ABSTRACT
A near-diploid mouse fibroblast cell line m5S/1M used in this study shows a high sensitivity to contact-dependent inhibition of growth, and the addition of EGF causes both morphological change and loss of contact-dependent inhibition of growth. The m5S/1M cell and its transformants obtained by x-ray irradiation have been used to search for the cell surface glycoproteins that are responsible for the growth regulation via cell-cell interactions. Lectin blotting analyses showed that the expression of the cell surface glycoprotein of 140 kD (140KGP) is highly sensitive to the transformation induced either by x-ray irradiation or by the EGF stimulation. We purified the 140KGP and found that it was composed of two glycoproteins. The major component of 140KGP was identified as neural cell adhesion molecule (NCAM) by amino acid sequence analyses of the peptide fragments and by the cross-reactivity with anti-NCAM mAb, clone H28.1.2.3. Monoclonal antibody against 140KGP (clone LN-10) recognizes all three isoforms of NCAM expressed on m5S/1M cell and showed that the expression of NCAM was highly sensitive to the transformation. Furthermore, the immobilized LN-10 strongly inhibited the growth of actively proliferating m5S/1M cells and the LN-10 in a soluble form showed a significant growth-stimulating effect on the confluent quiescent cultures of m5S/1M cells. The results show that NCAM plays a major role in the contact-dependent inhibition of growth of m5S/1M, and that NCAM might be involved in the regulation of cell growth during embryogenesis and formation of nervous systems.
Subject(s)
Cell Adhesion Molecules, Neuronal/physiology , Cell Division/physiology , Fibroblasts/cytology , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Bradykinin/pharmacology , Cell Adhesion Molecules, Neuronal/chemistry , Cell Adhesion Molecules, Neuronal/immunology , Cell Line , Diploidy , Electrophoresis, Polyacrylamide Gel , Epidermal Growth Factor/physiology , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Mice , Molecular Sequence Data , Tetradecanoylphorbol Acetate/pharmacology , Transformation, GeneticABSTRACT
Analysis of the amino acid sequence of one Bence Jones protein is almost comtplete. Many points of interchange occur in the amino terminal. portion of the molecule relative to partial-sequence data for other proteins. Most, but not all, are, compartible with one-step mutations. Such structural variation in immunoglobulin light chains may result from many related genes.
Subject(s)
Bence Jones Protein , Amino Acid Sequence , Genes , MutationABSTRACT
The amino acid sequence of a human lamba chain has been determined. There are many identities in sequence with human kappa chains, but this intraspecies homology is less than the interspecies homology of kappa light chains of man and mouse. Structural relationships suggest a common evolutionary origin and early differentiation of light- and heavy-chain genes.
Subject(s)
Amino Acid Sequence , Bence Jones Protein/analysis , Animals , Biological Evolution , Genes , Humans , Mice , MutationABSTRACT
Variation and conservation in the primary structure of human lambda light chains is revealed by complete amino acid sequence of three Bence Jones proteins. These proteins differ in amino acid sequence in from 38 to 48 positions; they are of unequal length in the amino-terminal half of the chain but have identical sequence in the last 105 amino acids.
Subject(s)
Amino Acid Sequence , Bence Jones Protein/analysis , Peptides/analysis , gamma-Globulins/analysis , Biological Evolution , Carboxypeptidases , Chymotrypsin , Humans , In Vitro Techniques , Leucyl Aminopeptidase , MutationABSTRACT
Activin, a member of the transforming growth factor beta protein family, was originally isolated from gonadal fluids and stimulates the release of pituitary follicle-stimulating hormone (FSH). Activin has numerous functions in both normal and neoplastic cells. Various cells synthesize activin and have a specific binding site for this peptide. However, the molecular basis for its actions is unknown. A binding protein for activin was purified from rat ovary and was identical to follistatin, a specific inhibitor of FSH release. It is likely that the binding protein participates in the diverse regulatory actions of activin.
