kD Polypeptide Research Articles

A Comparison of Murine and Human Factor XI

Factor XI is a plasma glycoprotein that is required for contact activation initiated fibrin formation in vitro and for normal hemostasis in vivo. In preparation for developing a mouse model of factor XI deficiency to facilitate investigations into this protease's contributions to coagulation, we cloned the complementary DNA for murine factor XI, expressed the protein in a mammalian expression system, and compared its properties with human recombinant factor XI. The 2.8-kb murine cDNA codes for a protein of 624 amino acids with 78% homology to human factor XI. Both recombinant murine and human factor XI are 160 kD homodimers comprised of two 80 kD polypeptides connected by disulfide bonds. Murine factor XI shortens the clotting time of human factor XI deficient plasma in an activated partial thromboplastin time assay, with a specific activity 50% to 70% that of the human protein. In a purified system, murine factor XI is activated by human factor XIIa and thrombin in the presence of dextran sulfate. Murine factor XI differs from human factor XI in that it undergoes autoactivation slowly in the presence of dextran sulfate. This is due primarily to murine factor XIa preferentially cleaving a site on zymogen factor XI within the light chain, rather than the activation site between Arg371 and Val372. Northern blots of polyadenylated messenger RNA show that murine factor XI message is expressed, as expected, primarily in the liver. In contrast, messenger RNA for human factor XI was identified in liver, pancreas, and kidney. The studies show that murine and human factor XI have similar structural and enzymatic properties. However, there may be variations in tissue specific expression and subtle differences in enzyme activity across species.

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (II. 2,6-Dichlorobenzonitrile Inhibition of Motility and Stalk Production in the Marine Diatom Achnanthes longipes).

The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059-1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158-162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.

MAPPING THE ADHESIVE DOMAINS OF THE C. ALBICANS GENE INT1. • 687

Virulence in Candida albicans correlates with adhesion to human epithelium. The C. albicans gene INT1 encodes a putative adhesin (Int1p) homologous to the mammalian integrins αM/αX. The nonpathogenic, nonadhesive, nonfilamentous yeast S. cerevisiae does not contain INT1. When INT1 is expressed in S. cerevisiae, germ tube formation and adhesion to human epithelium parallels that of C. albicans. Because there are multiple mediators of candidal adhesion to epithelium, we sought to map the adhesive domains of Int1p in nonadherent yeast S. cerevisiae. Sequence analysis of INT1 predicts a 188 kD polypeptide including several motifs common to αM/αX [an I domain, two divalent cation binding sites (CBS1& CBS2), a transmembrane domain (TM) and a cytoplasmic tail with a single tyrosine residue (CYTO)], as well as an internal RGD site not found in integrins. INT1 constructs involving deletions/substitutions of these sites were subcloned into a yeast shuttle vector (pBM272) and expressed in S. cerevisiae YPH500 under the control of a galactose-inducible promoter. All transformed yeast strains showed normal growth curves in standard and selective media. All mutants, save the RGD deletion, formed INT1-dependent germ tubes when grown in galactose. Western blot analysis using Int1p specific antibodies revealed a protein of 200 ± 20 kD in all transformants but the RGD mutant. Epithelial adhesion of S. cerevisiae expressing INT1 and each mutant gene was determined. Adhesion of the yeast expressing the CBS2 construct was reduced but not significantly different from that expressing native INT1(24.9±4.4 vs 32.6±1.4). Specific adhesion was significantly reduced for S. cerevisiae expressing the TMCYTO (14.9 ± 2.4) and RGD (12.5 ± 2.5) constructs (p<0.001). Adhesion of the S. cerevisiae transformed with vector alone was 4.9 ± 1.2. These results provide 1) confirmation of INT1-mediated adhesion in S. cerevisiae; 2) further evidence that INT1-mediated adhesion is not calcium dependent; 3) a novel role for the TMCYTO domain in epithelial adhesion.

