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A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin. [ABSTRACT FROM AUTHOR]

The DNA-binding protein HU from Escherichia coil is a heterodimer constituted of two poly- peptide chains termed HU-1 and HU-2, of 90 residues each. Their primary structures were established from structural data obtained from tryptic peptides of each monomer in addition to the structural data provided by the automated Edman degradation of the dimer and by peptides derived from cleavage of the dimer with trypsin, chymotrypsin, V8 staphylococcal protease and dilute acid. The results presented in this paper confirm the amino-terminal and carboxy-terminal sequences of the dimer HU reported previously [Laine et al. (1978) FEBS Lett. 89, 116–120]. The amino acid sequences of proteins HU-1 and HU-2 are identical to those of proteins NS-1 and NS-2 respectively, determined independently by Mende et al. [FEBS Lett. (1978) 96, 395–398]. The amino acid sequences of proteins HU-1 and HU-2 are closely related but differ by 28 residues. These proteins are characterized by their high content of hydrophobic residues represented mostly by alanine. In both proteins, half of the basic residues are scattered along the polypeptide chain and the remainder is found within two short sequences located in the carboxy-terminal part of the molecule. No sequence homology could be established between the proteins HU-1 and HU-2 and any one of the five histones from different eukaryotes. [ABSTRACT FROM AUTHOR]

We have incorporated chloroplast ribosomal proteins (R-proteins) L12 and S18 into Escherichia coli ribosomes and examined the hybrid ribosomes for their ability to form polysomes in vivo and perform poly(U)-dependent poly(Phe) synthesis in vitro. The rye chloroplast S18 used for the experiment is a highly divergent protein (170 amino acid residues; E. coli S18, 74 residues), containing a repeating, chloroplast-specific, heptapeptide motif, and has amino acid sequence identity of only 35% to E. coli S18. When expressed in E. coli, chloroplast S18 was assembled in E. coli ribosomes. The latter formed polysomes in vivo at about the same rate as the host ribosomes, indicating that the replacement of E. coli S18 with its chloroplast homologue has only a minor, if any, effect on function. The L12 protein is much more conserved in sequence and chain length, and is known to have a very important function. The Arabidopsis chloroplast L12 used in the experiment was incorporated into E. coli 50S subunits that associated with the 30S subunits to form ribosomes, but the latter were unable to form polysomes. This result indicates functional inactivation of E. coli ribosomes by a chloroplast R-protein. To further confirm this result, we overproduced chloroplast L12 through the use of a secretion, vector and purified the protein to homogeneity. Chloroplast L12 could be efficiently incorporated in vitro into L7/12-1acking E. coli ribosomes, but the hybrid ribosomes were totally inactive in poly(U)-dependent poly(Phe) synthesis. Computer modeling of the spatial structure of all known chloroplast L12 proteins (using E. coli L12 coordinates) indicated a 'chloroplast loop' present only in chloroplast L12. The presence of this loop might have a role in the observed inactivation. Taken together with previously reported results (summarized in this paper), it would appear that the features of chloroplast R-proteins concerned with specific functions are more divergent than their assembly properties. We have previously described methods suitable for overproduction and purification of chloroplast Rproteins that are encoded in organellar DNA (≈20), but that gave poor yield for those encoded in the nuclear DNA (≈45). Here we describe a method that overcomes this problem and allows the purification of nucleus-encoded chloroplast R-proteins in milligram quantities. [ABSTRACT FROM AUTHOR]

This paper describes a biochemical study on the protein composition of crystalline inclusions (cores) from plant peroxisomes. By SDS/PAGE and immunoblotting, a catalase of 59 kDa was identified as the predominant protein component in purified cores from sunflower (Helianthus annuus L.) cotyledons. A 55-kDa catalase was the only additional peptide detected. In contrast to in cores, the 55-kDa catalase was the major catalase protein in matrix fractions obtained from lysed peroxisomes. These findings suggested two peroxisomal populations of catalase differing in molecular structure and subperoxisomal compartmentation in sunflower cotyledons. Evidence for different amino acid sequences of the two catalases was found by peptide mapping with endoproteinase Glu-C, by expressing a eDNA encoding matrix catalase in Escherichia coli, and by partial amino acid sequencing of peptide fragments from 59-kDa core catalase. These results contradict the previous view that the formation of cores occurred via condensation of matrix catalase, and indicate that new concepts on the biogenesis and physiological function of plant peroxisomal cores need to be developed. [ABSTRACT FROM AUTHOR]