Purification of the antibacterial fragments of guinea-pig major basic protein

We have shown that serum levels of a molecule immunochemically similar to eosinophil granule major basic protein (MBP) are elevated in pregnant women throughout gestation. MBP levels increase during gestation and plateau at approximately 7,500 ng/ml by the 20th wk (greater than 10-fold above normal). Levels return to normal after delivery, with a T1/2 of 13.7 d. The MBP in pregnancy serum is remarkably similar to the eosinophil granule MBP in that: (a) pregnancy MBP fully inhibits the binding of radiolabeled MBP standard in a double antibody radioimmunoassay; (b) this inhibition reaction is specific for human MBP because pregnancy serum produces no inhibition of the binding of radiolabeled guinea pig MBP in the guinea pig MBP radioimmunoassay; (c) in a two-site immunoradiometric assay for MBP, slopes of dose-response curves for pregnancy serum, purified MBP, and serum from a patient with hypereosinophilic syndrome are identical, and maximal binding is comparable; (d) reduction and alkylation of pregnancy sera increases measured MBP 100-fold, as previously shown for eosinophil granule MBP in serum; and (e) the MBP in pregnancy serum demonstrates the same pattern of heat lability as has been previously reported for MBP. Four observations have raised the possibility that the eosinophil is not the source of the MBP in pregnancy serum: (a) no correlation between serum MBP level and peripheral blood eosinophil count exists in pregnant women, in contrast to previous studies of patients with eosinophilia; (b) levels of three other eosinophil-associated proteins are normal or low in pregnancy sera, whereas the serum levels of these proteins are elevated in patients with eosinophilia; (c) the slopes of dose-response curves for pregnancy sera and MBP standards differ in the double antibody radioimmunoassay; and (d) the molecule in pregnancy serum elutes from Sephadex G-50 columns at the void volume, while eosinophil granule MBP and the MBP in serum of patients with eosinophilia elute at a volume consistent with the previously established molecular weight of 9,300. These findings suggest that the MBP in pregnancy serum is derived from a source other than the eosinophil.

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Guinea pig eosinophil major basic protein as a potent histamine releaser. (II) Structure-activity relationships of GMBP1 and GMBP2

Agents and Actions ◽

10.1007/bf01997342 ◽

1992 ◽

Vol 36 (S2) ◽

pp. C246-C249 ◽

Cited By ~ 1

Author(s):

K. Tasaka ◽

M. Mio ◽

S. Nakai ◽

I. Aoki

Keyword(s):

Guinea Pig ◽

Basic Protein ◽

Major Basic Protein ◽

Structure Activity Relationships ◽

Structure Activity ◽

Eosinophil Major Basic Protein

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Localization of the guinea pig eosinophil major basic protein to the core of the granule

The Journal of Cell Biology ◽

10.1083/jcb.77.3.702 ◽

1978 ◽

Vol 77 (3) ◽

pp. 702-713 ◽

Cited By ~ 69

Author(s):

DM Lewis ◽

JC Lewis ◽

DA Loegering ◽

GJ Gleich

Keyword(s):

