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C G Knight

Publications and source records attributed to C G Knight.

At least 55 records · Page 3Linked to original sources
PubMed1993

The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays.

Two endopeptidases are present in the seeds of Vigna aconitifolia (moth bean), and their activities increase during germination. One enzyme, which we term "vignain," can be assayed with benzyloxycarbonyl-phenylalanyl-arginyl-7-(4-methyl)coumarylamide as substrate. The second is legumain (EC 3.4.22.34), which can be assayed with benzyloxycarbonyl-alanyl-alanyl-asparaginyl-7-(4-methyl)-coumarylamide. The enzymes were purified, and their specificities for substrates and inhibitors were examined. Vignain has properties expected of a cysteine endopeptidase of the papain family, with the exception of a remarkably low reactivity with iodoacetate. Legumain is a very atypical cysteine endopeptidase, being insensitive to inhibition by chicken cystatin and E-64 (L-3-carboxy-2,3-trans-epoxypropionyl-leucyl-amido(4-guanidino )butane), and reacting more rapidly with iodoacetamide than with iodoacetate. We discuss our findings in relation to the literature on the proteolytic enzymes of legume seeds.

Amino Acid Sequence↗
PubMed1992

A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases.

(7-methoxycoumarin-4-yl)Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L- 2,3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly fluorescent 7-methoxycoumarin group is efficiently quenched by energy transfer to the 2,4-dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3.4.24.23) cleaves the substrate at the Gly-Leu bond with a 190-fold increase in fluorescence (lambda cx 328 nm, lambda cm 393 nm). In assays of the human matrix metalloproteinases. Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 is about 50 to 100 times more sensitive than dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (kcat/Km) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.

Amino Acid Sequence↗
PubMed1992

CA074 methyl ester: a proinhibitor for intracellular cathepsin B.

The specificity of compound CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-pro line] for the inactivation of cathepsin B was quantified in in vitro measurements with cysteine endopeptidases from cattle, it being found that the compound is a very rapid inactivator of cathepsin B (rate constant 112,000 M-1.s-1), with barely detectable action on cathepsins H, L, and S or m-calpain. Conversion of the proline carboxyl group of the inhibitor to the methyl ester virtually abolished the effect on cathepsin B, and a possible explanation for the importance of the carboxyl is presented on the basis of the tertiary structure of cathepsin B. It was found that CA074 methyl ester (1 microM, 3 h) caused selective inactivation of the intracellular cathepsin B of human gingival fibroblasts in culture, in contrast to other available agents, and we suggest that CA074 methyl ester will be of value in the elucidation of the biological functions of cathepsin B.

Binding Sites↗
PubMed1992

The use of a chelating derivative of alpha melanocyte stimulating hormone for the clinical imaging of malignant melanoma.

A novel chelating derivative of alpha melanocyte stimulating hormone, bis MSH-DTPA, has been used for the diagnostic targeting of malignant melanoma. 15 patients were investigated of whom nine were shown by other means to have active disease at the time of the scan. Tumours were imaged in all of these nine patients. Of a total of 46 lesions over 10 mm encountered, 41 (89%) were imaged. There were no false positives and in two cases bisMSH-DTPA was instrumental in reversing diagnoses made using ultrasound. Derivatives of melanocyte stimulating hormone may be of considerable value in targeting melanomas.

Adult↗
PubMed1991

Structure/function relationships in the inhibition of thimet oligopeptidase by carboxyphenylpropyl-peptides.

Some novel N-[1(RS)-carboxy-3-phenylpropyl]tripeptide p-aminobenzoates have been synthesised as inhibitors of thimet oligopeptidase (EC 3.4.24.15). These compounds are considered to bind as substrate analogues with the Cpp group in S1 and the peptide portion in the S' sites. The most potent inhibitor is Cpp-Ala-Pro-Phe-pAb, which has a Ki = 7 nM. Substitution of Gly for Ala at P1' leads to weaker binding which can be ascribed to increased rotational freedom. Good substrates often have Pro at P2' and Pro is favoured over Ala at this position in the inhibitors, too. When P2' is Pro, Phe is preferred over Tyr and Trp in P3'. The p-aminobenzoate group makes an important contribution to the binding, probably by forming a salt bridge, and removal of the C-terminal negative charge results in much less potent inhibitors.

Alanine↗
PubMed1991

A quenched fluorescent substrate for thimet peptidase containing a new fluorescent amino acid, DL-2-amino-3-(7-methoxy-4-coumaryl)propionic acid.

DL-2-Amino-3-(7-methoxy-4-coumaryl)propionic acid, a new fluorescent amino acid (abbreviated to Amp), has been synthesized to provide an alternative to tryptophan in quenched fluorescent peptide substrates for peptidases. The model compound Ac-DL-Amp-NH2 was intensely fluorescent with an excitation maximum at 328 nm and an emission maximum at 392 nm. Fmoc (fluoren-9-ylmethoxycarbonyl)-DL-Amp was made to allow the solid-phase synthesis of Amp-containing peptides by the Fmoc-polyamide method. The peptide derivative Dnp (2,4-dinitrophenyl)-Pro-Leu-Gly-Pro-DL-Amp-D-Lys was cleaved by thimet peptidase at the Leu-Gly bond, with a 20-fold enhancement of fluorescence. The value of kcat./Km for thimet peptidase was 6.7 x 10(5) M-1.s-1, compared with the value of 2.4 x 10(5) M-1.s-1 for the tryptophan-containing analogue, Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys.

Alanine↗
PubMed1990

An alternative quenched fluorescence substrate for Pz-peptidase.