Subject(s)
Carrier Proteins , Inhibins/metabolism , Ovary/metabolism , Activins , Animals , Cells, Cultured , Female , Follicle Stimulating Hormone/metabolism , Inhibins/isolation & purification , Inhibins/pharmacology , Kinetics , Molecular Weight , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Protein Binding , RatsABSTRACT
We have purified and characterized the growth inhibitory factor (GIF) that is abundant in the normal human brain, but greatly reduced in the Alzheimer's disease (AD) brain. GIF inhibited survival and neurite formation of cortical neurons in vitro. Purified GIF is a 68 amino acid small protein, and its amino acid sequence is 70% identical to that of human metallothionein II with a 1 amino acid insert and a unique 6 amino acid insert in the NH2-terminal and the COOH-terminal portions, respectively. The antibodies to the unique sequence of GIF revealed a distinct subset of astrocytes in the gray matter that appears to be closely associated with neuronal perikarya and dendrites. In the AD cortex, the number of GIF-positive astrocytes was drastically reduced, suggesting that GIF is down-regulated in the subset of astrocytes during AD.
Subject(s)
Alzheimer Disease/metabolism , Brain/metabolism , Growth Inhibitors/metabolism , Membrane Proteins/metabolism , Metallothionein/metabolism , Aged , Aged, 80 and over , Amino Acid Sequence , Amino Acids/analysis , Antibodies , Astrocytes/metabolism , Astrocytes/ultrastructure , Brain/ultrastructure , Electrophoresis, Polyacrylamide Gel , Growth Inhibitors/analysis , Growth Inhibitors/genetics , Humans , Immunoblotting , Immunohistochemistry , Membrane Proteins/analysis , Membrane Proteins/genetics , Metallothionein/analysis , Metallothionein/genetics , Middle Aged , Molecular Sequence Data , Neurofibrils/metabolism , Neurofibrils/ultrastructureABSTRACT
We have investigated ubiquitinated paired helical filaments, which produce a proteinaceous smear in SDS-polyacrylamide gel electrophoresis and immunoblotting. The smear consisted largely of the carboxy-terminal portion of tau and ubiquitin. The ubiquitin-targeted protein was identified as tau in paired helical filaments, and the conjugation sites were localized to the microtubule-binding region. Most ubiquitin in paired helical filaments occurred as a monoubiquitinated form, and only a small proportion of ubiquitin formed multiubiquitin chains. There was a ubiquitin-negative smear, in which tau was much less processed in the amino-terminal portion. This strongly suggests that the amino-terminal processing of tau in paired helical filaments precedes its ubiquitination.
Subject(s)
Peptide Fragments/metabolism , Ubiquitins/metabolism , tau Proteins/metabolism , Amino Acid Sequence , Antibodies, Monoclonal , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Endopeptidases , Humans , Mass Spectrometry , Microtubules/metabolism , Molecular Sequence Data , Molecular Weight , Neurofibrillary Tangles/metabolism , Peptide Fragments/isolation & purification , Peptide Mapping , Protein Binding , Ubiquitins/isolation & purification , tau Proteins/isolation & purificationABSTRACT
A small but consistent proportion of the von Willebrand factor (vWF) in normal plasma is composed of 189, 176, and 140 kD fragments cleaved from the 225 kD subunit. A monoclonal antibody map of vWF, based on the reactivity of individual antibodies with cyanogen bromide and tryptic fragments of known carboxy and/or amino termini, showed that in normal and IIA von Willebrand disease (vWD) plasmas the 140 kD fragment was derived from the amino-terminal region, whereas the 176 kD fragment was derived from the carboxy-terminal region of the subunit. In type IIA vWD, however, the fragments comprised a greater proportion of circulating vWF. In contrast, plasmin cleaved a 176 kD fragment from the amino terminus and a 145 kD fragment from the carboxy terminus of the subunit. Species similar to these plasmin-cleaved fragments were demonstrated in plasmas from four patients treated with fibrinolytic agents, but not in IIA vWD.
