Biomedical subjects

A Komoriya

Publications and source records attributed to A Komoriya.

18 recordsLinked to original sources

PubMed1991

The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine.

Fibronectin contains at least two major domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types via the integrin alpha 5 beta 1. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). A dominant cell type-specific adhesion site within the IIICS has been localized to a peptide designated as CS1 comprising its amino-terminal 25 residues. The receptor for CS1 is the integrin alpha 4 beta 1. We have synthesized a variety of peptides with overlapping sequences in order to identify the minimum active amino acid sequence of this major cell adhesion site. A peptide comprising the carboxyl-terminal 8 amino acids of CS1, EILDVPST, was found to support melanoma cell spreading, while all peptides without this sequence had little or no activity. Two smaller overlapping pentapeptides, EILDV and LDVPS, were also active, whereas EILEV, containing a conservative substitution of Glu for Asp, was inactive. These data suggested that the minimum sequence for cell adhesion activity is Leu-Asp-Val, the tripeptide sequence common to both active peptides. This prediction was confirmed by the observed ability of the Leu-Asp-Val peptide itself to block spreading on fibronectin, whereas Leu-Glu-Val was inactive. Interspecies amino acid sequence comparison also supports the importance of the LDV sequence, since it is completely conserved in the IIICS regions of human, rat, bovine, and avian fibronectins.

Amino Acid Sequence↗

PubMed1991

The CS5 peptide is a second site in the IIICS region of fibronectin recognized by the integrin alpha 4 beta 1. Inhibition of alpha 4 beta 1 function by RGD peptide homologues.

The alternatively spliced type III connecting segment (IIICS) region of fibronectin contains two distinct sites that support the adhesion of melanoma cells. These sites are contained within the synthetic peptides CS1 and CS5 (residues 1-25 and 90-109 of the IIICS, respectively). Recently, the cellular receptor for the CS1 site has been identified as the integrin heterodimer alpha 4 beta 1. In this report, we have investigated the role of the CS5 sequence in melanoma cell adhesion and the identity of its receptor. Adhesion to CS5, when presented to cells as an immobilized IgG conjugate, was blocked by antifunctional monoclonal antibodies directed against either the alpha 4 or beta 1 integrin subunits, but not by antibodies against other subunits, implying that alpha 4 beta 1 is also the receptor for CS5. In peptide inhibition experiments, CS5 was inhibitory for melanoma cell spreading on both CS5-IgG and CS1-IgG conjugates; conversely, CS1 inhibited spreading on both CS1-IgG and CS5-IgG. In both cases, peptide inhibition could be outcompeted by increasing the concentration of substrate-bound conjugate. These results suggest that CS1 and CS5 are recognized by the same or overlapping sites on alpha 4 beta 1. The minimal active sequence within CS5, the tetrapeptide Arg-Glu-Asp-Val (REDV), is somewhat related to the Arg-Gly-Asp-Ser (RGDS) sequence that represents a major active site in the central cell-binding domain (CCBD) of fibronectin. When RGDS peptide homologues were tested for their ability to inhibit spreading of melanoma cells on CS1- and CS5-IgG conjugates, GRGDS, GRGES, and REDV were found to be inhibitory, while GRDGS had no effect. In contrast, spreading on a fibronectin fragment containing the CCBD was inhibited by GRGDS only. GRGDS was also able to elute alpha 4 beta 1 specifically from a CS1 affinity column, confirming directly that alpha 4 beta 1-IIICS interactions are sensitive to peptides containing this recognition motif. Because the minimal active sequence within CS1 is the tripeptide Leu-Asp-Val (LDV; Komoriya et al., manuscript submitted for publication), these findings together define a new adhesive recognition sequence, X-Asp-Y, used by alpha 4 beta 1 for binding to fibronectin. The central aspartate residue in this tripeptide is almost always essential, but some flexibility in the amino acid residues at X (glycine, leucine, or glutamic acid) and Y (serine or valine) is tolerated. Potential models for the interaction of the IIICS region with alpha 4 beta 1 are discussed.

