scholarly journals PHYSIOCHEMICAL AND BIOLOGICAL PROPERTIES OF THE MAJOR BASIC PROTEIN FROM GUINEA PIG EOSINOPHIL GRANULES

1974 ◽  
Vol 140 (2) ◽  
pp. 313-332 ◽  
Author(s):  
Gerald J. Gleich ◽  
David A. Loegering ◽  
Friedrich Kueppers ◽  
Satya P. Bajaj ◽  
Kenneth G. Mann
Keyword(s):  
Guinea Pig ◽  
Disulfide Bonds ◽  
Basic Protein ◽  
Biological Properties ◽  
Low Molecular Weight ◽  
Major Basic Protein ◽  
Guanidinium Chloride ◽  
E Coli ◽  
Single Polypeptide Chain ◽  
Single Polypeptide

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.

scholarly journals IDENTIFICATION OF A MAJOR BASIC PROTEIN IN GUINEA PIG EOSINOPHIL GRANULES

1973 ◽  
Vol 137 (6) ◽  
pp. 1459-1471 ◽  
Author(s):  
Gerald J. Gleich ◽  
David A. Loegering ◽  
Jorge E. Maldonado
Keyword(s):  
Molecular Weight ◽  
Guinea Pig ◽  
Peroxidase Activity ◽  
Basic Protein ◽  
Low Molecular Weight ◽  
Major Basic Protein ◽  
Cationic Protein ◽  
Major Band ◽  
Electrophoretic Patterns ◽  
Eosinophil Granule

Elucidation of the functions of the eosinophil might be accomplished by analysis of the granule constituents. We have purified eosinophils (93% or greater) from the peritoneal cavity of the guinea pig and have investigated a variety of methods to disrupt cells and liberate intact granules. Lysis in 0.34 M sucrose gave the best yield of granules and these had the characteristic morphology of eosinophil granules when examined by electron microscopy. Granules were solubilized by a variety of treatments and the solutions analyzed by polyacrylamide electrophoresis at pH 3 in 6 M urea. Comparison of the electrophoretic patterns of solubilized eosinophil and neutrophil granules revealed a difference: a major portion (53±3%; x ±1 SE) of the protein from the eosinophil granule migrated as a single component. This major band protein has a molecular weight between 6,000 and 12,000 daltons and a pI of 10 or greater. Analysis of eosinophil granule constituents on Sephadex G-50 revealed two main peaks; peak 1 possessed peroxidase activity and peak 2 contained the major band protein. These studies indicate that eosinophil granules contain a cationic protein of low molecular weight which lacks peroxidase activity and which accounts for greater than 50% of granule protein.

scholarly journals Full Capacity of Recombinant Escherichia coliHeat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity

2000 ◽  
Vol 68 (7) ◽  
pp. 4064-4074 ◽  
Author(s):  
Isabelle Batisson ◽  
Maurice Der Vartanian ◽  
Brigitte Gaillard-Martinie ◽  
Michel Contrepois
Keyword(s):  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Biological Properties ◽  
Leader Peptide ◽  
Dependent Manner ◽  
Reporter Protein ◽  
E Coli ◽  
Heat Stable

ABSTRACT We have successfully used the major subunit ClpG ofEscherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH2 or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface ofE. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

Measurement of guinea pig eosinophil major basic protein by radioimmunoassay

1979 ◽  
Vol 16 (9) ◽  
pp. 711-719 ◽  
Author(s):  
Donald L. Wassom ◽  
David A. Loegering ◽  
Gerald J. Gleich
Keyword(s):  
Guinea Pig ◽  
Basic Protein ◽  
Major Basic Protein ◽  
Eosinophil Major Basic Protein

scholarly journals Acidic precursor revealed in human eosinophil granule major basic protein cDNA.

1988 ◽  
Vol 168 (4) ◽  
pp. 1493-1498 ◽  
Author(s):  
R L Barker ◽  
G J Gleich ◽  
L R Pease
Keyword(s):  
Endoplasmic Reticulum ◽  
Nucleotide Sequence ◽  
Cdna Library ◽  
Basic Protein ◽  
Cell Types ◽  
Major Basic Protein ◽  
Human Eosinophil ◽  
Damage Cell ◽  
Eosinophil Granule ◽  
Single Polypeptide

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and various cell types, is a 13.8-kD single polypeptide rich in arginine with a calculated isoelectric point (pI) of 10.9. A cDNA for human MBP was isolated from a gamma GT10 HL-60 cDNA library. The nucleotide sequence of the MBP cDNA indicates that MBP is translated as a 25.2-kD preproprotein. The 9.9-kD pro-portion of proMBP is rich in glutamic and aspartic acids and has a calculated pI of 3.9, while proMBP itself has a calculated pI of 6.2. We suggest that MBP is translated as a nontoxic precursor that protects the eosinophil from damage while the protein is processed through the endoplasmic reticulum to its sequestered site in the granule core toxic MBP, and we present results from the literature suggesting that other cationic toxins, which damage cell membranes, may also be processed from nontoxic precursors containing distinct anionic and cationic regions.

scholarly journals Biosynthesis of pro-C3, a precursor of the third component of complement.

1977 ◽  
Vol 146 (3) ◽  
pp. 759-765 ◽  
Author(s):  
V Brade ◽  
R E Hall ◽  
H R Colten
Keyword(s):  
Disulfide Bonds ◽  
Polypeptide Chain ◽  
Peritoneal Macrophages ◽  
Tissue Cultures ◽  
The Third ◽  
Single Polypeptide Chain ◽  
Single Polypeptide ◽  
Polypeptide Chains ◽  
Third Component Of Complement

A precusor of the third component of complement, pro-C3, was detected in studies of cell-free synthesis and intracellularly in homogenates of liver tissue cultures. The molecular weight of pro-C3 was indistinguishable from that of intact native C3 secreted in vitro by liver or peritoneal macrophages, but its structure was different. Pro-C3 is a single polypeptide chain, whereas C3 secreted by cells in culture consists of two polypeptide chains (mol wt 120,000 and 76,000) linked by disulfide bonds.

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein

FEBS Letters ◽  
1991 ◽  
Vol 279 (2) ◽  
pp. 330-334 ◽  
Author(s):  
Ikuo Aoki ◽  
Yutaka Shindoh ◽  
Tsutomu Nishida ◽  
Satoru Nakai ◽  
Yeong-Man Hong ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Basic Protein ◽  
Major Basic Protein ◽  
Eosinophil Major Basic Protein
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