Subject(s)
Epitopes/analysis , Fibrinolysin/metabolism , von Willebrand Diseases/blood , von Willebrand Factor/immunology , Antibodies, Monoclonal , Humans , Macromolecular Substances , Molecular Weight , Peptide Fragments/analysis , Peptide Fragments/immunology , von Willebrand Factor/analysisABSTRACT
Qualitative and quantitative alterations of human tenascin (TN) expression in virally transformed lung fibroblasts and in lung tumor tissues were investigated using S1 nuclease protection analysis in comparison with those of fibronectin (FN). Transformed fibroblasts and fetal lung tissues expressed more TN mRNA with an extra sequence encoding the sixth FN type III repeat than did normal cells and adult tissues. The splicing pattern of TN mRNA was also altered in many lung cancer tissues, showing increased or sometimes decreased expression of the TN mRNA with the extra sequence when compared with their surrounding normal tissues. These results provide additional evidence for the oncodevelopmental regulation of alternative RNA splicing in human lung tissues, first observed with FN mRNA (F. Oyama, et al., Cancer Res., 50: 1075-1078, 1990). Quantitative analysis of the levels of TN and FN mRNAs showed that the ratio of TN mRNA to FN mRNA was significantly increased in transformed fibroblasts and in some lung tumor tissues, when compared with their normal counterparts. Among different types of lung tumors, a significant increase of the TN/FN ratio was observed with most squamous cell carcinoma but with only a small fraction of adenocarcinoma. Since TN has been shown to inhibit cell adhesion to FN, the altered ratio of TN mRNA to FN mRNA may well affect the adhesive and migratory properties of tumor cells in lung cancer tissues.
Subject(s)
Cell Adhesion Molecules, Neuronal/genetics , Extracellular Matrix Proteins/genetics , Fibronectins/genetics , Gene Expression Regulation, Neoplastic/physiology , Lung Neoplasms/genetics , Base Sequence , Cell Adhesion Molecules, Neuronal/metabolism , Cell Transformation, Viral/genetics , Extracellular Matrix Proteins/metabolism , Fibroblasts/physiology , Fibronectins/metabolism , Gene Expression Regulation/physiology , Humans , Lung/cytology , Molecular Sequence Data , RNA Splicing/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tenascin , Transcription, Genetic/geneticsABSTRACT
Alternative splicing of fibronectin pre-mRNA at the ED-A region has been shown to be deregulated in malignant human liver tumors (F. Oyama et al., J. Biol. Chem., 264: 10331-10334, 1989). In order to extend this observation to other human cancers, we investigated the splicing patterns of fibronectin pre-mRNA at both ED-A and ED-B regions in normal, fetal, and cancerous lung tissues. Unlike in the liver, the ED-A+ mRNA was constitutively expressed in the lung irrespective of ontogenic or oncogenic stages. Although fetal tissues expressed the ED-A+ mRNA slightly more than did adult tissues, there was virtually no significant difference between malignant and nonmalignant tissues in the level of the ED-A+ mRNA. In contrast, significant expression of the ED-B+ mRNA was observed with fetal and cancerous tissues but not with normal adult tissues. Increased expression of the ED-B+ mRNA was associated with all types of lung cancer including adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. These results indicate that it is the ED-B, but not the ED-A, region where the alternative splicing of fibronectin pre-mRNA is oncodevelopmentally regulated in the lung. Our results also suggest that deregulation of the tissue-specific alternative splicing of fibronectin pre-mRNA is not a unique phenotype of liver cancer but rather a general feature of naturally occurring human cancer.
