Biomedical subjects

G B Fields

Publications and source records attributed to G B Fields.

64 records · Page 4Linked to original sources

PubMed1991

Sequence specificities of human fibroblast and neutrophil collagenases.

The sequence specificities of human fibroblast and neutrophil collagenases have been investigated by measuring the rate of hydrolysis of 60 synthetic oligopeptides covering the P4 through P'5 subsites of the substrate. The choice of peptides was patterned after both known cleavage sites in noncollagenous proteins and potential cleavage sites (those containing Gly-Ile-Ala, Gly-Leu-Ala, or Gly-Ile-Leu sequences) found in types I, II, III, and IV collagens. The initial rate of hydrolysis of the P1-P'1 bond of each peptide has been measured under first-order conditions ([SO] much less than KM), and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P4 through P'4 all influence the hydrolysis rates for both collagenases. However, the effects of substitutions at each site are distinctive and are consistent with the view that human fibroblast and neutrophil collagenases are homologous but nonidentical enzymes. For peptides with unblocked NH2 and COOH termini, occupancy of subsites P3 through P'3 is necessary for rapid hydrolysis. Compared with the alpha 1(I) cleavage sequence, none of the substitutions investigated at subsites P3, P2, and P'4 produces markedly improved substrates. In contrast, many substitutions at subsites P1, P'1, and P'2 improve specificity. The preferences of both collagenases for alanine in subsite P1 and tryptophan or phenylalanine in subsite P'2, is noteworthy. Human neutrophil collagenase accommodates aromatic residues in subsite P'1 much better than human fibroblast collagenase. The subsite preferences observed for human fibroblast collagenase in these studies agree well with the residues found at cleavage sites in noncollagenous substrates. However, the sequence specificities of these collagenases cannot explain the failure of these enzymes to hydrolyze many potentially cleavable but apparently protected sites in intact collagens. This represents additional support for the notion that the local structure of collagen is important in determining the location of collagenase cleavage sites.

Amino Acid Sequence↗

PubMed1990

Proteolytic activities of human fibroblast collagenase: hydrolysis of a broad range of substrates at a single active site.

The action of human fibroblast collagenase (HFC) on six substrates of markedly different size, sequence, and conformation, including rat type I collagen, rat alpha 1(I) gelatin, beta-casein, and the three synthetic oligopeptides Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln, Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-Pro-Gly, and Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln, has been examined. The first peptide is a model for the collagenase cleavage site in the alpha 1(I) chain of type I collagen, while the latter two peptides are models for the autolytic activation and degradation sites in pro-HFC, respectively. The goal of these studies was to assess whether HFC hydrolyzes all of these disparate substrates at the same active site. Individual kinetic parameters for the hydrolysis of all six substrates have been determined. Gel zymography experiments using collagen, gelatin, and casein as substrates show that all three activities are associated solely with HFC rather than impurities. Recombinant HFC expressed in Escherichia coli also exhibits caseinase activity, reinforcing the view that this activity is not due to a contaminating protease from fibroblasts. The ratios of these activities agree within experimental error for several independent HFC preparations and do not change when two additional affinity purification steps are employed. The inhibition of the hydrolysis of these substrates by both 1,10-phenanthroline and Boc-Pro-Leu-Gly-NHOH is identical within experimental error. A series of assays carried out in the presence of pairs of these substrates clearly shows that they compete for the same active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids.

9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.

Amino Acids↗

PubMed1990

A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis.

The success of solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl (Fmoc) amino acids is often limited by deleterious side reactions which occur during TFA peptide-resin cleavage and side-chain deprotection. The majority of these side reactions modify susceptible residues, such as Trp, Tyr, Met, and Cys, with TFA-liberated side-chain protecting groups and linkers. The purpose of this study was to assess the relative effectiveness of various scavengers in suppressing these side reactions. We found that the cleavage mixture 82.5% TFA : 5% phenol : 5% H2O : 5% thioanisole : 2.5% EDT (Reagent K) was maximally efficient in inhibiting a great variety of side reactions. Synthesis and cleavage of 10 peptides, each containing 20-50 residues, demonstrated the complementarity of Fmoc chemistry with Reagent K for efficient synthesis of complex peptides.

Amino Acid Sequence↗

PubMed1989

Solid phase peptide synthesis of 15N-gramicidins A, B, and C and high performance liquid chromatographic purification.

Four single-site 15N-labeled molecules of gramicidin have been synthesized using the 9-fluorenylmethoxycarbonyl method of solid phase peptide synthesis. Formylvaline was coupled as the N-terminal amino acid, and the peptide was cleaved from the resin with ethanolamine. Each synthesized gramicidin was purified in one step by semipreparative reverse phase high performance liquid chromatography and obtained in overall yields as high as 86%. The peptide was characterized by comparison with natural gramicidin using amino acid analysis, u.v. spectroscopy, and analytical high performance liquid chromatography.

Chromatography, High Pressure Liquid↗

PubMed1988

Solid-phase peptide synthesis and solid-state NMR spectroscopy of [Ala3-15N][Val1]gramicidin A.

