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J D Robertus

Publications and source records attributed to J D Robertus.

At least 73 records · Page 4Linked to original sources
PubMed1987

Crystallization of ricin A chain obtained from a cloned gene expressed in Escherichia coli.

Ricin is a heterodimeric toxin of the form AB, where B is a lectin which binds cell surfaces, triggering endocytosis. The B chain then aids the A chain in escaping from the endosome. The A chain enzymatically attacks and inactivates ribosomes, thereby killing the intoxicated cell. We have recently solved the three-dimensional structure of whole ricin. Here we report that the A chain, expressed from a gene cloned into Escherichia coli has been crystallized in a suitable form for high resolution x-ray analysis. The crystals are monoclinic space group P2(1) with a = 42.6, b = 68.1, c = 50.2 A and beta = 112.9 degrees. There is evidence that the A chain undergoes a conformational change, resulting in activation, when it is released from the B chain. Comparison of the two structures should facilitate an analysis of this process.

Crystallization↗
PubMed1987

The intersubunit disulfide bridge of ricin is essential for cytotoxicity.

Alkylation of the cysteine residues which link the A and B chains of ricin through a disulfide bridge produces a molecule which still binds to HeLa cells and is toxic toward in vitro ribosome-directed translation, but which has little or no cytotoxicity toward cells in culture. This and similar observations on diphtheria toxin implicate the intersubunit disulfide bridge in the transport of the toxic subunits of these toxins into the cytoplasm.

Alkylation↗
PubMed1986

Cloning and nucleotide sequence of wild type and a mutant histidine decarboxylase from Lactobacillus 30a.

Prohistidine decarboxylase from Lactobacillus 30a is a protein that autoactivates to histidine decarboxylase by cleaving its peptide chain between serines 81 and 82 and converting Ser-82 to a pyruvoyl moiety. The pyruvoyl group serves as the prosthetic group for the decarboxylation reaction. We have cloned and determined the nucleotide sequence of the gene for this enzyme from a wild type strain and from a mutant with altered autoactivation properties. The nucleotide sequence modifies the previously determined amino acid sequence of the protein. A tripeptide missed in the chemical sequence is inserted, and three other amino acids show conservative changes. The activation mutant shows a single change of Gly-58 to an Asp. Sequence analysis up- and downstream from the gene suggests that histidine decarboxylase is part of a polycistronic message, and that the transcriptional promotor region is strongly homologous to those of other Gram-positive organisms.

Amino Acid Sequence↗
PubMed1986

Extracellular localization of pokeweed antiviral protein.

Pokeweed antiviral protein is an enzyme of Mr 29,000 known to inactivate a wide variety of eukaryotic ribosomes. We have used electron microscopy to show that the antibody specific for the protein is bound within the cell wall matrix of leaf mesophyll cells from Phytolacca americana. Any penetration or breakage of the cell wall and membrane could allow the enzyme to enter the cytoplasm, where it is likely to inhibit protein synthesis in the damaged cell. We speculate that pokeweed antiviral protein is a defensive agent whose principal function is probably antiviral.

Depression, Chemical↗
PubMed1984

Ricin-like plant toxins are evolutionarily related to single-chain ribosome-inhibiting proteins from Phytolacca.

A comparison has been made of the amino-terminal sequences of a number of ribosome-inhibiting proteins (RIPs) and cytotoxins. These include the monomeric enzymes PAP, PAP-S, PAP-II, and dodecandrin and the enzymatic A chains from the heterodimeric toxins ricin and modeccin. We show that these proteins have all evolved from a single ancestor. A statistical analysis is used to show the likely evolutionary relationship among the proteins. A similar analysis was performed on the amino-terminal sequences of ricin, Ricinus agglutinin, and modeccin B chains. These are galactoside-binding proteins associated with the A-chain enzymes. From the two comparisons we propose a scheme for the development of two major classes of proteins. The RIP and sugar-binding genes probably evolved independently. In some plant lines the genes never fused, although the RIP gene replicated and developed into several proteins expressed at various stages of plant maturation. In another line the RIP gene fused with a sugar binding (B-chain) gene to form the class of heterodimeric toxins. In some species this fused gene appears to have multiplied, one or more of the toxin genes mutating to code for a self-dimerizing agglutinin molecule.

Amino Acid Sequence↗
PubMed1984

Dodecandrin, a new ribosome-inhibiting protein from Phytolacca dodecandra.

