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R J Collier

Publications and source records attributed to R J Collier.

At least 253 records · Page 14Linked to original sources
PubMed1980

Ligand interactions of diphtheria toxin. I. Binding and hydrolysis of NAD.

Prior studies showed that diphtheria toxin could be separated into ATP-binding and nonbinding fractions (Fractions II and I, respectively) by affinity chromatography on ATP-Sepharose (Lory, S., and Collier, R. J. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 267-271). Here we show that the two fractions also differ in their interactions with NAD. Fraction II bound a single molecule of NAD (Kd about 9 microM) as assayed by flow dialysis or fluorescence quenching and catalyzed both NAD-glycohydrolase and auto-ADP-ribosylation reactions. Fraction I was deficient in NAD-binding and NAD-related reactions. The ratio of the two fractions vried widely among toxin preparations and was independent of the proportion of toxin in the nicked state. Properties of th NAD site on Fraction II were similar to, but not identical with, those of the corresponding site on free Fragment A.

Binding Sites↗
PubMed1980

Ligand interactions of diphtheria toxin. II. Relationships between the NAD site and the P site.

Prior studies have described two functionally distinct ligand-binding sites on whole diphtheria toxin, the NAD site, which catalyzes the intracellular ADP-ribosylation reaction, and the P site, which affects toxin binding to sensitive cells. Occupancy of the P site by ATP or other phosphorylated compounds inhibits toxin attachment to cells. Here we show that binding of NAD site and P site ligands is competitive; and we characterize ligand-binding properties of two mutant forms of the toxin, CRM 45 and CRM 197. The data suggest that the NAD site, on the A moiety, lies immediately adjacent to the P site, formed by a strongly cationic region on the COOH-terminal half of B. The cationic character of the P site slightly alters the substrate specificity of the NAD site, and occupancy of either of the sites blocks ligand binding to the other. Possible roles of the P site in toxin attachment are discussed.

Adenosine Diphosphate Ribose↗
PubMed1980

Ligand interactions of diphtheria toxin. III. Direct photochemical cross-linking of ATP and NAD to toxin.

The locations of ATP- and NAD-binding sites on diphtheria toxin were investigated by ultraviolet irradiation of ligand . toxin complexes. Illumination of ATP with ultraviolet light (253.7 nm) in the presence of various proteins resulted in photoinduced cross-linking only with Fraction II of diphtheria toxin. Under the same conditions, NAD was cross-linked most effectively to Fragment A, followed by Fraction II and CRM 45. For both ATP and NAD, the degree of protein labelling correlated well with binding data, suggesting that photoinduced cross-linking ocurred only at the high affinity binding sites for these ligands. Nonspecific labeling of unrelated proteins was consistently less than 5% of that observed for Fraction II. Analysis of nicked and reduced Fraction II . ligand complexes on SDS polyacrylamide gels demonstrated that essentially all of the cross-linked label migrated with the A fragment, whether photolysis was performed with ATP or NAD.

Adenosine Triphosphate↗
PubMed1980

A hybrid toxin containing fragment A from diphtheria toxin linked to the B protomer of cholera toxin.

We have constructed and characterized a hybrid toxin containing the A chain of diphtheria toxin linked via a disulfide bridge to the B protomer of cholera toxin. Cholera toxin B protomer, previously derivatized with 4-5 cystaminyl groups per pentameric protomer, was reacted with reduced diphtheria toxin chain A to give the desired hybrid, containing an average of 2 molecules of diphtheria toxin chain A per cholera toxin B protomer. A concentration of 0.3 nM hybrid inhibited protein synthesis by 50% in 24 h in several cultured cell lines; thus the hybrid was about 10-fold more toxic than of a (diphtheria toxin chain A)-SS-(concanavalin A) conjugate described previously. Evidence was obtained that toxicity of the hybrid was dependent on the functional contributions of both the diphtheria toxin chain A and cholera toxin B protomer moieties.

Animals↗
PubMed1980

Epidermal growth factor-toxin A chain conjugates: EGF-ricin A is a potent toxin while EGF-diphtheria fragment A is nontoxic.

We have prepared a 2-pyridyl-dithiopropionate derivative of epidermal growth factor (EGF) and conjugated the derivative by disulfide interchange to the A chain of ricin (RTA) or to fragment A of diphtheria toxin (DTA). The EGF-RTA conjugate was toxic to 3T3 cells at concentrations (10(-9)--10(-11) M) similar to those at which EGF exerts its biological activity and within an order of magnitude of the toxicity of ricin. Ricin A chain alone only exerted toxic effects at concentrations (10(-6)--10(-7) M) three to four orders of magnitude higher than required for the activity of the EGF-RTA conjugate or ricin. An unconjugated mixture of RTA and EGF had no greater effect than RTA alone. Toxicity of the EGF-RTA conjugate on 3T3 cells was competed by EGF and was blocked by antibodies to RTA, but not by lactose or antibodies to the ricin B chain (RTB). In contrast to the EGF-RTA conjugate, the EGF-DTA conjugate proved virtually nontoxic at concentrations as high as 3 X 10(-8) M. Control experiments showed that the EGF-DTA conjugate retained EGF receptor binding activity; the DTA moiety of the hybrid retained ADP-ribosyltransferase activity; and the disulfide bridge linking DTA to EGF was readily reducible.