[Ala3-15N][Val1]Gramicidin A has been prepared by solid-phase peptide synthesis and studied by solid-state 15N nuclear magnetic resonance spectroscopy. The synthesis of desformyl[Ala3-15N][Val1]gramicidin A employed N-hydroxysuccinimide esters of 9-fluorenylmethoxycarbonyl-N alpha-amino acids and completely avoided the use of acid. Since deblocking was done with piperidine and the peptide was removed from the resin by treatment with ethanolamine, this synthetic protocol prevented oxidation of the indole rings of this tryptophan-rich peptide and reduced truncations produced by acid hydrolysis. After formylation and purification by anion-exchange and high-pressure liquid chromatography, the peptide was obtained in an overall yield of 30%. Solid-state 15N nuclear magnetic resonance spectra of this peptide and uniformly labeled [15N]gramicidin A' oriented in hydrated lipid bilayers have been obtained, allowing unambiguous assignment of the [15N]Ala3 resonance in the latter. The solid-state 15N nuclear magnetic resonance experiments provide evidence that [Val1]gramicidin A is rotating about an axis that is perpendicular to the plane of the lipid bilayer and that the N--H axis is nearly parallel with the rotational axis. This study demonstrates that site-specifically labeled [15N]gramicidin A analogs prepared by solid-phase peptide synthesis are valuable tools in the study of the solid-state nuclear magnetic resonance spectra of samples in oriented lipid bilayers.

Amino Acid Sequence↗

PubMed1987

Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site.

The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.

Amino Acid Sequence↗

PubMedDate not supplied

Evaluation of radiolabeled type IV collagen fragments as potential tumor imaging agents.

The objective of this study was to examine radiopharmaceuticals that target the alpha3beta1 integrin to determine if these agents target tumors for diagnostic imaging and/or targeted radiotherapy of cancer. Prior studies had shown that residues 531-542 from the alpha1 chain of type IV collagen bind a variety of tumor cell alpha3beta1 integrins. A peptide mimic of this sequence containing all D-amino acids (designated D-Hep-III) was synthesized by solid-phase methods. The tetraazamacrocyclic chelator, TETA, was conjugated to the peptide while it was resin-bound. TETA-D-Hep-III and D-Hep-III were radiolabeled with 64Cu and 125I, respectively, in high specific activity and radiochemical purity. Heterologous competitive binding assays between D-Hep-III and either 125I-D-Hep-III or 64Cu-TETA-D-Hep-III indicated low micromolar affinity of D-Hep-III. The biodistribution of each radiolabeled analogue of D-Hep-III was carried out in rats and tumor-bearing mice. Both analogues were rapidly cleared from the blood in normal rats, with the kidneys receiving the highest accumulation of each. SKOV3 human ovarian tumor cells, known to strongly express alpha3beta1, were xenografted in SCID mice. Localization of 125I-D-Hep III and 64Cu-TETA-D-Hep III in the xenografts were low (<2% ID/g), and in the case of 125I-D-Hep III, not inhibited by a competitive dose of D-Hep III. The low tumor accumulation is likely not due to receptor down-regulation, but rather due to the weak affinity of the radioligands for the alpha3beta1 integrin.

Animals↗

PubMedDate not supplied

Synthetic peptides and tumor cell metastasis.

Synthetic peptides are used presently to screen a great variety of biological activities, including the adhesion, spreading and motility of metastatic tumor cells. By "mapping" extracellular matrix protein sequences, peptides have been identified that have discreet tumor cell activities such as inhibiting in vivo lung metastasis. These peptides are often found to have distinct secondary structures (beta-turns, beta-sheets, alpha-helices). Some sequences have been incorporated into tertiary structures (triple-helices, timeric alpha-helical coils) that enhance cellular activities. By combining information on the primary, secondary and tertiary structures of these peptides with the nature of cell-surface adhesion molecules, the processes by which tumor cells adhere, spread and invade can be better understood.

Amino Acid Sequence↗

PubMedDate not supplied

Edman degradation sequence analysis of resin-bound peptides synthesized by 9-fluorenylmethoxycarbonyl chemistry.

The efficacy of Edman degradation sequence analysis for evaluating the synthetic efficiency of peptide-resin assembly by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase methodology has been studied. Prior researchers have described the use of solid-phase "preview" sequence analysis for peptides synthesized by tertiary-butyloxycarbonyl (Boc) chemistry, where benzyl-based side-chain protecting groups and peptide-resin linkers are stable to the conditions of Edman chemistry. We have successfully sequenced a variety of resin-bound peptides synthesized by Fmoc chemistry, where tertiary-butyl-based side-chain protecting groups and peptide-resin linkers are labile to the conditions of Edman chemistry. Crude peptides are liberated from trifluoroacetic acid-labile linkers during the first cycle of Edman degradation and subsequently "embedded" in membranes. For peptides up to 20 residues, embedded sequencing repetitive yields were comparable to those of solid-phase sequencing. Preview sequencing of resin-bound Fmoc-synthesized peptides proved to be advantageous compared to other analytical methods, in that synthetic failures were detected and quantitated at the point of occurrence, regardless of whether incomplete Fmoc deprotection or incomplete coupling was responsible, and without interference from by-products formed during peptide-resin cleavage. Quantitative ninhydrin analysis, which previously has been found to give false positive results due to removal of the Fmoc group by a combination of reagents and high temperature, gave false negative results in this study, most probably due to incomplete removal of the Fmoc group prior to coupling. Quantitative sequence analysis results were supported by high-performance liquid chromatographic, amino acid and electrospray mass spectrometric analyses of the crude and purified peptides.

Amino Acid Sequence↗