Chromatography, Affinity↗

PubMed1990

VLA-4 mediates CD3-dependent CD4+ T cell activation via the CS1 alternatively spliced domain of fibronectin.

We previously showed that fibronectin (FN) synergized with anti-CD3 in induction of CD4+ T cell proliferation, and that VLA-5 acted as a functional FN receptor in a serum-free culture system. In the present study, we showed that VLA-4 is also involved in this CD3-dependent CD4 cell activation through its interaction with the alternatively spliced CS1 domain of FN. When highly purified CD4 cells were cultured on plates coated with anti-CD3 plus synthetic CS1 peptide-IgG conjugate, significant proliferation could be observed. Neither CS1 alone nor anti-CD3 alone induced this activation. This proliferation was completely blocked by anti-VLA beta 1 (4B4) and anti-VLA-4 (8F2), while anti-VLA-5 (monoclonal antibody [mAb] 16 and 2H6) had no effect. These data indicate that VLA-4 mediates CD3-dependent CD4 cell proliferation via the CS1 domain of FN. Anti-VLA-4 also partially (10-40%) inhibited CD4 cell proliferation induced by native FN plus anti-CD3, implying that the CS1 domain is active in the native plasma FN. However, this native FN-dependent proliferation was entirely abolished by addition of anti-VLA-5 alone. Moreover, when native FN-coated plates were pretreated with anti-FN (mAb 333), which blocks RGDS sites but not CS1 sites, no CD4 cell activation could be observed. These results strongly suggest that CD4 cell activation induced by plasma FN/anti-CD3 may be dependent on both VLA4/CS1 and VLA5/RGDS interactions, although the latter interaction may be required for function of the former.

Amino Acid Sequence↗

PubMed1990

Insulin binding to its receptor induces a conformational change in the receptor C-terminus.

Antibodies against peptides corresponding to sequences in the C-terminus of the insulin receptor beta-subunit were used to approach the putative role of this receptor domain in signal generation. Two sequences were chosen and correspond to peptide C1, comprising amino acids 1309-1326, and peptide C2, comprising amino acids 1294-1317. The two antibodies produced distinct immunoprecipitation patterns as a function of the insulin receptor form and recognized changes in the insulin receptor molecule induced by ligand binding and autophosphorylation. Both antipeptides, anti-C1 and anti-C2, showed an important decrease in their recognition capacity for the receptor occupied by insulin when compared to the empty receptor. Further, anti-C1 had a lower affinity for the phosphorylated receptor compared to the unphosphorylated receptor and failed to recognize a fraction of the phosphoreceptor population. In contrast, anti-C2 had similar affinities for the phosphorylated and unphosphorylated receptors but was unable to interact with part of the unphosphorylated receptors. Finally, using immunoblotting of the receptor to analyze the denatured molecules, we showed that the phosphorylation-induced changes detected by anti-C1 are retained, suggesting that they are likely not of a conformational nature. In contrast, the insulin-induced changes in the receptor molecule disappear with receptor denaturation which points to their reversible nature. We conclude from these data that (i) antipeptides against the receptor C-terminal sequence are able to distinguish between phosphorylated and unphosphorylated receptor forms and (ii) binding of insulin to its receptor leads to a reversible, phosphorylation-independent, and possibly conformational change at the level of the receptor C-terminal domain.

Animals↗

PubMed1990

Affinity chromatographic isolation of the melanoma adhesion receptor for the IIICS region of fibronectin and its identification as the integrin alpha 4 beta 1.

