The Resistance Protein Tm-1 Inhibits Formation of a Tomato Mosaic Virus Replication Protein-Host Membrane Protein Complex

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3′ untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis.     

Two distinct Gi-proteins mediate formyl peptide receptor signal transduction in human leukemia (HL-60) cells

In membranes of myeloid differentiated HL-60 cells, the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine stimulates phospholipase C via a pertussis toxin-sensitive G-protein but does not inhibit adenylyl cyclase. In these membranes, the chemotactic peptide markedly stimulates the cholera toxin-dependent [32P]ADP-ribosylation of two proteins with approximate molecular masses of 40 and 41 kDa, respectively. The radiolabeled proteins comigrate on sodium dodecyl sulfate-polyacrylamide gels with the two pertussis toxin substrates present in HL-60 membranes, alpha i2 and alpha i3. The effect of the chemotactic peptide is blocked by treatment of intact HL-60 cells with pertussis toxin. Peptide mapping studies using Staphylococcus aureus protease V8 reveal that the two radiolabeled proteins are structurally distinct. Thus, the agonist-activated formyl peptide receptor functionally interacts with two distinct pertussis toxin substrates, most likely with Gi2 and Gi3. As the third Gi protein, Gi1, appears to be absent from both HL-60 cells and from systems that clearly reveal hormonal inhibition of adenylyl cyclase, the results strongly suggest that primary structure alone does not suffice to determine which effector mechanism is regulated by a given Gi-protein.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

On the possible involvement of bovine serum albumin precursor in lipofection pathway

Protein factors involved in lipofection pathways remain elusive. Using avidin-biotin affinity chromatography and mass finger printing analysis technique, herein we report the identification of a 70 kDa size protein (bovine serum albumin precursor, BSAP) which binds strongly with lipoplexes and may play role in lipofection pathway. Using multiple cultured animal cells and three structurally different cationic transfection lipids, we show that the efficiencies of liposomal transfection vectors get significantly enhanced (by ~2.5- to 5.0-fold) in cells pre-transfected with lipoplexes of reporter plasmid construct encoding BSAP. Findings in the cellular uptake experiments in A549 cells cultured in DMEM supplemented with 10% (w/w) BODIPY-labelled BSAP are consistent with the supposition that BSAP enters cell cytoplasm from the cell culture medium (DMEM supplemented with 10% FBS) used in lipofection. Cellular uptake studies by confocal microscopy using BODIPY-labelled BSAP and FITC-labelled plasmid DNA revealed co-localization of plasmid DNA and BSAP within the cell cytoplasm and nucleus. In summary, the present findings hint at the possible involvement of BSAP in lipofection pathway.     

A heparin-binding protein involved in inhibition of smooth-muscle cell proliferation.

A heparin-binding protein was isolated from bovine uteri and purified to homogeneity. This protein appears as a double band of approx. 78 kDa in SDS/polyacrylamide-gel electrophoresis and has an isoelectric point of 5.2. The binding of heparin to this protein is saturable. No other glycosaminoglycan from mammalian tissue, such as hyaluronic acid, chondroitin sulphate, dermatan sulphate or keratan sulphate, binds to the 78 kDa protein. Dextran sulphate binds in a non-saturable fashion. Certain heparan sulphate polysaccharide structures are required for binding to the 78 kDa protein. Some proteoheparan sulphates, such as endothelial cell-surface proteoheparan sulphate, show only weak interaction with the 78 kDa protein in contrast with a basement-membrane proteoheparan sulphate from HR-9 cells. Antibodies against the 78 kDa protein inhibit binding of proteoheparan [35S]sulphate from basement membranes to smooth-muscle cells. Conventional antibodies, Fab fragments and some monoclonal antibodies, inhibit smooth-muscle cell proliferation in a similar range as that observed for heparin. The protein was detected in a variety of tissues and cells but not in blood cells. A possible role of this protein as a receptor for heparin or heparan sulphate and its function in the control of the arterial wall structure are discussed.     

Characterization of the outermembrane proteins of Vibrio cholerae expressed in in vivo culture

Two outer membrane proteins (Omps) of Vibrio cholerae O1, expressed in the intestine (in vivo) but not in culture media (in vitro), were investigated. The molecular masses of those proteins were 116 kDa and 15 kDa, and they were not associated with iron-regulated proteins. Convalescent cholera patients' sera reacted with the 15 kDa protein but not with the 116 kDa protein. The N-terminal amino acid sequence of the 15 kDa protein was homologous to V. cholerae OmpT. Anti-serum to the 15 kDa protein caused agglutination of the organisms grown in the intestine, but not the organisms in in vitro culture. The anti-serum was bactericidal, but it did not inhibit the adhesion of the organisms to the intestine and HEp-2 cells. These findings suggest the possibility that the 15 kDa protein could be involved in post-infection immunity.     

