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1.
Structure of an antifreeze polypeptide precursor from the sea raven, Hemitripterus americanus 总被引:4,自引:0,他引:4
The cystine-rich antifreeze polypeptides (AFP) from sea raven were fractionated by reverse-phase high performance liquid chromatography into several components, with SR2 (Mr 17,000) as the major AFP. Sea raven AFP cDNA clones were isolated from a liver cDNA library using a synthetic oligonucleotide, and the identity of one of the clones, C2-1, was confirmed by hybridization selection and cell-free translation. C2-1 encodes a pre-AFP of 195 amino acids with no evidence of any profragments. Comparison of the deduced amino acid sequence with partial peptide sequences from SR2 showed substitutions in at least four amino acid positions, suggesting that C2-1 cDNA codes for a minor component. Both the primary and the predicted secondary structures of sea raven AFP are completely different from those of other fish AFP. This further confirms that sea raven AFP belongs to a different class of antifreezes. The high frequency of reverse turns and the presence of paired hydrophilic amino acids in these structures are striking features of the protein and may contribute to their antifreeze action. 相似文献
2.
Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus 总被引:5,自引:0,他引:5
X M Li K Y Trinh C L Hew B Buettner J Baenziger P L Davies 《The Journal of biological chemistry》1985,260(24):12904-12909
Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes. 相似文献
3.
Three photosynthetic enzymes were characterised in extractsfrom leaves and aerial roots of Aranda Christine 130.The enzymes from both tissues were similar in activity and kineticproperties. Grana-containing chloroplasts were found in rootcells of Vanda suauis. Thus components crucial to photosynthesisare present in aerial roots of these leafy orchids. (Received March 22, 1983; Accepted July 7, 1983) 相似文献
4.
Antifreeze polypeptides from the Newfoundland ocean pout,Macrozoarces americanus: presence of multiple and compositionally diverse components 总被引:1,自引:0,他引:1
Choy L Hew Don Slaughter Shashikant B. Joshi Garth L. Fletcher V. S. Ananthanarayanan 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1984,155(1):81-88
Summary Eight major antifreeze polypeptides (AFP) were purified from the sera of Newfoundland ocean pout. Except for their approximately identical size (6,000 Dalton), these components were shown to be separate entities by their behaviour on polyacrylamide gel electrophoresis, ion exchange chromatography, gel permeation and reverse phase high performance liquid chromatography. They could also be divided into two cross-reactive, yet distinct, immunological groups. Amino acid analysis demonstrated that ocean pout AFP are different from all of the other antifreezes studied to date. The ocean pout AFP do not contain the abundance of alanine (60 mol%) found in winter flounder and shorthorn sculpin AFP nor the high half-cystine residues (8 mol%) observed in sea raven AFP. It is suggested that ocean pout AFP represent a new type of macromolecular antifreeze.Abbreviations
AFGP
antifreeze glycoprotein(s)
-
AFP
antifreeze polypeptide(s)
-
HPLC
high performance liquid chromatography
-
SDS
sodium dodecyl sulfate
-
PAGE
polyacrylamide gel electrophoresis 相似文献
5.
A Chakrabartty V S Ananthanarayanan C L Hew 《The Journal of biological chemistry》1989,264(19):11307-11312
The major antifreeze polypeptide (AFP) from winter flounder (37 amino acid residues) is a single alpha-helix. Aspartic acid and arginine are found, respectively, at the amino and carboxyl-termini. These charged amino acids are ideally located for stabilizing the alpha-helical conformation of this AFP by means of charge-dipole interaction (Shoemaker, K. R., Kim, P.S., York, E.J., Stewart, J. M., and Baldwin, R. L. (1987) Nature 326, 563-567). In order to understand these and other molecular interactions that maintain the AFP structure, we have carried out the chemical synthesis of AFP analogs and evaluated their conformations by circular dichroism spectroscopy. We synthesized the entire AFP molecule (37-mer) and six COOH-terminal peptide fragments (36-, 33-, 27-, 26-, 16-, and 15-mers). Peptides containing acidic NH2-terminal residues displayed greater helix formation and thermal stability compared to those peptides of similar size, but with neutral NH2-terminal residues. Helix formation was maximum above pH 9.2. The peptide conformations also displayed a pH-dependent sensitivity to changes in ionic strength. Helix formation was reduced in the presence of acetonitrile. We conclude that the AFP helix is most likely stabilized by: charge-dipole interactions between charged terminal amino acids and the helix dipole, a charge interaction between Lys18 and Glu22 (either a salt bridge or a hydrogen bond), and hydrophobic interactions. 相似文献
6.
7.
Differential recovery of membrane proteins after extraction by aqueous methanol and trifluoroethanol
Zhang H Lin Q Ponnusamy S Kothandaraman N Lim TK Zhao C Kit HS Arijit B Rauff M Hew CL Chung MC Joshi SB Choolani M 《Proteomics》2007,7(10):1654-1663
Cell membrane proteome analysis is limited by inherent membrane hydrophobicity. Conventional membrane protein extraction techniques use detergents, chaotropes and organic acids that require sample clean-up or pH adjustment, and are associated with significant sample loss. We extracted membrane proteins from red blood cells (RBCs) using methanol (MeOH), trifluoroethanol (TFE) and urea, and identified membrane proteins using 2-D LC coupled with MALDI-TOF/TOF-MS. We show that organic solvents MeOH- and TFE-based methods have membrane protein analysis efficiencies comparable to urea, and are complementary for the recovery of both hydrophilic and hydrophobic peptides. The mean grand average of hydropathicity (GRAVY) value of identified peptides from the TFE-based method (-0.107) was significantly higher than that of the MeOH-based method (-0.465) (p<0.001). Sequential and adjunctive use of the organic solvents MeOH and TFE increases the number of proteins identified, and the confidence of their identification. We show that this strategy is effective for shotgun membrane proteome analysis. 相似文献
8.
9.
Sulfate reduction and S-oxidation in a moorland pool sediment 总被引:1,自引:2,他引:1
In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO4
2– reduction rates in the sediment, (2) depletion of SO4
2– in the overlying water column and (3) release of35S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO4
2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core35SO4
2– injection. Rates of SO4
2– consumption in the overlying water were estimated by changes in SO4
2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m–2 d–1. Rates of SO4
2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m–2 d–1 (November) to 0.43–1.81 mmol m–2 d–1 (July, August and April). Maximum rates of oxidation to SO4
2– in July 1990 estimated by combination of SO4
2– reduction rates and rates of in situ SO4
2– uptake in the enclosed water column were 10.3 and 10.5 mmol m–2 d–1 at an organic rich and at a sandy site respectively.Experiments with35S2– and35SO4
2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO4
2– and (2) the presence of mainly non SO4
2–-S derived from reduced S transported from the sediment into the overlying water. A35S2– tracer experiment showed that about 7% of35S2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author 相似文献
10.
Tong L Lin Q Wong WK Ali A Lim D Sung WL Hew CL Yang DS 《Protein expression and purification》2000,18(2):175-181
HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) from the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium. Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG induction, a biologically active rAFP was expressed. The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm. After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC. Mass spectrometric and amino acid composition analyses confirmed the identity of the purified product. The rAFP, which lacked amidation at the C-terminal, was about 70% active when compared to the amidated wild-type protein, thus confirming the importance of C-terminal cap structure in protein stability and function. 相似文献