Eukaryotic cells adhere to at least two different regions of the fibronectin molecule: a central domain present in all fibronectin isoforms, and the type III connecting segment domain (IIICS), the expression of which is controlled by complex alternative splicing of precursor mRNA. Using affinity chromatography on a matrix containing a synthetic peptide ligand (CS1) representing the strongest active site within the IIICS, we have isolated the human melanoma cell receptor recognizing this region of fibronectin. The receptor is a complex of two polypeptides with subunit molecular masses of 145 and 125 kDa. This heterodimeric structure resembles that of receptors for other extracellular matrix proteins. Immunological analysis with specific antibodies identified these polypeptides as the integrin subunits alpha 4 and beta 1. In addition, antifunctional monoclonal antibodies directed against either alpha 4 or beta 1, but not against other integrin subunits, were potent inhibitors of CS1-mediated melanoma cell spreading. Furthermore, when the function of the central cell-binding domain was blocked, anti-alpha 4 and anti-beta 1 antibodies abolished spreading of A375-M cells on fibronectin, indicating that alpha 4 beta 1 is an authentic fibronectin receptor. Taken together, these results identify the human fibronectin IIICS receptor as the integrin heterodimer alpha 4 beta 1.

Amino Acid Sequence↗

PubMed1989

Phorbol ester and interferon-gamma modulation of epidermal growth factor receptors on human amniotic (WISH) cells.

In this study we report that pretreatment of human amniotic (WISH) cells with interferon gamma (IFN-gamma) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the down-modulation of epidermal growth factor (EGF) receptors with respect to both receptor number and affinity. Scatchard analysis of EGF binding in the absence of both IFN-gamma and TPA indicated biphasic binding whereas addition of TPA resulted in the loss of the higher affinity class of sites. Pretreatment with IFN-gamma for 24 h enhanced the TPA-induced inhibition of EGF binding whereas IFN-gamma alone had no effect on binding. Protein kinase C (Ca2+/phospholipid-dependent enzyme) was examined as a possible mediator of IFN-induced EGF-receptor modulation; pretreatment of cells with IFN-gamma affected neither the binding of [3H]phorbol 12,13-dibutyrate to membrane or cytosolic fractions nor the protein kinase C activity, suggesting that IFN-gamma pretreatment did not result in translocation or activation of protein kinase C.

Cell Compartmentation↗

PubMed1988

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1988

Neurite extension of chicken peripheral nervous system neurons on fibronectin: relative importance of specific adhesion sites in the central cell-binding domain and the alternatively spliced type III connecting segment.

Fibronectin contains at least two domains that support cell adhesion. One is the central cell-binding domain that is recognized by a variety of cell types, including fibroblasts. The second, originally identified by its ability to support melanoma cell adhesion, is located in the alternatively spliced type III connecting segment (IIICS). Using specific adhesive ligands and inhibitory probes, we have examined the role of each of these domains in fibronectin-mediated neurite extension of neurons from chick embryo dorsal root and sympathetic ganglia. In studies using explanted ganglia, both fl3, a 75-kD tryptic fragment of human plasma fibronectin containing the central cell-binding domain, and CS1-IgG, a synthetic peptide-IgG conjugate containing the principal cell adhesion site from the IIICS, supported neurite outgrowth after adsorption onto the substrate. The maximal activities of fl3 and CSl-IgG were 45-55% and 25-30% that of intact fibronectin, respectively. Co-coating of the substrate with f13 and CS1-IgG produced an additive stimulation of neurite outgrowth, the extent of which approached that obtained with fibronectin. Similar results were obtained with purified neuronal cell preparations isolated by tryptic dissociation of dorsal root ganglia. In complementary studies, blockage of the adhesive function of either the central cell-binding domain (with mAb 333, an antiadhesive monoclonal antibody) or the IIICS (with CS1 peptide), resulted in approximately 60 or 30% reduction in fibronectin-mediated neurite outgrowth, respectively. When tested in combination, the inhibitory activities of mAb 333 and CSl were additive. From these results, we conclude that neurons from the peripheral nervous system can extend neurites on both the central cell-binding domain and the IIICS region of fibronectin, and that these cells are therefore the first normal, embryonic cell type shown to adhere to the IIICS. These results suggest that spatiotemporal fluctuations in the alternative mRNA splicing of the IIICS region of fibronectin may be important in regulation of cell adhesive events during development of the peripheral nervous system.

Animals↗

PubMed1988

Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells.