Identification of different receptor types for toxic phospholipases A2 in rabbit skeletal muscle.

Oxyuranus scutellatus scutellatus toxins 1 (OS1) and 2 (OS2) are two phospholipase A2S (PLA2) isolated from the venom of the Australian Taipan snake. Their iodinated derivatives have been used to characterize PLA2 binding sites on rabbit skeletal muscle. Competition and cross-linking experiments indicate that 125I-labelled OS2 binding sites in rabbit skeletal muscle in vivo are distributed into two classes of receptors. One class binds OS2 and OS1 and is insensitive to the bee venom PLA2. It is composed of a 180 kDa binding protein. This class of PLA2 receptor is expressed at a high level in rabbit myotube membranes. The other class of PLA2 receptor identified with 125I-OS2 also binds with high affinity the bee venom PLA2 but not OS1 and is composed of major polypeptides of 34, 48 and 82 kDa. This second class of receptor is similar to the one found in brain membranes. The density of the two classes of receptors varies during muscle development.     

28 kDa adenosine-binding proteins of brain and other tissues.

Membranes prepared from calf brain were solubilized and chromatographed on a column containing 5'-amino-5'-deoxyadenosine covalently linked to agarose through the 5'-amino group. When the column was eluted with adenosine, a pure protein emerged with subunit molecular mass of 28 kDa. The protein was extracted from the membranes with sodium cholate, but not with 100 microM-adenosine or 0.5 M-NaCl. A similar 28 kDa protein was isolated from the soluble fraction of calf brain. The yield of membrane-bound and soluble 28 kDa protein per gram of tissue was about the same. The 28 kDa protein was also found in membrane and soluble fractions of rabbit heart, rat liver and vascular smooth muscle from calf aorta. The yield per gram of tissue fell into the order brain greater than heart approximately vascular smooth muscle greater than liver for the 28 kDa protein from the membrane fraction, and brain approximately heart greater than vascular smooth muscle greater than liver for the 28 kDa protein from the soluble fraction. Polyclonal antibodies to pure 28 kDa protein from calf brain membranes cross-reacted with the 28 kDa protein from calf brain soluble fraction and with 28 kDa proteins isolated from other tissues. The 28 kDa protein from calf brain membranes was also eluted from the affinity column by AMP and 2',5'-dideoxyadenosine, but at a concentration higher than that at which adenosine eluted the protein, but N6-(R-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine, ADP, ATP, GTP, NAD+, cyclic AMP and inosine failed to elute the protein at concentrations up to 1 mM. The 28 kDa protein from the soluble fraction was not eluted by 3 mM-AMP or 1 mM-N6-(R-phenylisopropyl)adenosine,-5'-N-ethylcarboxamidoadenosine or -cyclic AMP. Unexpectedly, the soluble 28 kDa protein was eluted by AMP in the presence of sodium cholate. Soluble 28 kDa protein from calf brain had a KD for adenosine of 12 microM. Membrane 28 kDa protein from calf brain had a KD of 14 microM in the presence of 0.1% sodium cholate. Amino acid compositions of the 28 kDa proteins were similar, but not identical.     

Differential effects of spermine on aggregation, inositol phosphate formation and protein phosphorylation in human platelets in response to thrombin, arachidonic acid and lysophosphatidic acid

Platelet aggregation stimulated by thrombin, arachidonic acid or lysophosphatidic acid is associated with rapid phosphorylation of two platelet proteins, myosin light chain and a 47 kDa protein. The polyamine, spermine, inhibited platelet aggregation stimulated by all three agents. Spermine inhibited thrombin-stimulated phosphorylation of myosin light chain and the 47 kDa proteins as well as thrombin-induced production of the inositol phosphates and phosphatidic acid. In contrast, spermine did not inhibit phosphorylation of either protein or the formation of inositol phosphates and phosphatidic acid in response to arachidonic acid or lysophosphatidic acid. Although spermine has been demonstrated to inhibit both phosphatidylinositol-specific phospholipase C and calcium-dependent protein kinases in cell free systems, these results suggest that, in the intact platelet, spermine does not directly inhibit these enzymes. Inhibition of aggregation stimulated by arachidonic acid and lysophosphatidic acid is secondary to interference with platelet-platelet interaction but not with platelet activation. In contrast, spermine inhibits thrombin-induced platelet activation. This thrombin-specific inhibition may be related to interference with the binding of thrombin to its receptor or to its catalytic substrate on the cell surface.     

beta-Granins: 21 kDa co-secreted peptides of the insulin granule closely related to adrenal medullary chromogranin A

Three closely related forms of a 21 kDa protein which is co-secreted with insulin have been purified and analysed. These differed in behaviour on ion-exchange chromatography but were indistinguishable by their susceptibility to staphylococcal V8 proteinase digestion, amino acid composition or N-terminal amino acid sequence. Their amino acid composition and N-terminal sequences were remarkably similar to adrenal medullary chromogranin A, a much larger protein (72 kDa). Antibodies to chromogranin A also reacted strongly with the 21 kDa protein in isolated insulin granules. It is concluded that the 21 kDa proteins either represent a repeated domain within the chromogranin molecule or a closely related gene product. The name beta-granin is proposed for these proteins.     

