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G B Fields

Publications and source records attributed to G B Fields.

At least 55 records · Page 3Linked to original sources
PubMed1995

Purification and analysis of synthetic, triple-helical "minicollagens" by reversed-phase high-performance liquid chromatography.

To better study collagen-mediated cellular and enzymatic activities, a generally applicable solid-phase methodology has been developed by which aligned triple-helical peptides (designated THPs or "minicollagens") ranging from 79 to 124 residues can be assembled. Reversed-phase HPLC is typically the purification method of choice following chemical synthesis of small proteins of this size, as well as one of the analytical techniques used to verify product purity. We have thus compared the effects of different stationary phases (C18, C4, or diphenyl), organic modifiers (acetonitrile or isopropanol), support pore sizes (120 angstroms, 300 angstroms, or nonporous), and counterions for the reversed-phase HPLC analysis of THPs. Large pore C18 or C4 reversed-phase HPLC gave broad peaks, resulting in poor resolution of the desired THP from synthetic impurities. Broad peaks were presumably due to conformational instability of THPs to reversed-phase conditions and subsequent slow cis-trans isomerization of the peptide bonds. Peak sharpness was improved greatly by use large-pore diphenyl reversed-phase HPCL. We found that THPs can be best resolved from synthetic impurities by diphenyl or non-porous C18 reversed-phase HPLC using water-acetonitrile gradients. These results most likely reflect conditions which maintain the native conformation of collagen-like triple-helices.

Amino Acid Sequence↗
PubMed1995

The collagen triple-helix: correlation of conformation with biological activities.

Collagens are distinguished from other extracellular matrix proteins by their triple-helical conformation. Triple-helical conformation has been proposed to be important for cellular activities, such as adhesion and activation, extracellular matrix assembly, and enzyme function, such as hydroxylation of collagen Lys and Pro residues and matrix metalloproteinase (MMP) catabolism of native collagens. A collagen-like triple-helix is also a necessary component of several macrophage cell surface receptors. Collagen-mediated cellular and/or enzymatic activities that (i) require an intact triple-helix, (ii) require a denatured triple-helix, or (iii) are "conformationally independent" of the state of the triple-helix have been documented. Recently developed synthetic protocols have allowed for the study of biological activities of specific collagen sequences in triple-helical conformation.

Amino Acids↗
PubMed1994

Promotion of cell adhesion by single-stranded and triple-helical peptide models of basement membrane collagen alpha 1(IV)531-543. Evidence for conformationally dependent and conformationally independent type IV collagen cell adhesion sites.

Several regions within the triple-helical domain of type IV collagen function as cellular recognition sites. We have demonstrated previously that melanoma cell activities promoted by the alpha 1(IV)1263-1277 sequence are enhanced by triple helicity (Fields, C. G., Mickelson, D. J., Drake, S.L., McCarthy, J.B., and Fields, G.B. (1993) J. Biol. Chem. 268, 14153-14160), whereas Eble et al. reached similar conclusions for alpha 1 beta 1 integrin-mediated fibrosarcoma cell adhesion to [alpha 1(IV)]2 alpha 2(IV)434-472 (Eble, J. A., Golbik, R., Mann, K., and Kühn, K. (1993) EMBO J. 12, 4795-4802). In the present study, we have examined the cell adhesion activities of a third region in type IV collagen. A single-stranded peptide (SSP) incorporating the alpha 1(IV)531-543 sequence promoted the adhesion of melanoma, ovarian carcinoma, and Jurkat cells in a dose-dependent manner, with 40% cell adhesion observed at [SSP] = 1.8, 11.5, and 42.2 microM, respectively. Nearly identical results were obtained for cell adhesion to an all-D-enantiomer of the SSP, suggesting that the cell surface receptor(s) for this site do not discriminate based on chirality. The alpha 1(IV)531-543 sequence maintained its cell adhesion promoting activity when incorporated into a homotrimeric triple-helical polypeptide, although relative levels of adhesion were either slightly enhanced or slightly diminished compared with the SSP. Triple-helical conformation was thus not critical for cellular recognition of the alpha 1(IV)531-543 sequence. Single-site substitution experiments of the SSP showed no overall correlation between the biological effects of substitutions and SSP conformation. The SSP, D-SSP, triple-helical polypeptide, and SSP substitution results suggest that cell recognition of the alpha 1(IV)531-543 sequence is generally independent of substrate conformation. The present and prior studies indicate that "conformationally dependent" and "conformationally independent" cellular recognition sites exist within the triple-helical domain of type IV collagen.