Dodecandrin, a newly discovered ribosome-inhibiting protein, has been isolated and purified from the leaves of the African endod plant, Phytolacca dodecandra. Dodecandrin has a molecular weight of approx. 29 000. It cross-reacts with antiserum prepared against pokeweed antiviral protein from Phytolacca americana and exhibits similar requirements for antiribosomal activity. It is more basic than pokeweed antiviral protein, and comparison of the first 30 amino-terminal residues of the two proteins reveals 83% homology. This level of homology is greater than that between pokeweed antiviral protein and pokeweed antiviral protein S, another antiviral protein found in P. americana. Such conservatism in sequence, coupled with the high efficiency of the proteins in deactivating ribosomes and with their abundance in plant tissue, suggests that they serve an important function in the life of the plant, probably as a defense against infection.

Immunodiffusion↗
PubMed1984

Ricin B chain and discoidin I share a common primitive protein fold.

The galactoside-binding B chain of the cytotoxic protein ricin is apparently derived from a conservative exon-sized 40-residue peptide which is repeated four times in the molecule. A very similar peptide can also be seen in the amino acid sequence of the slime mold lectin discoidin I, which itself appears to be the product of a gene duplication. There is presently no chemical or structural evidence concerning the function of this peptide region. Nevertheless, the size of this unit, its prominence in the structure of ricin B chain, and its apparent conservation in carbohydrate-binding proteins from widely divergent organisms suggest that it may represent an extremely ancient galactoside-binding exon unit.

Amino Acid Sequence↗
PubMed1983

Requirements for antiribosomal activity of pokeweed antiviral protein.

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K+ and 3 mM Mg2+ retards the reaction, while 20 mM K+ and 2 mM Mg2+ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300-400 mol/mol per min. in each case. The Km for ribosomes is 1 microM in the presence of low salt and 0.2 microM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pKa value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.

Adenosine Triphosphate↗
PubMed1981

Ricin B chain is a product of gene duplication.

The cytotoxin ricin is a heterodimer composed of an A chain which enzymatically inhibits protein synthesis on eukaryotic ribosomes and a lectin B chain which binds to cell surfaces and triggers uptake of the toxin. A low resolution (4 A) x-ray structure revealed that the B chain is a bilobal structure and that each domain binds a galactose sugar. These apparent structural similarities suggested that the protein might have internal symmetry and might have arisen by gene duplication. A subsequent search of the amino acid sequence provided strong evidence for homology between th NH2- and COOH-terminal halves of the B chain and suggested that they may have arisen from a common ancestor. There is no strong relationship between the halves of the A chain and little, if any, significant sequence homology between the A and B chains of ricin.

Amino Acid Sequence↗
PubMed1975

Structure of yeast phenylalanine transfer RNA at 2.5 A resolution.

The x-ray analysis of the monoclinic form of yeast tRNAPhe has been taken to a resolution of 2.5 A by the method of isomorphous replacement. The model proposed at 3 A has been confirmed and extended to reveal additional features of the tertiary structure and of the stereochemistry. An extensive hydrogen bonding network is described involving specific interactions between bases and the ribose-phosphate backbone. The structure of a G-U base pair has been solved.

Base Sequence↗
PubMed1974

Conservation of the molecular structure of yeast phenylalanine transfer RNA in two crystal forms.

A comparison is made between the electron density maps of the monoclinic and orthorhombic crystal forms of yeast tRNA(Phe) which have been obtained respectively by ourselves and by another group. It is concluded that the molecular structures are essentially the same in both crystals, although the models derived from the maps are not the same. The relation between the two molecular packings is discussed, and it is suggested that the intermolecular contact in the orthorhombic form which is not present in the monoclinic form, may arise through base pairing of the anticodons of neighboring molecules.

Models, Molecular↗
PubMed1974

Correlation between three-dimensional structure and chemical reactivity of transfer RNA.

The bases of yeast tRNA(Phe) which react with carbodiimide and methoxyamine have been determined and this information has been combined with chemical modification studies of other workers to produce a composite picture of base accessibility in this tRNA. The results are compared with the three-dimensional structure which we have recently determined. The bases which react chemically lie in exposed positions in the three-dimensional model and those which do not are either in the double helical stem regions or else are involved in maintaining the tertiary structure through pairing or stacking interactions.

Base Sequence↗