The gene for the A chain of ricin toxin was fused to a j8-galactosidase marker cistron via a DNA sequence encoding a short collagen linker, and the tripartite fusion protein was expressed in Escherichia coli. Site-specific mutagenesis was used to change glutamic acid residue 177 to aspartic acid or alanine. When the mutant proteins were expressed, purified, and tested quantitatively for enzymatic activity, the carboxylate function at position 177 was found not to be absolutely essential for ricin toxin A-chain catalysis.Ricin is a polypeptide toxin found in the seeds of the Ricinus communis (castor bean) plant. This 65-kilodalton (kDa) heterodimeric protein consists of a 32-kDa A chain (RTA) linked by disulfide bond to a 33-kDa B chain (RTB) (24,28). RTB is a lectin that binds to ,3-galactoside-terminated oligosaccharides on the surface of mammalian cells. After binding, the toxin is internalized and transported to an intracellular compartment (thought to be the trans-Golgi [25,30]), where the chains separate and RTA translocates across the vesicle membrane to the cytosol. In the cytosol, RTA catalytically inactivates protein synthesis by hydrolysis of the N-glycosidic bond of adenosine 4324 in the 28S RNA of the eucaryotic 60S ribosomal subunit (5). This depurination irreversibly inactivates the ribosome, possibly by altering the binding site for elongation factors (20). Under optimal conditions, a single molecule of RTA can inactivate 1,500 ribosomes per min, and a single molecule of RTA in the cytosol is sufficient to cause cell death (4).The three-dimensional structure of ricin determined by X-ray crystallography (21) showed a prominent deep cleft that was proposed as the enzyme-active site (21,27,28).

A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL] promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.Ricin is the toxic lectin from Ricinus communis (castor bean) seeds. The ricin molecule is a 65,000-dalton (Da) heterodimer consisting of an A chain and a B chain linked by a disulfide bond (16). The B chain is a galatose-binding lectin which causes ricin to bind to mammalian cells. Once bound to cells, the ricin is internalized and the A chain translocates across endocytic vesicle or Golgi membranes to the cytosol. In the cytosol the ricin toxin A chain (RTA) catalytically inactivates protein synthesis by hydrolysis of the N-glycosidic bond of adenosine residue 4324 in the 28S rRNA of the eucaryotic 60S ribosomal subunit (5). This modification irreversibly inactivates the ribosome, possible by altering the binding site for elongation factors (D. Moazed, J. Robertson, and H. Noller, Nature [London], in press). A single molecule of RTA in the cytosol is sufficient to cause cell death (3).The detailed mechanism for the RNA N-glycosidase activity of RTA is unknown. X-ray crystallography shows a cleft in the second domain of RTA which resembles the active sites of other proteins that bind large polynucleotide substrates (14). Furthermore, a number of amino acid residues located in the second domain of RTA are conserved among a group of several additional toxins and RNA-binding proteins (19), suggesting that this region may be critical for catalytic activity.The genomic ricin gene encodes both the A and B proteins. The gene lacks introns and encodes a 24-amino-acid signal peptide, the A chain, a 12-amino-acid linker peptide, and a B chain (6). Cloned RTA prepared from both the genomic ricin DNA and a cDNA clone of ricin has been expressed in Escherichia coli (15,17). In each case a soluble biologically functional molecule was produced. However, procaryotic ribosomes are not a substrate for RTA (15), and no mutants with defects in enzymatic activity or other RTA functions have been described.We have been studying the expression of cloned RTA in the yeast Saccharomyces cerevisiae, whose ribosomes are sensitive to RTA. When RTA was expressed in yeast cells, growth was arrested. Plasmids encoding mutant RTA were readily identified by their failure to kill S. cerevisiae. These observations were the basis for a positive selection for mutations that inactivated RTA. By extracting both protein * Corresponding author. and nucleic acid from the yeast cells, we determined the DNA sequence of mutant RTA genes and the enzymatic properties of the proteins they encode. Our results indicate that in...

These results indicate that the telomerase promoters have the capacity to drive the expression of the NAT. The potency of [(211)At]MABG is approximately three orders of magnitude greater than that of [(131)I]MIBG. Spheroids composed of only 5% of cells expressing NAT under the control of the RSV or hTERT promoter were sterilised by radiopharmaceutical treatment. This observation is indicative of bystander cell-kill.

Two peptides of potential utility for targeting melanoma cells, alpha-melanocyte-stimulating hormone (alpha-MSH) and its more potent analogue [Nle4,D-Phe7]-alpha-MSH, were radioiodinated in 45-65% yield using N-succinimidyl 3-[125I]iodobenzoate (SIB). To determine whether this labeling method resulted in improved in vitro and in vivo characteristics, these peptides also were labeled with 131I by direct iodination with the iodogen method. For alpha-MSH, the rapid tissue clearance of both radionuclides in mice was consistent with rapid degradation of the peptide; however, significantly lower levels of 125I were observed in thyroid and stomach, reflecting a greater inertness to deiodination. More extensive comparisons were performed with [Nle4,D-Phe7]-alpha-MSH. The in vitro binding of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH (prepared using SIB) to the murine B-16 melanoma cell line, 34.1 +/- 4.7%, was more than twice as high as that for [Tyr2(131I),Nle4,D-Phe7]-alpha-MSH (15.0 +/- 0.1%), and its KD was more than 10-fold lower than that for conventionally labeled peptide (10 +/- 5 versus 140 +/- 14 pM). The normal tissue clearance of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH in mice was faster than that of [Tyr2(131I),-Nle4,D-Phe7]-alpha-MSH. The 19-40-fold lower activity concentrations of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH in tissues accumulating free iodide (thyroid and stomach) suggest a greater inertness of this peptide to deiodination. The primary urinary catabolite of [Nle4,D-Phe7, Lys11-(125I)IBA]-alpha-MSH was the lysine conjugate of iodobenzoic acid, whereas radioiodide was the chief catabolite generated from [Tyr2(131I),Nle4,D-Phe7]-alpha-MSH. We conclude that further evaluation of [Nle4,D-Phe7,Lys11-(125I)IBA]-alpha-MSH for targeting alpha-MSH receptors is warranted and that SIB may be a useful method for the radioiodination of peptides.