We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-beta (TGF-beta), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-beta inhibited serum-induced proliferation of rat aortic smooth muscle cells (ED50 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-beta. TGF-beta concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47% increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-beta-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in G1 were hypertrophied with respect to the corresponding cells in vehicle-treated controls. Chronic treatment with TGF-beta (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-beta from these cultures was associated with the appearance of a significant fraction of cycling cells with greater than 4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-beta in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-beta.

Animals↗

PubMed1987

Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion.

The principal region of the human plasma fibronectin molecule mediating the adhesion of melanoma cells appears to be the alternatively spliced type III connecting segment (IIICS (Humphries, M. J., Akiyama, S. K., Komoriya, A., Olden, K., and Yamada, K. M. (1986a) J. Cell Biol., in press]. A series of overlapping synthetic peptides spanning the entire IIICS (CS peptides) were examined for their effects on B16-F10 melanoma cell adhesion to the parent fibronectin molecule. Two nonadjacent CS peptides, designated CS1 and CS5, were inhibitory. In contrast, neither inhibited fibronectin-mediated spreading of fibroblastic baby hamster kidney cells. When N-terminal cysteine derivatives of the CS peptides were conjugated to IgG by covalent cross-linking with N-succinimidyl-3(2-pyridyldithio)propionate, both the CS1 and CS5 conjugates promoted B16-F10 melanoma cell spreading. All conjugates were inactive for spreading of baby hamster kidney cells, confirming the cell type specificity of the IIICS adhesion site. Determination of the amounts of CS peptide required to support melanoma cell adhesion revealed that the activity of CS1 was only 2.4-fold lower than that of the intact fibronectin molecule. CS5 was approximately 320-fold less active than fibronectin, suggesting that the CS1 region may be the major site of interaction with the melanoma cell surface. The adhesion-promoting activities of CS1-IgG and CS5-IgG were additive as were the inhibitory activities of the free peptides for B16-F10 cell spreading on fibronectin. These findings suggest that both regions of the IIICS can function separately or together in mediating the interaction of melanoma cells with fibronectin. Since CS1 and CS5 are each found in separate alternatively spliced regions of the IIICS, it is conceivable that the adhesion-promoting activity of fibronectin for different cell types may be under complex regulation.

Amino Acid Sequence↗

PubMed1987

Mapping a region associated with Na channel inactivation using antibodies to a synthetic peptide corresponding to a part of the channel.

Antibodies to the synthetic peptide (carrier-coupled) corresponding to amino acids 210-223 of the primary sequence of eel Na channel (C1+ peptide) were generated. The antipeptide antibodies were used to identify functional roles as well as the accessibility from the external membrane surface of the C1+ domains. Rabbit antipeptide antibodies bound specifically to the C1+ synthetic peptide and to an eel membrane fraction bearing a high density of Na channels. When applied to the external surface of cultured dorsal root ganglion cells obtained from newborn rats, the antibodies modify Na channel inactivation by shifting the steady-state Na current-inactivation parameter, h infinity, curve to more negative potentials in fast and slow Na currents. The rate of inactivation of the slow channel is shown to be increased. The antibodies do not have a significant effect on activation of the channels. Part of the amino acid sequence corresponding to C1+ peptide is therefore accessible, in the mammalian Na channel, from the external membrane surface and is associated with the inactivation gate.

Action Potentials↗

PubMed1987

Inhibition of endothelial cell proliferation by gamma-interferon.

Endothelial cell growth factor (ECGF) is a potent polypeptide mitogen for endothelial cells and fibroblasts. The mitogenic effects of ECGF are inhibited by the lymphokine gamma-interferon (gamma-IFN) in a dose-dependent manner. Gamma-IFN also induces a unique change in endothelial cell morphology which is maximally expressed in the presence of ECGF. The antiproliferative and phenotypic modulatory effects of gamma-IFN on endothelial cells are reversible. Inhibition of ECGF-induced endothelial cell proliferation by gamma-IFN is accompanied by a concentration- and time-dependent decrease in binding of 125I-ECGF to the endothelial cell surface. Scatchard analyses of the binding data in the presence and absence of gamma-IFN demonstrate a decrease in the number of ECGF-binding sites rather than a decrease in ligand affinity for the receptor. Cross-linking experiments with disuccinimidyl suberate demonstrate a decrease in the 170,000 Mr cross-linked receptor-ligand complex. These data suggest that gamma-IFN inhibits endothelial cell proliferation by a mechanism which involves growth factor receptor modulation.