Amino Acid Sequence↗
PubMed1994

Generally applicable, convenient solid-phase synthesis and receptor affinities of octreotide analogs.

Octreotide, an analogue of the hormone somatostatin, has applications as a therapeutic and imaging agent for somatostatin-positive tumors. We have developed a generally applicable, convenient stepwise solid-phase synthetic protocol for octreotide (D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-threoninol). [Cys(Acm)2,D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6,Cys(Acm)7, des(threoninol)]-octreotide was assembled by Fmoc solid-phase synthesis and the intramolecular disulfide bond formed by treatment of the resin-bound peptide with thallium trifluoroacetate [Tl(Tfa)3]. Side-chain protection of Trp by the Boc group was found to preserve Trp integrity during Tl(Tfa)3 treatment. The protected peptide was cleaved from the resin by aminolysis with threoninol and purified by semipreparative RP-HPLC. Isolated [D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6]octreotide had the correct molecular mass ([M+H]+ = 1275 Da) and sequence and was obtained in 14% yield at > 98% purity. [D-Trp(Boc)4,Lys(Boc)5,Thr(tBu)6]octreotide was utilized for the solution-phase synthesis of CPTA-D-Phe1-octreotide, where CPTA is 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid. Cyclic dianhydride of diethylenetriaminepentaacetic acid (DTPA) was coupled to a portion of the protected peptide-resin following disulfide bond formation. The DTPA-conjugated, side-chain-protected peptide was cleaved from the resin by aminolysis with threoninol, side-chain deprotected with trifluoroacetic acid, and purified by semipreparative RP-HPLC. The isolated DTPA-D-Phe1-octreotide had the correct molecular mass ([M+H]+ = 1395 Da) and was obtained in 5% yield at > 90% purity. The efficiency of aminolysis was partially dependent upon the linkage between 4-(hydroxymethyl)phenoxy (HMP) handles and the resin and/or resin particle size. The somatostatin receptor binding affinities of synthetic DTPA-D-Phe1-octreotide and CPTA-D-Phe1-octreotide to AtT-20 mouse pituitary carcinoma cell membranes were examined by labeling with 111In and 64Cu, respectively, and performing Scatchard analyses. The dissociation constant (Kd) for our synthetic [111In]DTPA-D-Phe1-octreotide was 4.31 nM, which is comparable to a Kd = 5.57 nM obtained with commercially available DTPA-D-Phe1-octreotide. The Kd for [64Cu]CPTA-D-Phe1-octreotide was 78.5 pM. On the basis of the criteria of molecular mass, RP-HPLC elution time, sequence analysis, and somatostatin receptor binding affinity, our synthetic octreotide is identical to commercially available octreotide. The aminolysis protocol used here has distinct advantages over either reductive cleavage or preformed linker methods described previously for the preparation of octreotide.

Amines↗
PubMed1994

Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3).

Members of the matrix metalloproteinase (MMP) family have been implicated in disease states such as arthritis, periodontal disease, and tumor cell invasion and metastasis. Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. We examined known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) in order to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km approximately 11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation experiments were additive, we found discrepancies between predicted and experimental kcat/Km values, one on the order of 2000-5000. Thus, the design of additional discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.

Amino Acid Sequence↗
PubMed1994

Laminin peptides stimulate human neutrophil motility.

Laminin, isolated from Engelbreth-Holm-Swarm tumor, and 10 chemically synthesized peptides, corresponding to various regions of the laminin A and B1 chains, were compared for their abilities to stimulate human peripheral blood polymorphonuclear leukocyte (PMN) chemotaxis and chemokinesis through polycarbonate membrane filters in a 48-well microchemotaxis assay. Peptides F-9, F-11, F-12, and F-13 were derived from the B1 chain of laminin at the intersection of the cross, and six peptides were derived from the laminin A chain: peptide TG-1 from the amino-terminal top globule; peptides GD-1, GD-3, GD-6, and GD-7 from the carboxyl-terminal globular domain; and peptide AG-1 from above the carboxyl-terminal globular domain. Laminin and the peptides were evaluated over a concentration range of 1 to 200 micrograms/ml in motility assays. Six of the peptides, F-9, F-12, GD-1, GD-3, GD-6, and TG-1, stimulated human PMN migration in the absence of a gradient (chemokinesis). A fluorescein conjugate of the most active laminin peptide, GD-1, exhibited nonspecific, nonsaturable binding to PMN. Intact laminin and the other peptides failed to stimulate human PMN migration. In contrast, intact Engelbreth-Holm-Swarm laminin stimulated rabbit peripheral blood PMN chemokinesis. These results demonstrate that rabbit and human peripheral blood PMNs have divergent migratory responses to intact laminin. These findings suggest that intact basement membrane laminin does not directly stimulate human blood PMN motility in vivo, but that selected laminin peptide sequences, which may be generated during proteolytic digestion of laminin, can activate human PMN migration.