Subject(s)
Fibronectins/biosynthesis , Gene Expression Regulation, Neoplastic , Lung Neoplasms/metabolism , Lung/metabolism , RNA Splicing , RNA, Messenger/biosynthesis , Animals , DNA Probes , Fetus , Fibronectins/genetics , Humans , Lung Neoplasms/genetics , Mice , Mice, Nude , RNA, Messenger/geneticsABSTRACT
The molecular diversity of fibronectin arises from alternative RNA splicing at regions termed ED-A, ED-B, and IIICS. We investigated the splicing patterns of fibronectin pre-mRNA at both ED-B and IIICS regions in various human liver tissues with an emphasis on the expression of the alternative cell adhesive site CS1 within the IIICS region. The relative abundance of the fibronectin mRNA containing the CS1 sequence was significantly increased in both fetal and cancerous liver tissues, although it was not affected in nonmalignant tissues with chronic hepatitis and cirrhosis. Similarly, the relative abundance of the fibronectin mRNA containing the ED-B region was also increased in both fetal liver and liver tumors, showing a close parallelism with the splicing pattern at the ED-A region. Immunohistochemical examination of cancerous liver tissues with monoclonal antibodies directed to the ED-A and ED-B segments revealed that the fibronectin isoforms containing these extra peptide segments were specifically deposited in the tumor nodules. Other genes encoding kininogen, gamma chain of fibrinogen, and beta-amyloid protein precursor, all of which had been shown to be alternatively processed, did not show any significant alteration in the splicing pattern in cancerous liver tissues. These results indicate that the alternative splicing of fibronectin pre-mRNA at the ED-A, ED-B, and IIICS regions is coordinately modulated in both fetal and cancerous liver tissues toward inclusion of the extra peptide segments and that not all but only selected genes are susceptible for "fine tuning" of alternative RNA splicing in cancerous liver tissues.
Subject(s)
Fibronectins/genetics , Liver Neoplasms/genetics , Alternative Splicing , Amyloid beta-Protein Precursor/genetics , Base Sequence , Fibronectins/chemistry , Gene Expression , Humans , Kininogens/genetics , Liver/embryology , Liver Diseases/genetics , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , RNA, Messenger/genetics , RNA, Neoplasm/geneticsABSTRACT
Proteins binding to amyloid beta-protein (Abeta) may modulate the accumulation of Abeta in Alzheimer's disease (AD) brain. We developed a monomeric Abeta column for isolation of the proteins binding to Abeta from rat brain. By amino acid sequence analysis and immunoreactivity with specific antibodies, we identified three new Abeta-binding proteins, glutamine synthetase, hemoglobin alpha-chain, and macrophage migration inhibitory factor as well as serum albumin, beta-tubulin, and glyceraldehyde-3-phosphate dehydrogenase already identified as proteins bound to amyloid beta-protein precursor. In addition, the retained fraction contained both apolipoprotein E and alpha(1)-antichymotrypsin already known as Abeta binding proteins. Furthermore, we detected the complexes of these new binding proteins with Abeta in a soluble fraction of the cerebral cortex of AD brain by immunoprecipitation. Our results suggest that these binding proteins also associate with Abeta, leading to the clearance or the accumulation of Abeta and the neuronal cell damage in human brain.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Brain/metabolism , Chromatography, Affinity/methods , Glutamate-Ammonia Ligase/metabolism , Hemoglobins/metabolism , Macrophage Migration-Inhibitory Factors/metabolism , Peptide Fragments/metabolism , Alzheimer Disease/enzymology , Amino Acid Sequence , Amyloid beta-Peptides/chemistry , Animals , Brain/enzymology , Glutamate-Ammonia Ligase/isolation & purification , Hemoglobins/chemistry , Hemoglobins/isolation & purification , Humans , Macrophage Migration-Inhibitory Factors/isolation & purification , Male , Molecular Sequence Data , Peptide Fragments/chemistry , Precipitin Tests , Protein Binding , Rats , Rats, WistarABSTRACT
The structure and function of snake venom proteases are briefly reviewed by putting the focus on their effects on hemostasis and thrombosis and comparing with their mammalian counterparts. Up to date, more than 150 different proteases have been isolated and about one third of them structurally characterized. Those proteases are classified into serine proteases and metalloproteinases. A number of the serine proteases show fibrin(ogen)olytic (thrombin-like) activities, which are not susceptible to hirudin or heparin and perhaps to most endogenous serine protease inhibitors, and form abnormal fibrin clots. Some of them have kininogenase (kallikrein-like) activity releasing hypotensive bradykinin. A few venom serine proteases specifically activate coagulation factor V, protein C, plasminogen or platelets. The venom metalloproteinases, belonging to the metzincin family, generally show fibrin(ogen)olytic and extracellular matrix-degrading (hemorrhagic) activities. A few venom metalloproteinases show a unique substrate specificity toward coagulation factor X, platelet membrane receptors or von Willebrand factor. A number of the metalloproteinases have chimeric structures composed of several domains such as proteinase, disintegrin-like, Cys-rich and lectin-like domains. The disintegrin-like domain seems to facilitate the action of those metalloproteinases by interacting with platelet receptors. A more detailed analysis of snake venom proteases should find their usefulness for the medical and pharmacological applications in the field of thrombosis and hemostasis.