Isolation and identification of hevein as a major IgE-binding polypeptide in Hevea latex

Background: Polypeptides in Hevea latex are known as the major cause of latex type I sensitivities. So far, only a few of them have been characterized. Methods: Proteins with a molecular weight lower than 10 kd in fresh Hevea latex were separated by ultrafiltration and further characterized by liquid chromatography on-line–coupled electrospray mass spectrometry. Hevein in this fraction was then purified by preparative reverse-phase high-performance liquid chromatography and characterized by matrix-assisted laser desorption ionization mass spectrometry and protein sequencing. Skin prick tests, enzyme-linked allergosorbent tests, and inhibition immunoblotting were performed to show the allergenicity of the purified hevein. Results: Hevein, a 4.7 kd polypeptide, is the predominant component in the fraction with latex proteins of smaller than 10 kd. Specific IgE antibodies to hevein were detected by enzyme-linked allergosorbent test in 48 of 64 (75%) sera from health care workers allergic to latex and in three of 11 (27%) sera from patients with spina bifida and hypersensitivity reactions to latex. Inhibition immunoblotting demonstrated that the preincubation of 14 sera and a serum pool from patients allergic to latex with purified hevein completely inhibited IgE binding to the 20 kd protein, which has been recently reported to be a major allergen in latex (prohevein). Skin prick testing showed a positive reaction to hevein in 17 of 21 (81%) patients with latex allergy. Conclusions: The results clearly demonstrate that hevein is an important latex allergen, and the IgE-binding capacity of prohevein in latex is mostly attributed to hevein, the N-terminal domain of prohevein. ( J Allergy Clin Immunol 1997;99:402–9.)

Serum levels of the tumour-associated trypsin inhibitor in patients with endometriosis.

To show that in patients with endometriosis a 6 kD polypeptide, the tumour-associated trypsin inhibitor (TATI), can occur at elevated concentrations in serum. In a prospective study TATI serum levels were assessed prior to surgery in 368 consecutive patients suffering from benign gynaecological diseases (e.g. pelvic pain, infertility, elective sterilisation, uterine fibroids and pelvic masses) with (n = 71) and without (n = 297) endometriosis, who underwent laparoscopy or laparotomy for diagnosis and/or treatment. The TATI serum levels of patients suffering from endometriosis were significantly different from those in the control group and showed a positive correlation with the stage of endometriosis. The sensitivity of TATI was 0.34 with a specificity of 0.85 for all cases of endometriosis, with an increase of sensitivity (0.67) and slight decrease of specificity (0.82), considering only a group of Stage III/IV patients. Excluding patients with benign ovarian cysts, the specificity of TATI was 0.91 and 0.85, respectively. The combination of TATI and CA125 showed an increase of sensitivity to 0.59 for all cases of endometriosis and 0.89 for patients with Stage III/IV endometriosis. The sensitivity of TATI as a screening method for endometriosis is too low, but considering its high specificity, TATI in combination with CA125 could provide an additional diagnostic tool in diagnosis and follow up of patients with endometriosis.

The role of central apparatus components in flagellar motility and microtubule assembly

In order to generate the complex waveforms typical of beating cilia and flagella, the action of the dynein arms must be regulated. This regulation not only depends on the presence of multiple dynein isoforms, but also clearly involves other structures in the axoneme such as the radial spokes and central apparatus; mutants lacking these structures have paralyzed flagella. In this article, we review recent progress in identifying protein components of the central apparatus and discuss the role of these components in regulation of flagellar motility and central apparatus assembly. The central apparatus is composed of two single microtubules and their associated structures which include the central pair projections, the central pair bridges linking the two tubules, and the central pair caps which are attached to the distal or plus ends of the microtubules. To date, the genes encoding four components of the central apparatus have been cloned, PF15, PF16, PF20 and KLP1. PF16, PF20 and KLP1 have been sequenced and their gene products localized. Two additional components have been identified immunologically, a 110 kD polypeptide recognized by an antibody generated against highly conserved kinesin peptide sequence, and a 97 kD polypeptide recognized by CREST antisera. Based on a variety of data, one model that has emerged to explain the role of the central apparatus in flagellar motility is that the central apparatus ultimately regulates dynein through interactions with the radial spokes. The challenge now is to determine the precise mechanism by which the polypeptides comprising the central apparatus and the radial spokes interact to transduce a regulatory signal to the dynein arms. In terms of assembly, the central apparatus microtubules assemble with their plus ends distal to the cell body but, unlike the nine doublet microtubules, they are not nucleated from the basal bodies. Since some central apparatus defective mutants fail to assemble the entire central apparatus, their gene products may eventually prove to have microtubule nucleating or stabilizing properties. By continuing to identify the genes that encode central apparatus components, we will begin to understand the contribution of these microtubules to flagellar motility and gain insight into their nucleation, assembly, and stability. Cell Motil. Cytoskeleton 38:1–8, 1997. © 1997 Wiley-Liss, Inc.