Animals↗
PubMed1980

Diphtheria toxin: nucleotide binding and toxin heterogeneity.

We have used flow dialysis to demonstrate binding of ATP and related compounds to diphtheria toxin. The results define a new site on the toxin molecule (the P site), which has distinctly different properties from the NAD+-binding site of the fragment A moiety. The relative affinities of various compounds for the P site are similar to their capacities to inhibit toxin attachment to cell surfaces and its action on cells. This suggests that the P site may correspond to the binding site for cell surface receptors. Affinity of nucleotides for the toxin depends strongly on the number of phosphates, although both nucleoside and phosphate moieties contribute to the interaction. A substantial fraction of the toxin in any given preparation did not bind ATP in a rapidly reversible manner and was not retained on ATP-Sepharose. This fraction, which varied in magnitude from preparation to preparation, was isolated and shown to contain an endogenous, firmly bound nucleotide or nucleotide-like compound. The presence of this compound may explain some of the physical heterogeneity within individual preparations of purified toxin as well as variations in physical and biological properties among various preparations.

Adenine Nucleotides↗
PubMed1980

Antibody-directed cytotoxic agents: use of monoclonal antibody to direct the action of toxin A chains to colorectal carcinoma cells.

We have constructed cell-specific cytotoxic agens by covalently coupling the A chain from diphtheria toxin or ricin toxin to monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen. Antibody 1083-17-1A was modified by attachment of 3-(2-pyridyldithio)propionyl or cystaminyl groups and then treated with reduced A chain to give disulfide-linked conjugates that retained the original binding specificity of the antibody moiety. the conjugates showed cytotoxic activity for colorectal carcinoma cells in culture, but were not toxic in the same concentration range for a variety of cell lines that lacked the antigen. Under defined conditions virtually 100% of antigen-bearing cultured cells were killed, whereas cells that lacked the antigen were not affected. Conjugates containing toxin A chains coupled to monoclonal antibodies may be useful in studying functions of various cell surface components and, possibly, as tumor-specific therapeutic agents.

Animals↗
PubMed1980

Expression of enzymic activity by exotoxin A from Pseudomonas aeruginosa.

Exotoxin A from Pseudomonas aeruginosa is a single polypeptide chain (M(r), 66,000) containing little if any adenosine 5'-diphosphate ribosyltransferase or oxidized nicotinamide adenine dinucleotide glycohydrolase activity. These activities have been demonstrated in the reduced intact toxin and in a peptide (M(r), 26,000) isolated from culture fluids or toxin preparations after storage. In this report we describe methods for generating enzymically active fragments by cleaving the fully or partially reduced exotoxin by proteolytic or chemical methods. Incubation of reduced toxin with chymotrypsin in the presence of oxidized nicotinamide adenine dinucleotide yielded an enzymically active peptide (M(r), 26,000) similar to the fragment characterized previously. Chemical cleavage by treatment of the reduced molecule with CNBr or 2-nitro-5-thiocyanobenzoate yielded fragments (M(r), 50,000 and 30,000, respectively) with similar activities. Also both adenosine 5'-diphosphate ribosyltransferase and oxidized nicotinamide adenine dinucleotide glycohydrolase activities were maximally expressed by the intact exotoxin after reduction of only two of its four disulfide bridges. Kinetic constants for activated whole toxin were similar to those of fragment A of diphtheria toxin. It is evident that in the native toxin the catalytic center is buried or distorted and that alterations in the covalent structure permit the center to become exposed or assume an active configuration. It is unknown whether reduction, proteolytic processing, or both occur during the course of toxin action on whole cells.

Carbon Radioisotopes↗
PubMed1980

A model system involving anti-concanavalin A for antibody targeting of diphtheria toxin fragment A1.