Apolactoferrin inhibits the catalytic domain of matrix metalloproteinase-2 by zinc chelation.

Lactoferrin (LTF) is a multifunctional iron-binding protein that is also capable of binding other divalent metal cations, especially Zn2+. Recent investigations indicate that lactoferrin levels are elevated in many disease conditions in which matrix metalloproteinases (MMPs), particularly MMP-2, are also elevated, suggesting that the 2 proteins may interact. This possibility was examined by determining the effect of LTF in its holo (metal-bound) and apo (metal-free) forms on the proteolytic activity of MMP-2 and other similar zinc metalloproteases. Pre-incubation with apolactoferrin, but not hololactoferrin, greatly reduced the hydrolysis of a peptide substrate by MMP-2, but not by MMP-1, -8, -9, or -13. This inhibition was specific for the 42 kDa catalytic domain fragment of MMP-2 lacking the hemopexin domain, since the 66 kDa form was poorly inhibited by apolactoferrin. The inhibition of the MMP-2 catalytic domain was strongly temperature sensitive, indicating that the conformation of one or both proteins is crucial to this interaction. To ascertain the mechanism of inhibition, increasing concentrations of ZnCl2 and FeCl2 were added to the reaction. While addition of Fe2+ did not reverse inhibition, the addition of Zn2+ resulted in a recovery of MMP-2 activity, and furthermore, zinc-saturated LTF did not inhibit MMP-2. Together, these data strongly suggest that apolactoferrin is capable of removing the catalytic zinc from the active site of MMP-2, although an exosite-based interaction between the 2 proteins cannot be fully ruled out. This inhibitory activity suggests a novel function for LTF and may represent a novel regulatory mechanism that regulates proteolysis by MMP-2 in vivo.     

Protein synthesis in tobacco pollen tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins

One- and two-dimensional electrophoresis of Nicotiana tabacum pollen and pollen tube proteins confirmed that a new protein is preferentially synthesized during pollen germination and tube growth and becomes the most abundant protein in pollen tubes. Analysis of proteins extracted with sodium dodecyl sulfate (SDS) from different pollen tube fractions showed that it is the most abundant non-covalently bound wall protein, characterized by molecular mass of 69 kDa, pI between 7.9 and 8.2, and glycosylation with glucose and/or mannose. Amino acid analysis revealed relative abundance of serine, glutamic acid and glycine, but did not show the presence of hydroxyproline. According to all these characteristics, it cannot be classified as an extensin-like protein. Another prominent wall-bound glycoprotein has a molecular mass of 66 kDa and the same pI as the 69 kDa glycoprotein. These two glycoproteins are similar also in ConA binding, rate of synthesis, and rapid incorporation into pollen tube walls. Their synthesis is strongly reduced by tunicamycin and this inhibition results in the occurrence of new polypeptides in the range of 57–61 kDa. Tunicamycin also inhibited pollen tube growth. At 10 ng ml^-1 and 50 ng ml^-1 the inhibitor reduced pollen tube mass after 24 h of culture by 30% and 85%, respectively. This indicates that tobacco pollen presents a system highly sensitive to tunicamycin and that cotranslational N-linked glycosylation on the rough endoplasmic reticulum is required for 66 and 69 kDa glycoprotein formation and for pollen tube growth. Although other proteins appear during pollen germination and tube growth, the new proteins occur at low levels and seem to originate through modifications of preexisting polypeptides. In contrast to 69 and 66 kDa proteins, most proteins detected by [¹⁴C]amino acid incorporation and fluorography of gels were not revealed by Coomassie blue staining.     