Cell Division↗

PubMed1986

Site-directed labeling of a monoclonal antibody: targeting to a disulfide bond.

We have designed and synthesized crabescein, the first member of a class of fluorescent labels that add across disulfide bonds. Crabescein is a fluorescein derivative that reports the rotational correlation time of the immunoglobulin G (IgG) segment to which it is covalently bound. Chemical analysis of the IgG labeled with crabescein indicates that the fluorophore is inserted into the third disulfide bond (cysteine-229 of mouse IgG2a) in the hinge region. The rotational correlation time of this labeled macromolecule was measured as a single exponential with a decay constant of 26.8 ns. This is in contrast to the double exponential with decay constants of 14.3 and 0.2 ns for the same IgG when labeled with fluorescein via a conventional labeling reagent in which the probe is bound to the macromolecule by one-point attachments. Thus, crabescein is the prototype of a class of fluorescent and phosphorescent probes that, by virtue of their two-point attachments to proteins, faithfully report on the dynamics of the segment of macromolecule to which they are covalently bound.

Animals↗

PubMed1986

Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion.

We have compared the molecular specificities of the adhesive interactions of melanoma and fibroblastic cells with fibronectin. Several striking differences were found in the sensitivity of the two cell types to inhibition by a series of synthetic peptides modeled on the Arg-Gly-Asp-Ser (RGDS) tetrapeptide adhesion signal. Further evidence for differences between the melanoma and fibroblastic cell adhesion systems was obtained by examining adhesion to proteolytic fragments of fibronectin. Fibroblastic BHK cells spread readily on fl3, a 75-kD fragment representing the RGDS-containing, "cell-binding" domain of fibronectin, but B16-F10 melanoma cells could not. The melanoma cells were able to spread instead on f9, a 113-kD fragment derived from the large subunit of fibronectin that contains at least part of the type III connecting segment difference region (or "V" region); f7, a fragment from the small fibronectin subunit that lacks this alternatively spliced polypeptide was inactive. Monoclonal antibody and fl3 inhibition experiments confirmed the inability of the melanoma cells to use the RGDS sequence; neither molecule affected melanoma cell spreading, but both completely abrogated fibroblast adhesion. By systematic analysis of a series of six overlapping synthetic peptides spanning the entire type III connecting segment, a novel attachment site was identified in a peptide near the COOH-terminus of this region. The tetrapeptide sequence Arg-Glu-Asp-Val (REDV), which is somewhat related to RGDS, was present in this peptide in a highly hydrophilic region of the type III connecting segment. REDV appeared to be functionally important, since this synthetic tetrapeptide was inhibitory for melanoma cell adhesion to fibronectin but was inactive for fibroblastic cell adhesion. REDV therefore represents a novel adhesive recognition signal in fibronectin that possesses cell type specificity. These results suggest that, for some cell types, regulation of the adhesion-promoting activity of fibronectin may occur by alternative mRNA splicing.

Amino Acid Sequence↗

PubMed1985

Crystallographic structure of an active, sequence-engineered ribonuclease.