7-Methoxycoumarin-3-carboxylyl-Pro-Leu-Gly-Pro-D-Lys(2,4-dinitr oph enyl) is introduced as a new quenched fluorescence substrate for assaying Pz-peptidase (also known as soluble metallo-endopeptidase and endo-oligopeptidase). The value of Km for partially purified Pz-peptidase from rat muscle was 8.6 microM. High protein concentrations did not interfere with the assay, so that for the first time continuous assays of Pz-peptidase in crude tissue extracts became possible.

Amino Acid Sequence↗
PubMed1990

A chelating derivative of alpha-melanocyte stimulating hormone as a potential imaging agent for malignant melanoma.

A chelating derivative of alpha-melanocyte stimulating hormone (MSH) has been synthesised, in which two molecules of the hormone are cross-linked by diethylenetriamine pentaacetic acid (DTPA). This compound, bisMSH-DTPA, was equipotent with MSH in an in vitro tyrosinase assay with Cloudman S91 melanoma cells. When DBA/2 mice bearing the same tumour were injected with bisMSH-DTPA labelled with the gamma-emitting isotope indium-111 (111In), the radioactivity became rapidly associated with the melanoma tissue. By 24 h post-injection, radioactivity in tumour tissue was significantly higher (P less than 0.001) than in spleen, lung, brain, eye and skin. Uptake of radioactivity by the tumours was inhibited by a 200-fold molar excess of MSH, whereas uptake by liver, kidney, spleen, lung, brain, eye and skin was unaffected. We conclude that bisMSH-DTPA may offer an alternative to antibody targeting in the imaging of malignant melanoma.

Animals↗
PubMed1989

A continuous fluorimetric assay for clostridial collagenase and Pz-peptidase activity.

The peptide derivative N alpha-(2,4-dinitrophenyl)-L-prolyl-L-leucyl-glycyl-L-prolyl-L-tryptophanyl-D- lysine (Dnp-Pro-Leu-Gly-Pro-Trp-D-Lys) has been found to be a convenient substrate for the assay of clostridial collagenase and Pz-peptidase. The substrate shows a 25-fold enhancement of fluorescence (gamma ex. 283 nm, lambda em. 350 nm) following hydrolysis of the Leu2-Gly3 peptide bond. The value of Km for clostridial collagenase was 17 microM. The substrate for the first time makes possible continuous fluorimetric assays for Pz-peptidase and clostridial collagenase.

Clostridium↗
PubMed1989

Xanthene-dye-labelled phosphatidylethanolamines as probes of interfacial pH. Studies in phospholipid vesicles.

We have been developing the use of plasma-membrane-bound fluorescent probes to measure the pH values at the surfaces of living chondrocytes. For this purpose, three lipophilic pH indicators were made by covalently binding the xanthene dyes fluorescein, eosin or dichlorofluorescein to the amino group of phosphatidylethanolamine. The probes were incorporated into phospholipid vesicles and the effect of pH on the fluorescence was characterized. Fluorescence was measured at a single emission wavelength during excitation at two wavelengths, and the ratio of the intensities was calculated. The experimentally observed pKobs. values were determined by fitting the fluorescence ratios to the Henderson-Hasselbalch equation. All three probes acted as pH indicators, and the eosinyl-, dichlorofluoresceinyl- and fluoresceinylphosphatidylethanolamines had pKobs. values of 3.5, 6.3 and 7.5 respectively. At physiological salt concentrations, changes in the composition of the vesicle membrane had little effect on these values. We concluded that these probes were promising candidates for the measurement of pH values at cell surfaces.

Cell Membrane↗
PubMed1988

Antibodies to a short synthetic peptide cross-react with human recombinant interleukin 1 alpha.

An antiserum to human interleukin 1 alpha has been prepared by immunizing a sheep with a short synthetic peptide (Mr 1427) conjugated to keyhole limpet haemocyanin using the heterobifunctional cross-linking agent N-succinimidyl bromoacetate. The peptide was selected from a highly hydrophilic region corresponding to residues 169-179 of the cDNA-derived sequence. Two additional peptides corresponding to residues 194-207 and 224-233 failed to elicit cross-reacting antibodies.

Animals↗
PubMed1988

Assessing the preoperative adult.

A patient who is about to undergo surgery requires a presurgery evaluation that focuses on certain risks, including the risk of anesthesia. Even the generally healthy, ambulatory patient who is scheduled for an elective procedure should have a focused history and physical in order to anticipate and prevent complications. This article outlines the questions necessary to ask, the physical examination to be performed and the tests to be ordered during a preanesthesia assessment.

Adult↗
PubMed1986

Cortisol succinate is slowly hydrolysed in organ cultures of porcine articular tissues and is not equivalent to cortisol.

The pro-drug cortisol succinate is frequently used as a substitute for cortisol in organ cultures. We found, however, that in Dulbecco's modified Eagle's medium the time taken for the ester to undergo 50 per cent hydrolysis (t1/2) to cortisol was 75 h. When 15 per cent heat-inactivated normal rabbit serum was present t1/2 decreased to 47 h, but the rate of hydrolysis was not further increased in the presence of porcine articular cartilage or minced synovial tissue. When frozen and thawed synovium was present t1/2 decreased to 33 h, presumably due to the release of carboxyl-esterases from the disrupted cells. The level of tetrahydrocortisol was low in all of the cultures. The slow hydrolysis of cortisol succinate resulted in the exposure of the tissues to undesirable fluctuations in the concentration of active hormone, which decreased to low levels at each medium change. Thus, in co-cultures of porcine synovium and articular cartilage, cortisol had a greater inhibitory effect than cortisol succinate on the breakdown of cartilage matrix caused by synovial tissue.

Animals↗