Mannose-specific lectin activity of parasporal proteins from a lepidoptera-specific Bacillus thuringiensis strain

Lectin activity, agglutinating sheep erythrocytes, was associated with parasporal inclusion proteins from a Lepidoptera-specific isolate of Bacillus thuringiensis serovar galleriae (H5ab). The activity was generated when parasporal inclusions were solubilized in an alkaline condition. Proteolytic processing was not required for generation of the lectin activity; the activity level was not affected by the presence/absence of the three proteases (trypsin, chymotrypsin, and proteinase K). SDS-PAGE analysis revealed that (1) alkali-solubilized parasporal inclusion proteins consisted of two major components of 130 kDa and 65 kDa, and (2) proteinase K treatment of alkali-solubilized proteins yielded a single major protein of 60 kDa. Lectin activity of our isolate was strongly inhibited by preincubation with D-mannose, but not with the six other monosaccharides: D-galactose, D-glucose, L-fucose, N-acetyl- D-glucosamine, N-acetyl- D-galactosamine, and N-acetylneuraminic acid. In contrast, D-mannose did not inhibit the in vivo larvicidal activity of the proteins against the silkworm, Bombyx mori.     

Isolation and characterization of a fraction from brain that inhibits 1,4-[3H]dihydropyridine binding and L-type calcium channel current

Bovine brain was subjected to acid extraction and several purification steps. A fraction from brain that eluted from C18 reverse-phase columns at 30-35% acetonitrile inhibited [3H]nitrendipine binding to cardiac membranes. Further purification of this fraction on a sizing column in the presence of 40% acetonitrile yielded a low molecular mass fraction (less than 1 kDa) that produced a time- and voltage-dependent inhibition of L-type (but not T-type) Ca2+-channel current in GH3 cells. The results suggest that this fraction contains an endogenous substance that binds directly to slowly-inactivating Ca2+ channels and thereby inhibits current flow.     

The Repetitive Microtubule-Associated Proteins MARP-1 and MARP-2 of Trypanosoma brucei

The microtubular membrane skeleton of Trypanosoma brucei contains two closely related, repetitive, high-molecular-weight microtubule-associated proteins, MARP-1 and MARP-2 (MARP for icrotubule- ssociated epetitive roteins). Their structure is unusual in that they consist of tandemly arranged, strongly conserved 38-amino-acid repeat units over almost their entire length of about 320 kDa. Their nonrepetitive N and C ends are comparatively short. The predicted amino acid sequences reveal a gradient of similarity between MARP-1 and MARP-2 which increases from the N-terminus (no significant similarity) through the repeat domain (50% similarity) to the C-terminus (94.5% similarity). Transfection of mammalian cell lines with recombinant fragments of MARP-2 demonstrate that the nonrepetitive C-terminus of MARP-2 binds specifically to microtubules. This C-terminus does not show sequence similarity with any other microtubule-associated proteins and thus appears to represent a novel type of microtubule-binding domain.     

Identification of a processed protein related to the human chaperonins (hsp 60) protein in mammalian kidney.

The chaperonin family of proteins, which includes GroEL protein of E. coli, yeast heat shock protein (hsp-60) and the ribulose-1-5-bisphosphate carboxylase (Rubis Co.) subunit binding protein of plant chloroplasts, shows strong sequence homology to the Chinese hamster ovary (CHO) mitochondrial P1 protein. We have identified a 60 kDa protein from bovine kidney which by N-terminal sequencing gives the amino acid sequence AKDVKFGADARALLMLQGVDLLADA. Bovine whole kidney membranes were delipidated, solubilized with octyl glucoside and fractionated over an affinity column using the amiloride analog 5-N pyrazine amiloride as the ligand. After extensive washing with 200 mM NaCl, the column was eluted with pH 4.0 buffer. Analysis of column fractions on a 7.5% polyacrylamide gel revealed 3-4 bands with a predominant band at 60,000 Da. Amino acid analysis after transfer to immobilon membranes demonstrated sequence identity to the human HSP (60), extending 24 amino acids from the N-terminus, but lacking the leader sequence. These data indicate that a processed form of a protein related to the human HSP (60) chaperonin is associated with a membrane fraction in the mammalian kidney, and that the processed form of the protein binds strongly to an amiloride affinity support.     

Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.

Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.     

Comparison of tyrosine phosphorylation of proteins by membrane fractions from mouse liver, Ehrlich ascites tumor and MH134 hepatoma

When membrane fractions from mouse liver, Ehrlich ascites tumor and MH134 hepatoma were incubated with [gamma-32P]ATP at 0 degree C in the presence of MnCl2, ZnCl2 and NaVO3, proteins were phosphorylated on tyrosines to a larger extent in liver membranes than in tumor membranes. Separation of labelled proteins by SDS-gel electrophoresis showed phosphorylated alkali-resistant bands of 170, 140, 130, 80, 56, 53 and 46 kDa proteins in Ehrlich ascites tumor membranes; liver membranes exhibited more strongly phosphorylated bands of 170, 56, 53 and 46 kDa proteins. Epidermal growth factor stimulated the tyrosine phosphorylation of only a 170 kDa protein, which was more significant in liver membranes. Liver membranes exhibited slightly higher levels of tyrosine protein kinase activity compared to tumor membranes.