Results obtained in a model system strongly suggest that antibodies to cell surface determinants may be used to direct the toxic potential of the A chain of diphtheria toxin (DTA). The A chain (M.W. 21,000) was covalently attached to antibody against concanavalin A (anti-Con A) by means of a disulfide-containing cross-bridge. This DTA-SS-(anti-Con A) conjugate was toxic for 3T3 cells containing Con A on their surface but was not toxic in the same concentration range for: (a) cells lacking Con A; (b) Con A-treated cells washed with buffer containing alpha-methyl-D-mannoside; (c) cells containing wheat germ agglutinin on their surface; or (d) Con A-treated mutant Chinese hamster ovary cells containing altered, toxin-insensitive elongation factor 2. Conjugates containing DTA disulfide linked to anti-wheat germ agglutinin antibody or to nonspecific rabbit immunoglobulin G were not toxic for cells coated with Con A. The results suggest a new approach to the construction of antibody-directed, tumor-specific chemotherapeutic agents. Conjugates containing DTA disulfide linked to antibody against specific cell surface antigens may also be generally useful as specific selective agents for the isolation of mutant cell lines.

Animals↗
PubMed1979

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. I. The tryptic peptides from the maleylated protein.

Six tryptic peptides ranging in size from 3 to 126 residues were isolated from maleylated Fragment A of diphtheria toxin after tryptic hydrolysis. These peptides accounted for all 193 residues found by amino acid analysis. After demaleylation, the six peptides were purified by chromatography on Sephadex G-50, coupled with paper chromatography and electrophoresis, and were analyzed by various methods. The compositions and properties of the peptides are reported. Almost 70% of the residues were positioned within these peptides.

Adenosine Diphosphate Sugars↗
PubMed1979

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. II. The cyanogen bromide peptides.

Cyanogen bromide cleavage of Fragment A from diphtheria toxin at the four methionines present in each molecule resulted in five major peptides which were isolated and studied by sequence methods. These five peptides of 4, 11, 14, 63, and 101 residues account for all 193 residues in Fragment A and provide overlaps for the tryptic peptides from the maleylated protein. Two additional peptides were isolated and shown to be shorter forms (8 and 10 residues) of the COOH-terminal cyanogen bromide peptide (11 residues).

Adenosine Diphosphate Sugars↗
PubMed1979

The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. III. The chymotryptic peptides, the peptides derived by cleavage at tryptophan residues, and the complete sequence of the protein.

Fragment A (21,145 daltons in its longest known form) may be derived from diphtheria toxin (60,000 daltons) by mild tryptic digestion and reduction. Purified Fragment A consists of a mixture of 3 molecules of 190, 192, and 193 residues; the first 190 residues are in common and correspond to the NH2-terminal region the toxin. All three species of Fragment A are active in catalyzing ADP ribosylation of elongation factor 2, an essential component of protein synthesis. This reaction inactivates the factor and is responsible for the toxin's action in inhibiting protein synthesis in animal cells. It is believed that Fragment A or similar enzymically active fragments released into the cytosol of toxin-treated cells mediate this inhibition. The complete amino acid sequence of Fragment A has been determined from 32 chymotryptic peptides, three peptides derived by chemical cleavage of Fragment A at its 2 tryptophan residues, five cyanogen bromide peptides, and six tryptic peptides from the maleylated protein.

Adenosine Diphosphate Sugars↗
PubMed1979

Enzymic activity of cholera toxin. II. Relationships to proteolytic processing, disulfide bond reduction, and subunit composition.

Cholera toxin containing intact A chain (Mr = 29,000) was isolated, and its enzymic properties were characterized. The "unnicked" form of the toxin, produced by a protease-deficient, hypertoxinogenic mutant of Vibrio cholerae 569B, had greatly reduced activity in catalyzing the NAD+-glycohydrolase and ADP-ribosyltransferase reactions as compared to the naturally nicked form commonly isolated. In the latter, the intact A chain has been cleaved by bacterial proteases to yield disulfide-linked A1 and A2 chains (Mr = 23,000 and 6,000, respectively). Digestion of unnicked toxin with trypsin or elastase yielded a nicked form similar to or identical with the naturally nicked toxin, but chymotryptic digestion did not. Disulfide bond reduction was necessary for expression of enzymic activity by naturally nicked or trypsin-nicked toxin, or the A1A2 protomer. Fractionation of thiol-treated, nicked cholera toxin by ion exchange, molecular exclusion, or affinity chromatography gave results suggesting that the reduced toxin displays enzymic activity while remaining structurally intact.

Adenosine Diphosphate Sugars↗
PubMed1979

Enzymic activity of cholera toxin. I. New method of assay and the mechanism of ADP-ribosyl transfer.