X-ray diffraction methods were used to test a synthetic-modeling approach to the sequence engineering of bovine pancreatic ribonuclease. A model of RNase S-peptide (residues 1-20), having a simplified amino acid sequence but retaining elements deduced to be essential for conformation and function, was previously synthesized and found to form a catalytically active and stable complex with native S-protein (residues 21-24). We have now obtained a 3-A-resolution electron density map of this semisynthetic complex which reveals that the conformation of model peptide closely mimics that of native S-peptide, as intended by sequence design. Some small differences from the native structure are observed: Glu-2 and Arg-10 of the model complex are not close enough to form a salt bridge, the position of the His-12 imidazole ring is slightly shifted in the active site, and the peptide's amino terminus is reoriented. Nonetheless, the major structural features predicted to be essential by computer-aided peptide-design analysis are preserved in the model peptide portion of the complex. These include (i) the alpha-helical framework involving residues 3-13, (ii) the catalytically competent orientation of His-12, and (iii) complex-stabilizing non-bonding interactions involving Phe-8 and Met-13 of S-peptide and hydrophobic residues in the cleft region of S-protein. Further, sequence simplification has not introduced any non-native, potentially stabilizing contacts between the model peptide and S-protein. The results emphasize the usefulness, in redesigning native proteins, of categorizing sequence into residues providing conformational framework and those determining intra-and intermolecular surface recognition.

Amino Acid Sequence↗

PubMed1979

Local interactions as a structure determinant for protein molecules: II.

Van der Waals interactions between sidechains are indicated to be important in determining the native state of the proteins of known structure by the following observations: 1. the average radial distribution of polarity increases continuously from the center of the molecule to its periphery. 2. nonpolar sidechains tend to occur in clusters. 3. the frequencies of long-range nearest-neighbor pairs are markedly non-random; each type of sidechain seeks nearest-neighbors of similar polarity. To investigate how these interactions affect the overall structure of the protein molecule, three simplified models are treated: a sheath-core model composed of independent residues, a modification accounting approximately for the connected nature of the chain, and a model consisting of three concentric spherical phases.

Amino Acids↗

PubMed1979

Local interactions as a structure determinant for protein molecules: III.

Investigation of the known protein structures has led to the generalization that the native folding permits each sidechain to select those nearest-neighbors which maximize stabilization from van der Waals interactions. With regard to secondary structure: 1. Helical and beta regions exhibit characteristic patterns of short-range contacts (residue numbers k and k + t with [t] less than or equal to 4) due to the geometries of these secondary structures. However, these are not strictly obligatory, and preferred short-range contacts which would result in unfavorable van der Waals interactions are replaced by favorable long-range contacts. 2. The generalization mentioned at the outset holds for individual proteins, both for short-range and long-range contacts, and without regard for the type or amount of secondary structure present. 3. These observations imply that van der Waals interactions arising from short-range contacts partially determine secondary structure, and this is demonstrated by tests based upon assignment of regions of secondary structure in the known proteins. The principle of optimizing van der Waals stabilization from long-range contacts is applied to predict the structure of the complex formed by the S-peptide and S-protein of ribonuclease-S. The formation of favorable pairs is found to be more important than the total number of intermolecular contacts, and 40 to 50% of this stabilization is contributed by two residues of the S-peptide, Phe-8 and Met-13.

Computers↗

PubMedDate not supplied

A structure function study of C-terminal extensions of bombesin.

Synthetic C-terminal extensions of BN were synthesized and the biological potency evaluated using Swiss 3T3 and small cell lung cancer cells. BN, which has an amidated C-terminal, inhibited specific [125I-Tyr4]BN binding activity to Swiss 3T3 cells with an IC50 value of 1 nM, whereas the IC50 of BN-OH, which has a free C-terminal, was 1800 nM. The IC50 values of BNG, BNGK and BNGKK were 1400, 4700 and 500 nM, respectively. Similar binding data were obtained using SCLC cell line NCI-H345 and the bombesin analogues functioned as agonists based on the ability to elevate cytosolic Ca2+ in Fura-2 AM loaded SCLC cells. Also, the bombesin analogues stimulated 3H-thymidine uptake in Swiss 3T3 cells and the ED50 values for BN, BNG, BNGK and BNGKK were 1, 1300, 3900 and 400 nM. These data suggest that an amidated C-terminal is essential for high affinity binding and potency of BN.

3T3 Cells↗