Amino Acid Sequence and Disulphide-bridge Pattern of three gamma-Thionins from Sorghum bicolor

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A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P 1 -P 1 H reactive site bond (where residues forming the reactive site have been identified as P n ¼P 1 and P 1 H ¼P n H , where P 1 -P 1 H is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1 H-NMR data indicates the presence of aaNOEs and 3JaNH coupling constants, typical of the b-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine b-trypsin and bovine a-chymotrypsin are 3.3 £ 10 9 m 21 and 2.4 £ 10 6 m 21 , respectively, at pH 8.0 and 21.0 8C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.Keywords: trypsin inhibitors; oil-rape seed; Brassica napus var. oleifera; amino acid sequence determination; disulphide bridge location; 1 H-NMR investigation; inhibitory properties.Serine proteinase inhibitors are widespread in the plant kingdom being found particularly in seeds of Cruciferae, Graminaceae and Leguminosae, as well as in tubers of Solanaceae. Their physiological roles include the regulation of endogenous proteinases during seed dormancy, the reserve protein mobilization, and the protection against the proteolytic enzymes of parasites and insects. Moreover, they may also act as storage or reserve proteins. Plant serine proteinase inhibitors are grouped into Soybean (Kunitz), Bowman-Birk, potato I and II, and squash families. Several other inhibitor families, such as barley, ragi 1 and 2, and thaumatin, were also suggested [1±10]. Different types of serine proteinase inhibitors have been identified in the same plant, suggesting that these proteins have evolved separately to perform distinct physiological roles. In this respect, oil-rape (Brassi...

Section: Methodsmentioning

confidence: 99%

Characterization of low‐molecular‐mass trypsin isoinhibitors from oil‐rape (Brassica napus var. oleifera) seed

Ascenzi

Ruoppolo

Amoresano³

et al. 1999

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“…Protein samples were reduced and alkylated with iodoacetic acid according to the procedure described previously [17]. Briefly, samples were reduced in 0.25 m Tris/HCl pH 8.5, 1.25 m m EDTA, containing 6 m guanidinium chloride, by incubation with a 10:1 molar excess of dithiotheitol over ‐SH groups at 37 °C for 2 h. Alkylation was carried out using a 5:1 molar excess over the total ‐SH groups at 37 °C for 30 min in the dark.…”

Section: Methodsmentioning

confidence: 99%

N‐Linked glycans of proteins from mitral valves of normal pigs and pigs affected by endocardiosis

Amoresano¹,

Amedeo²,

D’Andrea³

et al. 2000

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Endocardiosis, a degenerative and dystrophic process affecting cardiac valves and described in many mammalian species, is characterized by the accumulation of glycosaminoglycans, in particular hyaluronic acid, in the extracellular matrix. The glycoprotein patterns of pig mitral valves in normal animals and animals affected by endocardiosis were investigated. A different N-linked glycosylation pattern of glycoproteins was detected in affected valves compared with normal ones. In either normal or pathological species, the detected N-linked glycans were of the complex type. However, in samples from affected valves, sialic acid showed a prevalence of the a2,6 linkage to the galactosyl residue, whereas in normal samples the most frequent linkage was of the a2,3 type. In normal valves, the majority of complex oligosaccharides presented two outer branches with different degrees of fucosylation and sialylation, whereas in pathological samples we noted an increased number of glycans having up to four outer branches.

“…The separated isoforms of the three proteins (300 µg) were reduced and alkylated as previously described [20]. The reduced and carboxymethylated RfBPs were digested with trypsin and endoproteinase GluC in 50 m m ammonium bicarbonate, pH 8.5 at 37 °C overnight using an enzyne to substrate ratio of 1 : 50.…”

Section: Methodsmentioning

confidence: 99%

The carbohydrates of the isoforms of three avian riboflavin‐binding proteins

Amoresano¹,

Brancaccio²,

Andolfo³

et al. 1999

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The carbohydrate chains of nine isoforms of chicken egg-white riboflavin-binding protein (RfBP) and six isoforms each of quail egg-white and yolk RfBP have been structurally characterized. The two N-glycosylation sites, Asn36 and Asn147, of the most abundant isoform of each of the three proteins were analyzed in further detail leading to the identification of different glycosylation patterns. In both chicken and quail egg-white RfBP the carbohydrates attached to position 36 had a lower degree of branching and, in the case of the quail protein, this site was only partially glycosylated. A very heterogeneous mixture of complex structures was characteristic of the other glycosylation site. Analysis of the two sites in quail yolk RfBP confirmed this result which agrees with what has been established for hen yolk RfBP. The presence in the three proteins of a highly heterogeneous mixture of differently branched glycans suggests that the differences in isoelectric points, which is a peculiarity of the different isoforms, are probably indeed due to differences in carbohydrate structure.Keywords: riboflavin-binding protein; phosphoglycoprotein; oligosaccharide; structural characterization; MS.The best characterized riboflavin-binding proteins are those of avian origin and in particular those of the hen [1]. Three different proteins have been characterized in this species and they have been shown to be the products of the same gene and to have undergone different post-translational modifications [2]. The three proteins have a single high-affinity site for riboflavin and are found in the plasma of laying hens and in the egg yolk and white. The first two molecules are synthesized by the liver and the third is a product of the oviduct [3,4]. The function of plasma RfBP is that of vitamin transport to the developing oocytes, into which it is incorporated loosing in the process the last 11±13 amino acids which are thus absent from the yolk protein [5]. Egg white RfBP is believed to serve mainly as a storage protein and it is not clear why most of it is found in the apo form. The presence of estrogen-induced pregnancy-specific RfBPs in the plasma of several mammalian species has been demonstrated [6±9] although none of these proteins, which cross-react immunologically with hen RfBP, have been sufficiently characterized. Hen egg-white and plasma RfBP share the same 219 aminoacid sequence; in yolk RfBP the last 11±13 amino acids are missing [10]. The three molecules are phosphoglycoproteins with the carbohydrates attached at the same positions, Asn36 and Asn147. The structure of the oligosaccharides in hen egg yolk and plasma RfBPs has been determined [11,12]. Although from the sugar composition the carbohydrates of hen egg-white RfBP are known to be different from those of plasma and yolk RfBP, their structure has not been reported. A very special characteristic of the three hen proteins is the presence of a highly phosphorylated region that extends between amino acids 186 and 197, where there can be up to eight phosphates attac...