We tested various methods of assaying the ADP-ribosyltransferase activity of cholera toxin using artificial acceptors of the ADP-ribosyl group. Any of several proteins or poly(L-arginine) could be used with [adenine-14C]NAD+ as ADP-ribosyl donor, but this method was not ideal because of the heterogeneity of potential acceptor groups and the necessity of using costly labeled NAD+. We, therefore, developed an alternative assay using a synthetic low molecular weight acceptor, 125I-N-guanyltyramine (125I-GT). 125I-GT was specifically ADP-ribosylated by thiol-treated cholera toxin or its A1 peptide in the presence of beta-NAD. ADP-ribosyl-125I-GT was quantified after separation from unreacted 125I-GT by batch absorption of the latter to cation exchange resins. Analysis of the kinetics of ADP-ribosylation of 125I-GT indicated that the reaction proceeds by a sequential rather than a ping-pong mechanism. The Km values for NAD+ and 125I-GT were 3.6 mM and 44 microM, respectively. L-Arginine was a competitive inhibitor of 125I-GT (KI = 75 mM), but was at least 1000-fold less active than 125I-GT as an ADP-ribose acceptor.

Adenosine Diphosphate Sugars↗
PubMed1979

Serum glucocorticoids, growth hormone and insulin and plasma glucose in bulls given prostaglandin E2 or F2 alpha 1.

Plasma glucose and serum insulin, growth hormone and glucocorticoid concentrations were determined in five yearling bulls given (im) 5, 15 or 30 mg prostaglandin E2 (PGE2), 30 mg prostaglandin F2 alpha(PGF2 alpha) or saline. Jugular blood was collected at frequent intervals around the time of injection and at .5--hr intervals from 1 to 9 hr after injections. Thirty milligrams PGE2 and 30 mg PGF2 alpha each caused 15- to 20-fold increases in serum glucocorticoids. Glucocorticoids increased with increasing doses of PGE2. Although PGE2 and PGF2 alpha each increased blood growth hormone, this effect was about twofold larger after PGE2. By contrast, PGE2 depressed serum insulin about 50% for 1 hr, then insulin increased about sixfold until 3 to 4 hours. Blood serum insulin increased after PGF2 alpha, but this effect only approached significance (P less than .10). Plasma glucose increased about 10 mg/100 ml after PGE2, but was not affected significantly by PGF2 alpha. Thus, the effects of PGE2 and PGF2 alpha on hormones which control glucose metabolism differ markedly. We speculate that PGE2 caused a twofold increase in growth hormone secretion within 10 to 20 min, that increased growth hormone induced increased blood glucose within 1 to 2 hr and that increased glucose caused increased insulin secretion at 2 to 4 hr, but we cannot rule out a transitory (1 hr) suppressive effect of PGE2 directly on the pancreas.

Animals↗
PubMed1979

Season and treatment effects on serum prolactin and milk yield during induced lactation.

Nineteen nonpregnant, nonlactating dairy cows were allotted to three treatments to induce lactation during winter, 1976, or spring, 1977. All groups received 17 beta-estradiol (.1 mg/kg) days 1 to 7. Groups 2 and 3 also received progesterone (.25 mg/kg) days 1 to 7. Groups 1 and 2 were given reserpine (5 mg intramuscular) on days 8, 10, 12, and 14. Group 3 received reserpine (5 mg intramuscular) on days 2, 5, 8, 11, and 14. Blood samples were collected for prolactin analysis just prior to and 3 h after reserpine injection. Mean daily temperatures were 11.9 C for spring group and -6.5 C for winter group. Comparisons of spring with winter for basal prolactin concentrations, reserpine-stimulated prolactin concentrations, and 100-day milk yields were 44 with 10 ng/ml, 482 with 199 ng/ml, and 1991 with 862 kg. Differences in prolactin concentrations and milk yields among hormone and reserpine treatments could not be detected, but cows on treatment 3 in the spring gave the largest yield of milk. Prolactin concentrations were correlated with milk yields among cows and among cows within seasons. Seasonal differences demonstrate the critical role of prolactin in the treatment to induce lactation.

Animals↗
PubMed1979

Structure-activity relationships in diphtheria toxin and exotoxin A from Pseudomonas aeruginosa.

Diphtheria toxin and exotoxin A from Pseudomonas aeruginosa (Pseudomonas toxin) block protein synthesis in sensitive animal cells by virtually identical mechanisms. Both toxins are proenzymes that, after activation, catalyze attachment of the adenosine diphosphate ribose (ADP-ribose) moiety of NAD to elongation factor 2 (EF-2) by covalent linkage. EF-2 is thereby inactivated. In the case of diphtheria toxin (60,000 daltons) the ADP-ribosylation of EF-2 is catalyzed by a 21,000-dalton peptide (fragment A) released after mild tryptic digestion and reduction of the toxin. The complementary B moiety of the toxin (39,000 daltons) is required for toxic activity and functions by attaching the toxin to oligosaccharide-containing cell surface receptors. In the case of the Pseudomonas toxin, the ADP-ribosylation reaction may be catalyzed either by the intact 66,000-dalton chain after reduction, or by a 26,000-dalton peptide released after mild proteolysis. Current approaches to study of the mechanisms of entry of the two toxins in active form into animal cells are reviewed.

Adenosine Diphosphate Ribose↗