The role of central apparatus components in flagellar motility and microtubule assembly.

In order to generate the complex waveforms typical of beating cilia and flagella, the action of the dynein arms must be regulated. This regulation not only depends on the presence of multiple dynein isoforms, but also clearly involves other structures in the axoneme such as the radial spokes and central apparatus; mutants lacking these structures have paralyzed flagella. In this article, we review recent progress in identifying protein components of the central apparatus and discuss the role of these components in regulation of flagellar motility and central apparatus assembly. The central apparatus is composed of two single microtubules and their associated structures which include the central pair projections, the central pair bridges linking the two tubules, and the central pair caps which are attached to the distal or plus ends of the microtubules. To date, the genes encoding four components of the central apparatus have been cloned, PF15, PF16, PF20 and KLP1. PF16, PF20 and KLP1 have been sequenced and their gene products localized. Two additional components have been identified immunologically, a 110 kD polypeptide recognized by an antibody generated against highly conserved kinesin peptide sequence, and a 97 kD polypeptide recognized by CREST antisera. Based on a variety of data, one model that has emerged to explain the role of the central apparatus in flagellar motility is that the central apparatus ultimately regulates dynein through interactions with the radial spokes. The challenge now is to determine the precise mechanism by which the polypeptides comprising the central apparatus and the radial spokes interact to transduce a regulatory signal to the dynein arms. In terms of assembly, the central apparatus microtubules assemble with their plus ends distal to the cell body but, unlike the nine doublet microtubules, they are not nucleated from the basal bodies. Since some central apparatus defective mutants fail to assemble the entire central apparatus, their gene products may eventually prove to have microtubule nucleating or stabilizing properties. By continuing to identify the genes that encode central apparatus components, we will begin to understand the contribution of these microtubules to flagellar motility and gain insight into their nucleation, assembly, and stability.

The relationship between the accumulation of a 28 kd polypeptide and that of indole alkaloids in Catharanthus roseus cell suspension cultures

We previously found that the accumulation of the indole alkaloid ajmalicine and that of a polypeptide of 28kD were induced by 2,4-dichlorophenoxyacetic acid removal in periwinkle (Catharanthus roseus) cell cultures and further increased by zeatin addition to the culture medium. In this work, we compared the effect of various plant growth regulators and stresses on the accumulation patterns of both compounds. In a first series of experiments, the periwinkle cells were subcultured in auxin-free medium for five passages and their contents in ajmalicine and 28 kD polypeptide analysed. The maximal biomass progressively decreased along subcultures; the 28 kD polypeptide and ajmalicine accumulated concomitantly in the cells (treated with or without zeatin) up to the third passage but fully disappeared afterwards. In a second series of experiments, treatment with gibberellic acid deeply inhibited, whereas NaCl treatment (given together with or without zeatin) increased the accumulation of both ajmalicine and the 28 kD polypeptide. Moreover, growing the cells in hypoxia for 2 days inhibited the accumulation of both compounds. These concomitant changes in the accumulation pattern of ajmalicine and the 28 kD polypeptide suggest that the latter can play a (direct or indirect) regulatory role in the indole alkaloid pathway of periwinkle cells.

Immunochemical studies of the cellulose synthase complex inAcetobacter xylinum

An immunochemical method was used to analyse the 83 and 93 Kd polypeptides of cellulose synthase from Acetobacter xylinum.Polyclonal antibodies were raised against the LDS-PAGE-fractionated 83 and 93 Kd polypeptides isolated from A. xylinum.Using these antibodies, the 83 and 93 Kd polypeptides were localized in the different fractions during purification of cellulose synthase, and the ratio of these two polypeptides was determined to be 1∶1. A differential solubilization of the 83 and 93 Kd polypeptides from the cell strongly suggested that the mechanism by which these two polypeptides originate from a single acsAB gene product (Saxena et al.,1994) must be via a post-translational cleavage. The results of trypsin treatment of the membrane fraction used in the purification of cellulose synthase were analysed to determine the fate of these two polypeptides and their relationship to the enzyme activity.

Autoimmunity to RNA polymerase II is focused at the carboxyl terminal domain of the large subunit.