Amino Acid Sequence↗
PubMed1994

Promotion of human platelet adhesion and aggregation by a synthetic, triple-helical "mini-collagen".

Platelet activation and aggregation by fibrillar collagens are based on substrate primary, secondary, tertiary, and quaternary structure. Although several peptides incorporating sequences from the triple-helical domains of types I and III collagen inhibit collagen-mediated platelet aggregation, none independently promote platelet activation and aggregation. It is believed that the absence of these platelet activities is due to the lack of proper substrate tertiary and quaternary structures. We have utilized a synthetic, triple-helical "mini-collagen" that incorporates a known cell adhesion site (alpha 1(IV)1263-1277) to better understand the relationship between substrate primary, secondary, tertiary, and quaternary structure and platelet activation and aggregation. The promotion of platelet adhesion, activation, and aggregation was compared for this triple-helical polypeptide (THP), fibrillar and type IV collagens, and a single-stranded peptide (SSP) incorporating the alpha 1(IV)1263-1277 sequence. Glass-coated fibrillar and type IV collagens and the THP supported platelet adhesion at substrate concentrations of 0.33 nM, 0.20 nM, and 0.89 microM, respectively. When platelets were stirred with 10 micrograms/ml of fibrillar (0.33 nM) and type IV (0.20 nM) collagen, SSP (2.1 microM), and THP (0.89 microM), only the fibrillar collagen caused 3H-labeled arachidonic acid release, elevation of cytosolic calcium, irreversible aggregation, and secretion of granule contents. The THP (0.45-1.8 microM) effectively inhibited fibrillar collagen-mediated platelet aggregation, while the SSP did not. At a substrate concentration of 40 micrograms/ml, the THP (3.6 microM) was as effective as fibrillar collagen (1.3 nM) at inducing 3H-labeled oleic acid-labeled platelet activation and microaggregate formation, while the SSP and type IV collagen were relatively ineffective. Rotary shadowing images indicated that aggregates of the THP could form distinct quaternary structures, while the type IV collagen used here could not. These results are the first demonstrations of a synthetic peptide promoting platelet adhesion, activation, and aggregation and suggest that the combination of THP primary, secondary, tertiary, and quaternary structural features are required for platelet aggregation.

Adult↗
PubMed1993

Melanoma cell adhesion and spreading activities of a synthetic 124-residue triple-helical "mini-collagen".

A great variety of cells, such as melanoma cells, fibroblasts, platelets, keratinocytes, and epithelial cells, adhere to and migrate on specific regions within the triple-helical domains of types I, III, and IV collagen. The relative importance of collagen primary, secondary, and tertiary structures on these cellular activities has not been ascertained, as no general synthetic methodology exists to allow for the study of peptides incorporating biologically active sequences in triple-helical conformation. We have thus developed a novel, generally applicable solid-phase branching methodology for the synthesis of aligned, triple-helical collagen-model polypeptides (i.e. "mini-collagens"). Three nascent peptide chains are carboxyl-terminally linked through one N alpha-amino and two N epsilon-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. A homotrimeric triple-helical polypeptide (THP) of 124 amino acids, incorporating residues 1263-1277 of alpha 1 (IV) collagen, was synthesized. Highly metastatic mouse melanoma cells showed a profound preference for adhesion to this THP as compared with a single-stranded peptide (SSP) incorporating the same type IV collagen sequence or a branched peptide containing eight repeats of Gly-Pro-Hyp (designated GPP*). Specifically, 50% cell adhesion occurred at a THP concentration of 1.12 microM, while comparable levels of adhesion required [SSP] = 170 microM or [GPP*] > 100 microM. Melanoma cells also spread on the THP to a greater extent than on the SSP or GPP*. These results are the first direct demonstrations of the significance of triple helicity for cell adhesion to and spreading on a specific collagen sequence and support earlier conclusions of conformational dependency for cell adhesion to and migration on types I and IV collagen. In addition, the melanoma cell THP activities support the concept that tumor cell adhesion and spreading on type IV collagen involves multiple, distinct domains in triple-helical conformation. The triple-helical peptide synthetic protocol developed here will allow eventually for the study of both structure and biological activity of specific, glycosylated collagen sequences in homotrimeric and heterotrimeric forms.

