Immunological comparison of the NADH:nitrate reductase from different cucumber tissues

Soluble nitrate reductase from cucumber roots (<i>Cucumis sativus</i> L.) was isolated and purified with blue-Sepharose 4B. Specific antibodies against the NR protein were raised by immunization of a goat. Using polyclonal antibodies anti-NR properties of the nitrate reductase from various cucumber tissues were examined. Experiments showed difference in immuno-logical properties of nitrate reductase (NR) from cotyledon roots and leaves.

Tissue Distribution and Purification of Prophenoloxidase in Larvae of Asian Corn Borer, Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae)

Using ammonium sulphate precipitation, Blue-Sepharose CL-6B, Phenyl-Sepharose CL-4B, prophenoloxidase (PPO) was isolated and purified from hemolymph of Ostrinia furnacalis larvae. This zymogen was a heterodimer, and composed of two subunits with the relative molecular mass ranging from 66.2 kD to 97.4 kD determined by SDS-PAGE. Western blotting and indirect immunofluorescence test showed that PPO was present in integument, hemolymph plasma and cell membrane of granular hemocytes and oenocytoids of O. furnacalis larvae.

Improved resolution of human cerebrospinal fluid proteins on two-dimensional gels

Proteomics combines two-dimensional gel electro phoresis and peptide mass fingerprinting and can potentially identify a protein(s) unique to disease. Such proteins can be used either for diagnosis or may be relevant to the pathogenesis of disease. Because patients with multiple sclerosis (MS) have increased amounts of immunoglobulin (Ig) G in their cerebrospinal fluid (C SF) that is directed against an as yet unidentified protein, we are applying proteomics to MS C SF, studies that require optimal separation of proteins in human C SF. We found that recovery of proteins from C SF of MS patients was improved using ultrafiltration, rather than dialysis, for desalting. Resolution of these proteins was enhanced by aceto ne precipitatio n of desalted C SF before electrophoresis and by fractionation of C SF using C ibacron Blue sepharose affinity chromatography. Improved protein recovery and resolution will facilitate excision from gels for analysis by peptide mass fingerprinting.

acs1 of Haemophilus influenzae Type a Capsulation Locus Region II Encodes a Bifunctional Ribulose 5-Phosphate Reductase– CDP-Ribitol Pyrophosphorylase

ABSTRACT The serotype-specific, 5.9-kb region II of the Haemophilus influenzae type a capsulation locus was sequenced and found to contain four open reading frames termed acs1 toacs4. Acs1 was 96% identical to H. influenzaetype b Orf1, previously shown to have CDP-ribitol pyrophosphorylase activity (J. Van Eldere, L. Brophy, B. Loynds, P. Celis, I. Hancock, S. Carman, J. S. Kroll, and E. R. Moxon, Mol. Microbiol. 15:107–118, 1995). Low but significant homology to other pyrophosphorylases was only detected in the N-terminal part of Acs1, whereas the C-terminal part was homologous to several short-chain dehydrogenases/reductases, suggesting that Acs1 might be a bifunctional enzyme. To test this hypothesis, acs1 was cloned in an expression vector and overexpressed in Escherichia coli. Cells expressing this protein displayed both ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities, whereas these activities were not detectable in control cells. Acs1 was purified to near homogeneity and found to copurify with ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities. These had superimposable elution profiles from DEAE-Sepharose and Blue-Sepharose columns. The dehydrogenase activity was specific for ribulose 5-phosphate and NADPH in one direction and for ribitol 5-phosphate and NADP+ in the other direction and was markedly stimulated by CTP. The pyrophosphorylase showed activity with CTP and ribitol 5-phosphate or arabitol 5-phosphate. We conclude that acs1 encodes a bifunctional enzyme that converts ribulose 5-phosphate into ribitol 5-phosphate and further into CDP-ribitol, which is the activated precursor form for incorporation of ribitol 5-phosphate into the H. influenzae type a capsular polysaccharide.

