蛋白质氯胺-T双相碘标法的建立及其应用

常规的蛋白质碘标方法易引起被标细胞因子的失活,是受体配基竞争结合实验失败的原因之一.试用氯胺-T双相碘标法标记rhG-CSF和rhEPO,并应用受体配基竞争结合分析法测定NFS-60细胞G-CSF受体及BET-2细胞EPO受体的特性.结果显示所获 ¹²⁵I-EPO和 ¹²⁵I-G-CSF放射比活度均较高;发现BET-2细胞有高、低两种亲和力的EPO受体,NFS-60细胞只有一种高亲和力的G-CSF受体,所获结果与文献资料相一致.说明氯胺-T双相碘标法是细胞因子同位素碘标记的理想方法之一.     

Egg-white and blood-serum proteins functioning by noncovalent interactions: Studies by chemical modification and comparative biochemistry

Some of the more interesting and important proteins are those that function by forming associations or complexes with other substances. The structure-function relationships of three of these with very different substances are transferrins, which chelate metal ions; avian ovomucoids, which form complexes with proteolytic enzymes; and antifreeze glycoproteins, which interact at the ice-solution interface. Interrelating studies on the comparative biochemistry with studies using chemical modification have helped identify the side-chain groups of the proteins involved in function as well as to be useful for studies on general protein chemistry. The most strongly associated interaction is the chelation of iron by transferrin, with an association constant of 10²¹; tyrosines, histidines, and sometimes aspartate are involved. For ovomucoids, individual substratelike residues such as lysine are involved in a Michaelis-like complex, and association constants are as high as 10¹⁰. By contrast, the antifreeze glycoproteins appear to function by a polymeric interaction at the surface of ice, wiht a much weaker association.This paper comes at the time of the senior author's 75th birthday and also at the time of 28 years of still ongoing research between both authors, who have worked in close association in their laboratory at the University of California, Davis since 1960.     

Analysis of sequence requirements for protein tyrosine sulfation.

We analyzed sequences surrounding known tyrosine sulfation sites to determine the characteristics that distinguish these sites from those that do not undergo sulfation. Tests evaluated the number and position of acidic, basic, hydrophobic, and small amino acids, as well as disulfide and N-glycosylation (sugar) sites. We determined that composition-based tests that select close to 100% of known tyrosine sulfation sites reject 97% of the non-sulfated tyrosines. The acidic test, by far the most selective, eliminated 95% of the non-sulfated tyrosine residues and none of the sulfated tyrosines. Including the basic, hydrophobic, and disulfide tests increased the elimination rate to 97%. Whereas no position flanking the tyrosine residues had the same amino acid always present, imperfectly conserved amino acids found in some positions will improve the specificity of the tests.     

Bacitracin A: Structure-activity relationship

The single imidazole nucleus of L-histidine residue in bacitracin-A seems to be important for the anti-bacterial activity of the molecule, since iodination, carboxymethylation and coupling of diazobenzene sulphonic acid to the histidine residue in the antibiotic caused 90–94% loss of antibacterial activity of the antibiotic. In contrast, the bacitracin sulphone and sulphoxide derivatives are as active as the parent antibiotic.     

Analysis of thyroglobulin iodination by tandem mass spectrometry using immonium ions of monoiodo- and diiodo-tyrosine

Peptides containing a monoiodo- or diiodo-tyrosine residue (monoiodo-Y, diiodo-Y) were found to generate abundant immonium ions following collision-induced dissociation at m/z 261.97 and 387.87 Da, respectively. These residue-specific marker ions are between about 140 mDa (monoiodo-Y) and 300 mDa (diiodo-Y) mass deficient relative to any other peptide fragment ions of unmodified peptides, qualifying them as highly specific marker ions for tyrosine iodination when analyzed by high resolution tandem mass spectrometry (MS/MS). Two new iodination sites (Y-364 and Y-2165) were pinpointed in bovine thyroglobulin by MS/MS using these iodotyrosine-specific marker ions and combined tryptic/chymotryptic digestion.     