Subject(s)
Endopeptidases/metabolism , Snake Venoms/metabolism , Amino Acid Sequence , Animals , Endopeptidases/chemistry , Hemostasis/drug effects , Humans , Metalloendopeptidases/chemistry , Metalloendopeptidases/metabolism , Models, Molecular , Molecular Sequence Data , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Snake Venoms/chemistry , Thrombin/chemistry , Thrombin/metabolism , Thrombosis/etiologyABSTRACT
Binding specificities of ABO blood group-recognizing lectins toward blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides were studied by lectin-blotting analysis, enzyme linked immunosorbent assay and lectin-conjugated agarose column chromatography. Human serum albumin conjugated with A- and B-trisaccharides was clearly recognized by Helix pomatia (HPA), Phaseolus lunatus, Dolichos biflorus agglutinins, and Griffonia simplicifolia I agglutinin B(4), respectively. Almost the same results were obtained for human group A and B ovarian cyst and A-active hog gastric mucins, but Glycine max agglutinin only reacted to the group A hog mucin. When human plasma von Willebrand factor (vWF), having Asn-linked blood group antigens, was tested, HPA was highly sensitive to blood group A antigen on the vWF. Ulex europaeus agglutinin I (UEA-I) preferentially bound to the vWF from blood group O plasma. Within the GalNAc-recognizing lectins examined, a biantennary complex-type oligosaccharide having the blood group A structure retarded on an HPA-agarose column, and the affinity was diminished after digestion with alpha-N-acetylgalactosaminidase. This product bound to UEA-I agarose column. These results indicate that HPA and UEA-I are most sensitive for detection of glycoproteins possessing small amounts of blood group A and H antigens and also useful for fractionation of complex-type oligosaccharides with blood group A and H antigens, respectively.
Subject(s)
ABO Blood-Group System/metabolism , Lectins/metabolism , ABO Blood-Group System/chemistry , ABO Blood-Group System/immunology , Adult , Animals , Carbohydrate Sequence , Glycoproteins/chemistry , Glycoproteins/immunology , Glycoproteins/metabolism , Humans , In Vitro Techniques , Molecular Sequence Data , Mucins/chemistry , Mucins/immunology , Mucins/metabolism , Oligosaccharides/chemistry , Oligosaccharides/immunology , Oligosaccharides/metabolism , von Willebrand Factor/chemistry , von Willebrand Factor/immunology , von Willebrand Factor/metabolismABSTRACT
The effect of follistatin on activin-induced granulosa cell differentiation was investigated in freshly harvested granulosa cells from diethylstilbestrol (DES)-treated rats. Activin induced a remarkable change in granulosa cellular morphology from elongated fibroblast-like to round cells, which follistatin prevented. Follistatin itself had no influence on the cellular morphology. We studied the action of follistatin on activin-induced differentiation of granulosa cells cultured in a chemically defined medium. Addition of activin (30 ng/ml) to the culture increased the FSH binding site approximately 2-fold compared with the control (spontaneous expression) level, whereas follistatin reduced the activin-induced expression level to the control level in a concentration-dependent manner. Activin (30 ng/ml) markedly augmented FSH-induced hCG binding and progesterone production by approximately 20-fold, and these effects were suppressed by follistatin in a concentration-dependent manner. Similarly, addition of follistatin to the culture induced a concentration-dependent decrease of activin-enhanced inhibin-producing activity, but had no effect on FSH-induced inhibin production. These results suggest that follistatin/activin-binding protein binds to activin stoichiometrically to suppress the activin-induced differentiation of rat granulosa cell in vitro, but follistatin itself has no direct effect on activin-independent reactions.