Previous studies have demonstrated antibodies to the large (220 kd) polypeptide subunit of RNA polymerase II (Pol II) in sera from certain patients with scleroderma. In the present study, we sought to identify the autoantigenic region on this polypeptide. A recombinant fusion protein, corresponding to the 52-heptapeptide repeat found in the carboxyl terminal domain (CTD) of the large Pol II subunit, was used to identify 15 patient sera that contained autoantibodies. Synthetic peptides CTD7 (representing a single heptapeptide) and CTD18 (representing 2 1/2 heptapeptide repeats) were used in a competitive inhibition assay to define the specificity of these sera and the importance of the CTD as an autoantigen. All 15 sera immunoprecipitated the Pol II subunit from radiolabeled cell extracts, and 11 of them bound the CTD fusion protein in immunoblots. Immunoprecipitation of Pol II was completely inhibited by CTD18 in 5 sera and partially inhibited in 4 additional sera. These results indicate that the CTD heptapeptide repeat is a focal point for autoimmune responses in scleroderma. It is likely that the repetitive sequence and high content of charged residues of this structure contribute to its role as an autoantigen.

CHANGES IN PROTEIN COMPOSITION DURING STRAWBERRY (Fragaria × ananassa Duch.) FRUIT RIPENING1

The changes in strawberry native proteins and polypeptides during ripening were studied. Analysis of protein extracts by nondenaturing electrophoresis showed the presence of a protein species only in the ‘25% Red’ and later ripening stage. This 40 kD polypeptide was hardly detected in ‘Large green’ and ‘White’ fruit extracts. The polypeptide profile, obtained with SDS-PAGE, also changed during ripening. While most polypeptides were found in all ripening stages, the distribution of some of them varied during ripening. Protein synthesis (measured by35S-methionine labelling) in strawberry was mainly directed towards the regeneration of previously existing proteins. As ripening proceeded, the synthesis of 67 and 63 kD polypeptides increased, while those of 82, 56 and 25 kD decreased. Three very-abundant polypeptides (36, 24 and 23 kD) were located in the achenes and were not labelled in any ripening stage.

Sam68 from an immortalised B-cell line associates with a subset of SH3 domains

The binding of proteins from an immortalised B-cell line to a panel of SH3 domains was investigated in vitro. One of the most prominent SH3 domain binding proteins was a 68 kD polypeptide which strongly associated with the SH3 domains of c-src, p85α and p47 phox and weakly with the SH3 domain of PLCγ and n-src with undetectable binding to the other SH3 domains tested. Immunoblotting identified this protein as human Sam68. The ability of proline-rich peptides homologous to the Sam68 primary sequence to inhibit the binding of Sam68 to SH3 domains was investigated. Only one peptide inhibited binding of Sam68 to the p85a SH3 domain, whereas several peptides inhibited binding of Sam68 to c-src SH3 domain, suggesting that Sam68 uses different proline-rich motifs to bind to different SH3 domains. A peptide derived from residues 32–44 of Sam68 which fits the class H SH3 domain binding consensus sequence inhibited binding of Sam68 to both p85a SH3 domain and c-src SH3 domain, but with differential potency, suggesting a differential affinity of these SH3 domains for this proline-rich motif.

The Search for Chilling-Responsive Proteins in Blueberry Continues

ABSTRACT Previously, we identified three proteins in blueberry floral buds of 65, 60, and 14 kD that increase dramatically in abundance during chill-unit accumulation and cold acclimation and decrease during deacclimation and resumption of growth. These proteins are related immunologically to a group of proteins called dehydnns. Here we report results from immunoblot experiments using antise-rum raised against molecular chaperones, CAP 79 from spinach and a cyclophilin from Arabidopsis, in addition to dehydrin antiserum. Using these antisera, two new chilling-responsive polypeptides have been identified in blueberry floral buds. One is a 22 IcD polypeptide immunologically related to dehydnns and the other is a 17.5 kD polypeptide immunologically related to cyclophilins. The 17.5 kD polypeptide was purified and partially sequenced. Sequence information confirmed the 17.5 kD polypeptide is, indeed, cyclophilin and is very similar in sequence to cyclophilins identified in other plants.

