Bryan Dunbar

Evidence for a novel class of microbial 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase in Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa

The tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthases from Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa have been purified to homogeneity. All three enzymes have a subunit Mr of 54,000. The S. coelicolor DAHP synthase was physically and kinetically characterized and the N-terminal amino acid sequence was obtained. The N-terminal amino acid sequence could not be obtained for the enzymes from S. rimosus and N. crassa, their N-termini apparently being blocked. However, following proteolytic digestion, internal amino acid sequences were obtained from both enzymes. A comparison with the known DAHP synthase sequences indicated that these DAHP synthases are unrelated to other microbial DAHP synthase sequences but are similar to plant DAHP synthases. Up until now, two distinct classes of DAHP synthase have been described, one comprising exclusively enzymes from plants, the other restricted to enzymes from micro-organisms. These studies indicate that the class containing the plant DAHP synthases also contains enzymes from a microbial eukaryote and from several bacteria.

Component X of mammalian pyruvate dehydrogenase complex: Structural and functional relationship to the lipoate acetyltransferase (E2) component

The lipoate acetyltransferase (E2, M r 70 000) and protein X (M r 51 000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N‐terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C‐labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly‐arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N‐terminus.

The characterisation of the shikimate pathway enzyme dehydroquinase from Pisum sativum

Peptides accounting for 157 residues of the bifunctional shikimate pathway enzyme, dehydroquinase/shikimate dehydrogenase, of Pisum sativum were sequenced. Three of the peptides were homologous to regions in Escherichia coli dehydroquinase and two to E. coli shikimate dehydrogenase. The pea dehydroquinase activity was inhibited by treatment with dehydroquinate plus sodium borohydride, establishing it as a type I dehydroquinase. Synthetic oligonucleotides designed from the amino acid sequence were used as PCR primers to amplify fragments of P. sativum cDNA. DNA sequence analysis showed that these amplified products were derived from dehydroquinase/shikimate dehydrogenase CDNA. The complete amino acid sequence of the dehydroquinase domain has been defined; it is homologous to all other type I dehydroquinases and is N‐terminal.

Primary structure of the helical domain of porcine collagen X

The entire primary structure of the collagen X helical region is presented, including identification of the extensive post-ribosomal modifications by amino acid sequencing and mass spectrometry. As in collagen I, a single residue of 3-hydroxyproline was identified, but for collagen X this was located near the N-terminal end of the helix. Lysine residues in collagen X are extensively hydroxylated/glycosylated: at least 11 sites were localized and shown to be fully glycosylated, exclusively as glucosyl-galactosyl derivatives. The lysine-derived crosslinks, dihydroxylysinonorleucine and hydroxylysinonorleucine, were shown to be present in a 3:2 molar ratio primarily within the C-terminal portion of the helix.

Purification and characterization of fatty acid-binding proteins from brown adipose tissue of the rat

Fatty acid-binding proteins (FABPs) have been identified and purified from interscapular brown adipose tissue of the rat. The proteins were characterized and their properties compared with the FABP present in white adipose tissue. FABP was purified to electrophoretic homogeneity from brown adipose tissue by a procedure involving precipitation with 70% ammonium sulphate, followed sequentially by ion-exchange chromatography and gel filtration chromatography. The purified fraction migrated as a single band on SDS-PAGE with an apparent molecular mass of 14,200. Scatchard analysis of [14C]oleate-binding to purified FABP gave a Kd value of approx. 0.80 +/- 0.02 microM and a maximal binding of 0.65 +/- 0.03 mol per mol of protein; these values were similar to that found with the FABP purified from white fat. The FABP concentration in brown adipose tissue was almost twice that of FABP in white adipose tissue. Fatty acid analysis of FABP from brown adipose tissue revealed that the intrinsic arachidonic acid content was proportionately higher than that present in FABP of white adipose tissue. Isoelectric focusing of delipidated FABP indicated that it existed with two charge isoforms (pI 6.85 and 7.35). The purified FABP additionally emerged in two peaks (FABP-I and FABP-II) from a reverse phase HPLC column. Amino acid analysis showed that Gly, Thr, and Ser residues in FABP-I were almost twice as high as in FABP-II. The N-terminals of both FABP-I and -II were not blocked. These components have been partially sequenced and showed a sequence homology only between 25-31 residues from the N-terminal. Further studies are required to elucidate the precise function of the two different isoforms of FABP in brown adipose tissue.

Muscle and liver pyruvate kinases are closely related: amino acid sequence comparisons

Previous evidence has shown that the M1 and L pyruvate kinase isozymes differ markedly in kinetic and immunological properties, amino acid compositions and peptide maps. However, the amino acid sequence results we present here for the N‐terminal region and for a region of the C domain show that the M1 and L isozymes are very similar. The variable length of the N‐terminal sequences also explains the difference in regulation by phosphorylation between the M1 and L isozymes. The M1 isozyme lacks the serine residue that has been shown to be phosphorylated in the L isozyme.

Identification of plant mitochondrial proteins: A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a FastBlot cycle

Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a 2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing, and evaluates its performance. The paper also describes a “FastBlot” sequencing cycle with the cycle time for protein sequencing from PVDF membranes reduced to less than 29 min with femtomole sensitivity. Other benefits of the cycle include reduced lag, reduced background, reduced loss of labile residues, and increased initial and repetitive yields. The procedure gave excellent results with maize mitochondrial proteins: of six protein spots that we tried to sequence, only one was blocked. The other spots yielded considerable sequence information. One spot was identified from the sequence as superoxide dismutase, while another spot corresponded to an unidentified cDNA from rice. The results of these experiments show that modifications of our previous procedures can provide good N-terminal protein sequencing from individual spots on 2D gels. The technique makes it possible to obtain sequence data, prepare gene probes, and identify many of the polypeptides in the 2D-gel map for plant mitochondria.

A catalase from Streptomyces coelicolor A3(2)

Catalase was purified from the Gram-positive bacterium Streptomyces coelicolor A3(2) in a three-step purification procedure comprising (NH4)2SO4 fractionation, Phenyl-Sepharose chromatography and Mono Q chromatography. The purification of catalase, as judged by the final specific activity of 110,000 U mg-1, was 250-fold with a 35% yield. The native protein was a homotetramer with a subunit M(r) 55,000. N-terminal and internal peptide sequence analyses showed that there was a high degree of sequence similarity between the S. coelicolor catalase and other microbial and mammalian catalases. Southern blot analysis indicated that there was a single catalase gene in S. coelicolor. The specific activity of catalase throughout the growth of batch cultures was investigated and elevated catalase activity was found in stationary-phase cells.

Evidence for a new cytochrome P450 form induced by 3-methylcholanthrene in rats

Evidence is presented for a new 3-methylcholanthrene (3MC)-induced form of cytochrome P450, P450MCX, in rat liver microsomes. P450MCX was co-purified with CYP1A1 from 3MC-treated male Sprague-Dawley rats but was resolved by gel electrophoresis. The M(r) of P450MCX (56,700) was intermediate between CYP1A1 (57,000) and CYP1A2 (54,800). Monoclonal antibodies showed that P450MCX was immunorelated to both CYP1A1 and CYP1A2 but not to CYP2B1, CYP2C6 or CYP3A1. Immunoreactive P450MCX was not detectable in liver microsomes from untreated rats but was highly induced by 3MC and Aroclor 1254, although not induced by isosafrole. The N-terminal amino acid sequence of P450MCX, obtained from an electroblotted sample resolved on SDS-PAGE, did not match any known cytochrome P450 or other protein. P450MCX may be a new member of the CYP1 family.