Structure and substrate recognition of the Staphylococcus aureus protein tyrosine phosphatase PtpA

Phosphosignaling through pSer/pThr/pTyr is emerging as a common signaling mechanism in prokaryotes. The human pathogen Staphylococcus aureus produces two low-molecular-weight protein tyrosine phosphatases (PTPs), PtpA and PtpB, with unknown functions. To provide the structural context for understanding PtpA function and substrate recognition, establish PtpA's structural relations within the PTP family, and provide a framework for the design of specific inhibitors, we solved the crystal structure of PtpA at 1 Å resolution. While PtpA adopts the common, conserved PTP fold and shows close overall similarity to eukaryotic PTPs, several features in the active site and surface organization are unique and can be explored to design selective inhibitors. A peptide bound in the active site mimics a phosphotyrosine substrate, affords insight into substrate recognition, and provides a testable substrate prediction. Genetic deletion of ptpA or ptpB does not affect in vitro growth or cell wall integrity, raising the possibility that PtpA and PtpB have specialized functions during infection.     

Peculiarities of glycosylation of proteins in spores of microsporidia Paranosema (Antonospora) grylli

The long-term adaptation of microsporidia, a large group of fungi-related unicellular microorganisms, to intracellular parasitism has led to extreme minimization of the cell functional apparatus. For instance, diversity of carbohydrates in the composition of parasite glycoproteins and proteoglycans seems to be restricted to the presence of O-bound chains composed of mannose residues. This suggestion is based on the discovery in the genome of the human microsporidian Encephalitozoon cuniculi of three genes responsible for the O-mannosylation of proteins with a lack of enzymes participating in N-glycosylation. In the present work, peculiarities of protein glycosylation in spores of the microsporidian Paranosema grylli infecting the fat body of the Mediterranean field cricket Gryllus bimaculatus was studied. SDS-PAGE analysis of spore proteins with subsequent staining by periodate and Schiff reagent has shown that individual glycoproteins of P. grylli are highly glycosylated, while the maximal stain intensity was seen in the major polar-tube protein PTP1. Treatment of the extracted material with N-glycosidase F and hybridization with WGA lectin conjugated with horseradish peroxidase showed no presence of glycosylated proteins in the P. grylli spores. At the same time, the selectively extracted major protein of the exospore p40 was specifically recognized by lectin GNA conjugated with agarose balls. Pretreatment of p40 with α-and β-mannosidases decreased considerably the efficiency of binding. Since lectin GNA is specific towards mannose terminal residues, this indicates the O-mannosylation of the microsporidial exospore major protein. In spite of the intensive PTP1 glycosylation, extracted proteins of the P. grylli polar-tube had no specific binding with GNA-agarose, so the issue of peculiarities of their glycosylation remains an open question. Comparison of the obtained data with results of deciphering of the E. cuniuculi genome allows for the conclusion to be made that the minimization of the glycosylation apparatus of microsporidial proteins is the common peculiarity of this group of parasites.     

In vivo effect of vitamin E on serum and tissue glycoprotein levels in perchloroethylene induced cytotoxicity

The antioxidant efficacy of vitamin E on Perchloroethylene (PER) induced cytotoxicity has been studied in rats. Feeding PER to rats for 42 days using sesame oil as vehicle alters total protein and protein bound carbohydrate components in liver and kidney of experimental animals. Supplementation of vitamin E prevented the changes observed in total protein and protein bound carbohydrate components of PER administered rats. Histopathological studies also show the effectiveness of vitamin E on PER administered rats in protecting the cellular architecture of liver and kidney from PER induced cytotoxicity.     

Protein-polysaccharide linkages in glycoproteins from Phaseolus vulgaris

Serine and hydroxyproline participate in protein-polysaccharide linkages in hydroxyproline-poor glycoproteins from Phaseolus vulgaris cv Pinto. Most substituted hydroxyproline residues contain arabinose, galactose and glucose, but some have arabinose only. Serine residues contain arabinose, galactose and glucose.     

Variation In Surface Proteins In Tetrahymena*

SYNOPSIS. Surface proteins of Tetrahymena were identified by lactoperoxidase iodination, and comparisons were made between a number of strains and species within the genus. an adequate procedure for strain comparisons was found to be solubilization of whole cells following iodination, separation of total cell protein using polyacrylamide gel electrophoresis, and identification of surface proteins by autoradiography of dried gels. the results obtained in the present study show the existence of both interspecific and intraspecific variation in surface proteins of Tetrahymena, but the differences tend to be small within species and large between species. the relation of these cell surface fingerprints to the present taxonomic designations within the genus is discussed. Questions are raised about the functional significance of these surface proteins.