Phosphatase activities in spinach thylakoid membranes-effectors, regulation and location

The dephosphorylation of seven phosphoproteins associated with Photosystem II or its chlorophyll a/b antenna in spinach thylakoids, was characterised. The rates were found to fall into two distinct groups. One, rapidly dephosphorylated, consisted of the two subunits (25 and 27 kD) of the major light harvesting complex of Photosystem II (LHC II) and a 12 kD polypeptide of unknown identity. A marked correlation between the dephosphorylation of these three phosphoproteins, strongly suggested that they were all dephosphorylated by the same enzyme. Within this group, the 25 kD subunit was consistently dephosphorylated most rapidly, probably reflecting its exclusive location in the peripheral pool of LHC II. The other group, only slowly dephosphorylated, included several PS II proteins such as the D1 and D2 reaction centre proteins, the chlorophyll-a binding protein CP43 and the 9 kD PS II-H phosphoprotein. No dephosphorylation was observed in either of the two groups in the absence of Mg(2+)-ions. Dephosphorylation of the two LHC II subunits took place in both grana and stroma-exposed regions of the thylakoid membrane. However, deposphorylation in the latter region was significantly more rapid, indicating a preferential dephosphorylation of the peripheral (or 'mobile') LHC II. Dephosphorylation of LHC II was found to be markedly affected by the redox state of thiol-groups, which may suggest a possible regulation of LHC II dephosphorylation involving the ferredoxin-thioredoxin system.

Beta cap73: a novel beta actin-specific binding protein.

Whereas actin-binding proteins (ABPs) regulate network formation during the cell cycle, it is not known whether ABPs also function to sequester or target isoactins to specific subcellular compartments. Recently, we have shown that ezrin indirectly associates with beta, but not alpha actin filaments in a calcium- and cytochalasin-sensitive manner [Shuster and Herman, 1995: J. Cell Biol. 128:837-848]. To identify the beta actin-specific binding protein that fosters ezrin-beta actin interactions, we developed an isoactin affinity fractionation and F-isoactin overlay/Western blotting technique. Results reveal that a 73 kd polypeptide that co-precipitates with ezrin and beta actin [Shuster and Herman, 1995: J. Cell Biol. 128:837-848] can also binds directly to filaments of beta, but not alpha actin by isoactin overlay. In an effort to establish whether p73 plays a role in regulating beta actin dynamics in cells, we produced monoclonal antibodies by immunizing BALB/c mice with p73-containing lamellar lysates or high salt elutions from beta actin affinity columns. Two monoclonal antibodies were cloned that react with p73 present in fractions released from beta actin Sepharose-4B or purified to homogeneity by DEAE chromatography. Anti-p73 Western blots reveal that there is a 16-fold difference in p73 binding to beta actin vs. alpha actin affinity columns when experiments are performed in physiological salts. To characterize p73-beta actin binding in vitro and establish whether p73 binds along the lengths or at the barbed end of the beta actin filament, we asked whether cytochalasin D (CD) could displace p73 pre-bound to beta actin-Sepharose 4B. Anti-p73 Western blotting reveals that nanomolar concentrations of CD are capable of selectively eluting p73 and ezrin from beta actin Sepharose 4B, indicating that p73 binds beta actin via the barbed end. Simultaneous double antibody localization studies using anti-beta actin IgG and anti-p73 IgM reveal that p73 and beta actin are co-localized in the forward aspects of motile cytoplasmic domains, in close proximity to the plasma membrane. Because of its isoform-specific interactions with the barbed end of beta actin filaments, we have named this molecule beta cap73. These results indicate that isoform-specific actin-binding proteins can be identified from cortical cytoplasm, and suggest that beta cap73 may not only act to spatially regulate the intracellular distribution of isoactins, but may also facilitate forward protrusion formation through the regulated release of free filament ends during cell motility.

A NOVEL 43 KD PROTEIN BINDS A CONSERVED MAMMALIAN CACCC MOTIF WITHIN THE DROSOPHILA RAS2/ROP BIDIRECTIONAL PROMOTER

The Drosophila ras2 promoter is an authentic bidirectional promoter governing the expression of both the Dras2 and rop genes by a single mechanism. Characterisation of the Dras2/rop promoter has revealed that a unitary complex (M) interacts with two promoter sub-domains (regions A and B). Two distinct transcription factors (factors A and B),which make up the major complex (M), bind regions A and B, respectively. We have analyzed the putative CACCC element and AP-1-Iike sequence contained within region B (-41 to -20) of the Dras2/rop promoter. It was found that AP-1 is not involved in Dras2 expression as is the case for the human Ha-ras1 gene. The entire CACCC motif (-34 to -21) shares 83% homology with the conserved mammalian element. Detailed mutational analysis has however revealed that the CACCC core sequence (-27 to -23) is vital for Dras2/rop recognition by factor B. The cytosine residues at positions -27, -25, -24 and -23 were observed to play a critical role in factor B recognition. Factor B has been purified as a 43 kD polypeptide as measured by SDS-PAGE and the relative mass was confirmed by photo-chemical crosslinking. Our findings are the first report of the conservation of the mammalian CACCC motif in Drosophila.

