dictyostelium and Acanthamoeba MIHCK, suggesting that the signaling pathways that control myosin I are conserved.

Identification of a 374 amino acid protein encoded by RNA segment 6 of influenza C virus.

Unspliced mRNA from RNA segment 6 of influenza C virus contains a single open reading frame that potentially encodes a polypeptide of 374 amino acids. This polypeptide, which has not been identified as yet, is predicted to contain the complete amino acid sequence of the matrix protein, M1, as well as that of a small integral membrane protein, CM2. Here, we found that small amounts of two previously unrecognized proteins with apparent molecular masses of 42 (P42) and 44 kDa (P44) were immunoprecipitated with immune serum against CM2. The electrophoretic mobilities of P42 and P44 varied depending on virus strain, indicating that they are virus-coded. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with various endoglycosidases revealed that P42 is modified by the addition of a high-mannose oligosaccharide chain to generate P44. A monoclonal antibody against M1, like anti-CM2 serum, was able to immunoprecipitate both the P42 and P44 proteins. Furthermore, the tryptic peptide map of either P42 or P44 was indistinguishable from the map of the mixture of M1 and CM2. These results, taken together, suggest strongly that P42 and P44 correspond to the 374 amino acid protein encoded by unspliced RNA segment 6 mRNA and its N-glycosylated form, respectively.

Correct assembly of human normal adult hemoglobin when expressed in transgenic swine: chemical, conformational and functional equivalence with the human-derived protein.

Structural and functional investigations of recombinant human hemoglobin A (HbA) isolated from the erythrocytes of transgenic swine coexpressing human alpha- and beta-globins have been carried out to authenticate its correct expression, post-translational processing and assembly. The HbA expressed in transgenic swine (TgHbA) is indistinguishable from the human-derived HbA in terms of its isoelectric pH, mass and elution pattern on a Mono S column. The chemical identity of the alpha- and beta-globin chains of TgHbA with the corresponding chains from human-derived HbA has been established by tryptic peptide mapping and amino acid sequencing. The proton NMR spectra of TgHbA have demonstrated that the conformational aspects of the protein around the heme pocket are indistinguishable from those of the control sample of HbA. The equivalence of the hydrogen bond pattern of TgHbA (in particular the inter-subunit surfaces) with that of authentic HbA has also been established by NMR studies. Consistent with these structural and conformational analyses, the TgHbA also exhibits complete functional equivalence with the human-derived HbA with respect to oxygen affinity, cooperativity, Bohr effect and allostery. Hence the studies presented here demonstrate that the transgenic swine system correctly transcribes the alpha- and beta-globin transgenes, translates the respective alpha- and beta-globin mRNA to generate the corresponding globin chains, carries out the correct cotranslational processing of the translated globin chains, inserts the heme into the globin chains in the same orientation as in the human-derived HbA and assembles the alpha- and beta-subunits into a functionally cooperative tetramer that exhibits a response to allosteric effectors identical with that of human-derived HbA. Thus, in the transgenic swine system, in vitro chemical manipulation steps such as those needed in the Escherichia coli and the yeast systems, to convert the rHbA expressed in these systems into forms functionally identical with that of the human-derived protein, are not needed. An additional advantage of the transgenic swine system is the stability of the transgenes over many generations. Hence the transgenic swine could serve as an excellent system for the production of human HbA (or its variants) for structure-function studies and for therapeutic applications.