Subject(s)
Cell Differentiation/drug effects , Glycoproteins/pharmacology , Granulosa Cells/cytology , Inhibins/pharmacology , Activins , Animals , Cells, Cultured , Chorionic Gonadotropin/metabolism , Diethylstilbestrol/pharmacology , Dose-Response Relationship, Drug , Female , Follicle Stimulating Hormone/metabolism , Follistatin , Granulosa Cells/drug effects , Granulosa Cells/metabolism , Inhibins/antagonists & inhibitors , Inhibins/metabolism , Kinetics , Progesterone/metabolism , Rats , Rats, Inbred Strains , Receptors, FSH/metabolism , Receptors, LH/metabolism , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/pharmacologyABSTRACT
A complementary DNA (cDNA) clone of dihydrolipoamide acetyltransferase (E2) of the rat pyruvate dehydrogenase complex (PDC) was isolated from a lambda gt11 rat heart cDNA library. The amino acid sequence of a full mature protein of rat PDC-E2 was predicted by combination of the cDNA nucleotide sequence and the N-terminal amino acid sequence determined chemically. The amino acid sequence of rat PDC-E2 was well consistent with those of the E2 components of other alpha-ketoacid dehydrogenase complexes. These E2 components possess the sequence G-X-G-X-X-G, which is the consensus sequence for nucleotide binding sites of nucleotide binding proteins, in the E3 and/or E1 binding domains. The E2 components of the three alpha-ketoacid dehydrogenase complexes are suggested to be classified into three clusters separated during evolution.
Subject(s)
Acetyltransferases/genetics , Myocardium/enzymology , Pyruvate Dehydrogenase Complex/genetics , Acetyltransferases/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA/isolation & purification , Dihydrolipoyllysine-Residue Acetyltransferase , Gene Library , Models, Biological , Molecular Sequence Data , Pyruvate Dehydrogenase Complex/chemistry , Rats , Restriction MappingABSTRACT
L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 microM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.
Subject(s)
Amino Acid Oxidoreductases/chemistry , Platelet Aggregation Inhibitors/chemistry , Agkistrodon , Amino Acid Oxidoreductases/genetics , Amino Acid Sequence , Animals , DNA, Complementary , Humans , L-Amino Acid Oxidase , Molecular Sequence Data , Platelet Aggregation/drug effects , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
Two high molecular mass proteins, flavocetin-A and flavocetin-B, were purified from Trimeresurus flavoviridis venom. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the apparent molecular mass of flavocetin-A and -B were 149 and 139 kDa, respectively, under nonreducing conditions. On reduction, flavocetin-A showed two distinct subunits (17 and 14 kDa), and flavocetin-B three distinct subunits (17, 15 and 14 kDa). At 1 microgram/ml, flavocetin-A and -B (flavocetins) inhibited the von Willebrand factor (vWF)-dependent aggregation of fixed human platelets. However, flavocetins (10 micrograms/ml) had no effect on ADP- and collagen-induced platelet aggregation in PRP. Flavocetins (3 micrograms/ml) also inhibited shear-induced platelet aggregation at high shear stress. Furthermore, flavocetin-A completely inhibited the aggregation of and ATP release from washed platelets stimulated with a low concentration of thrombin. Flavocetin-A specifically bound to platelet with high affinity (Kd = 0.35 +/- 0.13 nM) at 21,500 +/- 1760 binding sites per platelet. The N-terminal amino acid sequences of the subunits of flavocetin-A show a high degree of homology with those of echicetin, botrocetin, alboaggregin-B and factor IX/factor X-binding protein. These results suggest that flavocetins may be a useful tool for further investigation of the GPIb-vWF interaction.