Guinea Pig ◽

Basic Protein ◽

Core Protein ◽

Sodium Dodecyl ◽

Rabbit Antiserum ◽

Staining Procedure ◽

Normal Rabbit Serum ◽

Major Basic Protein ◽

The Core ◽

Eosinophil Major Basic Protein

The localization of the guinea pig eosinophil major basic protein (MBP) within the cell was investigated by the use of immunoelectron microscopy and by isolation of the granule crystalloids. First, by immunoperoxidase electron microscopy, we found that the MBP of eosinophil granules is contained within the crystalloid core of the granule. Specific staining of cores was present when rabbit antiserum to MBP was used as the first stage antibody in a double antibody staining procedure, whereas staining was not seen when normal rabbit serum was used as the first stage antibody. Second, crystalloids were isolated from eosinophil granules by disruption in 0.1% Triton X-100 and centrifugation through a cushion of 50% sucrose. Highly purified core preparations yielded essentially a single band when analyzed by electrophoresis on polyacrylamide gels containing 1% sodium dodecyl sulfate (SDS). The E1%1cm of the core protein was 26.8 +/- 1.0 (X +/- SEM); the E1%1cm for the MBP was 26.3. The core protein could not be distinguished from the MBP by radioimmunoassay (RIA) and essentially all of the protein in the core preparations could be accounted for as MBP. The results indicate that the MBP is contained in the core of the guinea pig eosinophil granule and that it is probably the only protein present in the core.

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Antiserum to the major basic protein of guinea pig eosinophil granules

Immunochemistry ◽

10.1016/0019-2791(76)90194-4 ◽

1976 ◽

Vol 13 (9) ◽

pp. 743-746 ◽

Cited By ~ 14

Author(s):

Daniel M. Lewis ◽

David A. Loegering ◽

Gerald J. Gleich

Keyword(s):

Guinea Pig ◽

Basic Protein ◽

Major Basic Protein

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Prostaglandin secretion by guinea pig tracheal epithelial cells caused by eosinophil major basic protein

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.265.3.l234 ◽

1993 ◽

Vol 265 (3) ◽

pp. L234-L242 ◽

Cited By ~ 5

Author(s):

S. R. White ◽

K. S. Sigrist ◽

S. M. Spaethe

Keyword(s):

Epithelial Cells ◽

Guinea Pig ◽

Basic Protein ◽

Primary Cultures ◽

Thromboxane B2 ◽

Major Basic Protein ◽

Tracheal Epithelial Cells ◽

Subsequent Response ◽

Tracheal Epithelial ◽

Eosinophil Major Basic Protein

We examined the effect of eosinophil major basic protein (MBP) on prostaglandin (PG) secretion from guinea pig tracheal epithelial (GPTE) cells. Primary cultures of GPTE cells were incubated with 10(-6) M MBP for up to 6 h and then stimulated with 10(-6) M bradykinin (BK). PGE2, 6-ketoprostaglandin F1 alpha (PGF1 alpha), PGF2 alpha, and thromboxane B2 (TxB2) concentrations in media were determined by enzyme-linked immunoabsorbent assay (EIA). Incubation with MBP for 6 h caused secretion of both PGE2 (17,614 +/- 4,416 vs. 1,426 +/- 555 pg/10(6) cells at baseline, P < 0.001, n = 7) and PGF2 alpha (20,303 +/- 5,724 vs. 3,790 +/- 1.075 pg/10(6) cells at baseline, P < 0.002, n = 7). Secretion of PGE2 and PGF2 alpha stimulated by MBP required at least 2 h. Incubation with MBP for 6 h also augmented the subsequent response to BK: PGE2 secretion was 29,215 +/- 6,853 vs. 3,445 +/- 1,041 pg/10(6) cells for BK alone (P < 0.0001), and PGF2 alpha secretion was 25,407 +/- 6,237 vs. 5,213 +/- 1,535 pg/10(6) cells for BK alone (P < 0.0001). MBP did not change 6-keto-PGF1 alpha and TxB2 secretion. Incubation of GPTE cells from seven animals with polylysine, a protein with mass and ion charge similar to MBP, for 2 h, both caused secretion of PGE2 (8,579 +/- 3,244 vs. 788 +/- 419 pg/10(6) cells at baseline, P < 0.01) and augmented the response to BK (12,732 +/- 4,788 vs. 1,653 +/- 680 pg/10(6) cells after BK alone, P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

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IDENTIFICATION OF A MAJOR BASIC PROTEIN IN GUINEA PIG EOSINOPHIL GRANULES

Journal of Experimental Medicine ◽

10.1084/jem.137.6.1459 ◽

1973 ◽

Vol 137 (6) ◽

pp. 1459-1471 ◽

Cited By ~ 127

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.

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