Isolation and Partial Characterization of Buffalo Plasma Fibronectin (Fn)

Abstract Jagati, R., Paliwal, S., Siddiqui, M.Z., Kumar, S., Sharma, A. K. and Kumar, A. 1995. Isolation and partial characterization of buffalo plasma fibronectin (Fn). J. Appl. Anim. Res., 8: 97–104. Fibronectin (Fn) a large extracellular adhesive glycoprotein was isolated from buffalo plasma using gelatin-sepharose affinity chromatography and characterized by analytical SDS-PAGE, GPC-HPLC and amino acid analysis. Analytical GPC-HPLC revealed homogeneity of the isolate with a molecular weight of 460 kD and SDS-PAGE analysis of Fn in reduced form depicted presence of two 230 kD polypeptide chains linked by disulphide bonds. Microchemical analysis of the Fn hydrolysate using 6N HCl gave the amino acid composition similar to that of other species except a higher content of arginine (R) i.e. 8.2% as compared to 5.3% in other species. Staphylococcus aureus cells showed preferential binding to Fn coated surface.

Molecular and Physiological Analysis of a Thylakoid K+ Channel Protein.

Transport studies identified a K+ channel protein in preparations of purified spinach (Spinacea oleracea) thylakoid membrane. This protein was solubilized from native membranes and reconstituted into artificial proteoliposomes with maintenance of functional integrity. A 33-kD thylakoid polypeptide was identified as a putative component of this thylakoid protein. This identification was made using an antibody raised against a synthetic peptide representing a highly conserved region of K+ channel proteins. K+ channel activity co-migrated with the immunoreactive 33-kD polypeptide when solubilized thylakoid membrane protein was fractionated on a Suc density gradient. The antibody was used to immunoprecipitate the 33-kD polypeptide. Physiological function of this thylakoid membrane protein was elucidated by measuring photosynthetic electron transport of thylakoid preparations in the presence and absence of a K+ channel blocker. Results indicated that K+ efflux from the thylakoid lumen through this channel protein is required for the optimization of photosynthetic capacity. The effect this protein has on photosynthetic capacity is likely due to the requirement for K+ efflux from the thylakoid lumen to charge-balance light-induced proton pumping across this membrane.

Regulation of Phosphorylation of Wheat Mitochondrial Proteins by Divalent Cations

Mitochondria isolated from 4-day-old dark-grown wheat seedlings were purified by self-generating Percoll gradient. Phosphorylation reaction was carried out in vitro with the addition of [ c-32P]ATP and polypeptides resolved by 50S-PAGE were subjected to autoradiography. Amongst endogenous polypeptides phosphorylated, four polypeptides of 120, 66, 43 and 21 kD were prominent. Addition of Mg2+ (5 mM) caused dephosphorylation of 120 and 66 kO polypeptides but, simultaneously, induced/enhanced the phosphorylation of some polypeptides, with the effect being more pronounced on a 67 kD species. The phosphorylation of 120 kD species and a few other polypeptides was also down-regulated and that of a 18 kD polypeptide was up-regulated by Ca2+. The present study provides evidence that phosphorylation status of mitochondrial proteins is regulated by Mg2+ and/or Ca2+-dependent phosphatase(s) and protein kinase(s).

Evidence Against Dephosphorylation of Insulin-Elicited Phosphotyrosine Proteins in Vivo by the Phosphatase PTP2C

In order to determine whether the tyrosine phosphatase PTP2C dephosphorylates insulin-elicited phosphotyrosine proteins in vivo, we have compared the patterns of protein tyrosine phosphorylation and its reversal in the kidney 293 cell line with those in 293 cell lines overexpressing PTP2C and a catalytically inactive point mutant of PTP2C. In all three cell types insulin caused the rapid tyrosine phosphorylation of a 160 kD protein, which was shown not to be the insulin receptor substrate 1 (IRS-1) and may be the recently described IRS-2 as well as that of a 100 kD polypeptide, which is probably a mixture of the β subunits of the insulin and insulinlike growth factor I receptors. There was no difference among the three cell lines in the extent of tyrosine phosphorylation or in the rate of its reversal upon insulin withdrawal. These results indicate that PTP2C does not function to dephosphorylate these proteins significantly in vivo.