Subject(s)
Carrier Proteins/pharmacology , Crotalid Venoms/chemistry , Platelet Aggregation Inhibitors/pharmacology , Trimeresurus , Adenosine Triphosphate/blood , Amino Acid Sequence , Animals , Biophysical Phenomena , Biophysics , Blood Platelets/drug effects , Blood Platelets/metabolism , Carrier Proteins/chemistry , Carrier Proteins/isolation & purification , Erythrocytes/metabolism , Humans , Molecular Sequence Data , Molecular Weight , Neutrophils/metabolism , Platelet Aggregation , Sequence Alignment , Thrombin/pharmacology , von Willebrand Factor/metabolism , von Willebrand Factor/pharmacologyABSTRACT
CAP-23/NAP-22, a neuron-specific protein kinase C substrate, is Nalpha-myristoylated and interacts with calmodulin (CaM) in the presence of Ca2+ ions. Takasaki et al. (1999, J Biol Chem 274:11848-11853) have recently found that the myristoylated N-terminal nonapeptide of CAP-23/NAP-22 (mC/N9) binds to Ca2+ -bound CaM (Ca2+/CaM). In the present study, small-angle X-ray scattering was used to investigate structural changes of Ca2+/CaM induced by its binding to mC/N9 in solution. The binding of one mC/N9 molecule induced an insignificant structural change in Ca2+/CaM. The 1:1 complex appeared to retain the extended conformation much like that of Ca2+/CaM in isolation. However, it could be seen that the binding of two mC/N9 molecules induced a drastic structural change in Ca2+/CaM, followed by a slight structural change by the binding of more than two but less than four mC/N9 molecules. Under the saturated condition (the molar ratio of 1:4), the radius of gyration (Rg) for the Ca2+/CaM-mC/N9 complex was 19.8 +/- 0.3 A. This value was significantly smaller than that of Ca2+/CaM (21.9 +/- 0.3 A), which adopted a dumbbell structure and was conversely 2-3 A larger than those of the complexes of Ca2+/CaM with the nonmyristoylated target peptides of myosin light chain kinase or CaM kinase II, which adopted a compact globular structure. The pair distance distribution function had no shoulder peak at around 40 A, which was mainly due to the dumbbell structure. These results suggest that Ca2+/CaM interacts with Nalpha-myristoylated CAP-23/NAP-22 differently than it does with other nonmyristoylated target proteins. The N-terminal amino acid sequence alignment of CAP-23/NAP-22 and other myristoylated proteins suggests that the protein myristoylation plays important roles not only in the binding of CAP-23/NAP-22 to Ca2+/CaM, but also in the protein-protein interactions related to other myristoylated proteins.
Subject(s)
Calmodulin/chemistry , Calmodulin/metabolism , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , Myristic Acid/metabolism , Peptide Fragments/chemistry , Amino Acid Sequence , Animals , Binding Sites , Calcium/metabolism , Calmodulin-Binding Proteins , Models, Molecular , Nerve Tissue Proteins , Neurons/metabolism , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/metabolism , Protein Structure, Secondary , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , X-Ray Diffraction/methodsABSTRACT
We investigated the relationship between the differential expression of beta amyloid protein precursor (APP) and tau mRNA, and the extent of beta and tau deposition in three regions from each of the 38 aged brains obtained from consecutive autopsied cases. Remarkable variabilities were noted in the ratios of APP-770/-751/-695 and four-repeat tau among elderly individuals. There was no consistent alteration in the APP differential expression among beta plaque (-), (+), and (++(-) ) groups. Also, no differences in the four-repeat tau ratios were noted among tangle (-), (+), and (++) groups. Despite these great individual variabilities, APP-751 was found to be well-correlated with four-repeat tau. It is possible that APP-751 and four-repeat tau are increasing during aging, while APP-695 and three-repeat tau are decreasing.