PYRUVATE KINASE ISOENZYMES FROM SUBMAXILLARY AND SUBLINGUAL SALIVARY GLANDS OF RAT (Rattus rattus norvaegicus, Berkenhout). I PURIFICATION AND PHYSICOCHEMICAL PROPERTIES

Piruvatoquinase de glândula salivar submaxilar e sublingual de rato (Rattus rattus norvaegicus, Berkenhout) foi purificada até homogeneidade por “salting out” por precipitação com sulfato de amônio seguida de cromatografia de coluna, primeiro com fosfocelulose e eluição com solução 0,5M de KCl e depois com Blue Sepharose CL-6B eluida com PEP 5mM e ADP 5mM. Obteve-se atividade específica final de 324,5 UI/mg com rendimento global de 20,1% para SM-PK e de 427,4 UI com rendimento global de 9,5% para SL-PK, com pesos moleculares de 60.500 e 50.000 Daltons determinado em eletroforese do tipo PAGE com e sem SDS, para SM-PK e de 242.000 e 200.000 para SL-PK, sugerindo, com isso que se tratam de homotetrâmeros. Por eletroforese em gel de acetato demonstrou-se que tanto SM-K como SL-K possuem somente uma forma isoenzimática com mobilidade eletroforética similar à PK tipo L de fígado de rato e do tipo M2 do rim de rato. verificou-se que o pH ótimo para ambas as enzimas é de 7,4. Abstract Pyruvate kinase from rat (Rattus rattus norvaegicus) submaxillary and sublingual salivary glands was purified to homogeneity by a 3-step process. One step involved salting out by ammonium sulfate precipitation and two steps, column chromatography, first with phosphocellulose and elution with 0.5M KCl and then with Blue Sepharose CL-6B eluted with 5mM PEP and 5 mM ADP. The final specific activity of SM-PK was 324.5 IU/mg with an overall yield of 20.1%. The values for SL-PK were 427. 4 IU/mg and a yield of 9.5%. The molecular weights of the native enzymes and their subunits, as determined by PAGE electrophoresis with or without SDS were 60.500 and 50.000 Daltons respectively, for SM-PK and 242.000 and 200.000 for SL-PK, suggesting that these enzymes were present as homotetramers. By means of cellulose acetate electrophoresis it has been demonstrated that both SM-PK and SL-PK possess only one isozymic form displaying eletrophoretic mobility similar to that of the L-type PK from rat liver and M2- type PK form rat kidney. Optimum pH for both SM-PK and SL-PK was found to be 7. 4 in Tris-HCl buffer.

Amino-acid composition of pyruvate kinase M2 isoenzyme variants from rat liver and Morris hepatoma 7777.

Cytosolic fractions B (salted out between 51-70% ammonium sulphate saturation) from rat liver and Morris hepatoma 7777, containing pyruvate kinase (EC 2.7.1.40) M2 isoenzymes, were purified by affinity chromatography on Blue Sepharose CL-6B. When compared by polyacrylamide gel electrophoresis at pH 8.3, all three M2 pyruvate kinase variants from Morris hepatoma 7777 had lower mobilities (alpha2, beta2, gamma3) than the three corresponding variants (alpha1, beta1, gamma2) from normal rat liver. Using an automatic amino-acid analyser, significant differences in selected amino-acid content have been found in corresponding highly purified gamma3 and gamma2 variants from Morris hepatoma and normal rat liver, respectively. The gamma3-variant of the Morris hepatoma M2 isoenzyme had twice the amount of L-tyrosine and L-cysteine, and a content of L-serine higher by 20% than the corresponding gamma2 variant of the normal rat liver M2 isoenzyme. It contained, however, significantly less dicarboxylic amino acids which explains its lower electrophoretic mobility. It showed also a decrease (by about 10%) in several other amino-acid content, corresponding to a 10% decrease in the tumour enzyme molecular mass.

Δ-1-Piperideine-6-carboxylate dehydrogenase, a new enzyme that forms α-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes

Δ-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into α-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 μM) and NAD+ (apparent Km 115 μM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6-aminotransferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but not in actinomycetes that do not produce β-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to α-aminoadipic acid.