Amino Acid Sequence↗
PubMed1993

Solid-phase synthesis and stability of triple-helical peptides incorporating native collagen sequences.

A generally applicable solid-phase methodology has been developed for the synthesis of triple-helical polypeptides incorporating native collagen sequences. Three nascent peptide chains are C-terminal linked through one N alpha-amino and two N epsilon-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. Different protecting group strategies, including several three-dimensionally orthogonal schemes, have been utilized for the synthesis of four homotrimeric triple-helical polypeptides (THPs) of 79-124 residues, three of which incorporate native type IV collagen sequences. Highly efficient assemblies were achieved by 9-fluorenylmethoxycarbonyl (Fmoc) N alpha-amino group protection, in situ 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate mediated couplings, and 1,8-diazabicyclo [5.4.0] undec-7-ene mediated Fmoc group removal. THPs were characterized by Edman degradation sequencing, size-exclusion chromatography, mass spectrometry, reversed-phase high performance liquid chromatography, and CD spectroscopy. THP thermal stabilities ranged from 35 to 59 degrees C, with chain length and Hyp content being the influential factors. Melting temperatures and van't Hoff enthalpies for peptide triple-helical denaturation could be correlated well to Hyp content. The THP synthetic protocol developed here will allow for the study of both structure and biological activity of specific collagen sequences in homotrimeric and heterotrimeric forms.

Amino Acid Sequence↗
PubMed1992

The development of high-performance liquid chromatographic analysis of allyl and allyloxycarbonyl side-chain-protected phenylthiohydantoin amino acids.

Ten phenylthiohydantoin (PTH) amino acids possessing allyl (Al) or allyloxycarbonyl (Aloc) side-chain-protecting groups have been characterized by high-performance liquid chromatography for use in Edman degradation sequence analysis. Optimized separation of side-chain-protected and -unprotected PTH amino acids was achieved on a C-18 reversed-phase column with a two-step gradient spanning 32 min. Five of the side-chain-protected amino acids [Cys(Al), Cys(Aloc), Lys(Aloc), Thr(Aloc), Tyr(Al)] were completely stable to the conditions of PTH derivatization, four [Asp(OAl), Arg(Aloc)2, Glu(OAl), Ser(Aloc)] were partially deprotected during PTH derivatization, and one [His(Aloc)] was completely deprotected during PTH derivatization. All allyl-based derivatives were well resolved from their side-chain-unprotected counterparts. Studies on the stability to piperidine treatment showed Asp(OAl), Cys(Al), Glu(OAl), Lys(Aloc), Thr(Aloc), and Tyr(Al), and possibly Arg(Aloc)2 and Ser(Aloc), to be suitable for peptide synthesis by 9-fluorenylmethoxycarbonyl (Fmoc)-based chemistry. Edman degradation of Al and Aloc side-chain-protected Conus geographus Lys9-alpha-conotoxin GI synthesized on 4-methylbenzhydrylamine-copoly(styrene-1%-DVB)-resin demonstrated the usefulness of these derivatives for solid-phase preview sequence analysis.

Amino Acid Sequence↗
PubMed1991

A model for interstitial collagen catabolism by mammalian collagenases.

Mammalian collagenases cleave all three alpha chains of native, triple-helical types I, II, and III collagens after the Gly residue of the partial sequence Gly-[Ile or Leu]-[Ala or Leu] at a single locus approximately three-fourths from the amino terminus. There are an additional 31 sites in the triple-helical regions of types I, II, III, and IV collagens that contain the same partial sequence but are not hydrolyzed. A model has been developed to explain this remarkable specificity. The mammalian collagenase cleavage site in interstitial collagens is distinguished by: (a) a low side-chain molal volume-, high imino acid (greater than 33%)-containing region that is tightly triple-helical, consisting of four Gly-X-Y triplets preceding the cleavage site, (b) a low imino acid-containing (less than 17%), loosely triple-helical region consisting of four Gly-X-Y triplets following the cleavage site, and (c) a maximum of one charged residue for the entire 25 residue cleavage site region, which is always an Arg that follows the cleavage site in subsite P'5 or P'8. In addition, the high imino acid-containing region cannot have an imino acid adjacent to the cleaved Gly-[Ile or Leu] bond (i.e. in subsite P2). Careful scrutiny of the 31 non-cleaved sequences reveals that none of those sites shares all of the characteristics of the cleavage site. The criterion of this model thus explain both cleaved and non-cleaved sequences in the triple-helical regions of types I, II, III, and IV collagen, and are supported by all known experimental and theoretical results on collagen catabolism and structure.

Amino Acid Sequence↗