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Molecular and Cellular Biochemistry

, Volume 120, Issue 2, pp 95–102 | Cite as

Disulfide linked αoLH-gelonin conjugate failed to recombine with βoLH subunit to generate bioeffective hormonotoxin

  • Vinod Singh
  • Roy CurtissIII
Article
  • 21 Downloads

Abstract

Since, linking of ovine luteinizing hormone (oLH) to ribosome inactivating protein gelonin (in oLH-gelonin conjugate) occur via the alpha-subunit, αoLH, an attempt has been made to develop a ‘universal’ hormonotoxin for selective targeting to specific cells in the gonads. Four different molar ratios of oLH and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) were used to activate the epsilon amino (ψ-NH2) groups of αoLH. The αoLH-SPDP derivatives recombine to native beta subunit of oLH (βoLH) and the purified recombinants retained substantial receptor binding, steroidogenic activity and immunoreactivity to native oLH. The disulfide linked αoLH-S-S-gelonin conjugates prepared by SPDP method were purified by gel filtration chromatography and analysed by reverse-phase high performance liquid chromatography (RP-HPLC). In order to obtain specificity and bioeffectivity, the αoLH-S-S-gelonin conjugates were allowed to recombine to native βoLH and the recombination mixture was further purified by gel-filtration chromatography. The RP-HPLC analysis of these recombinants indicated that αoLH-S-S-gelonin did not recombine to βoLH. The failure of recombination may be due to the reasons. (i) The site of ε-NH2 activation by SPDP may be different in the αoLH than the native oLH. (ii) The activation site may be in close proximity to the annealing site which facilitates the recombination of β-subunit but failured to reassociate to αoLH-S-S-gelonin conjugate. (iii) The introduction of gelonin (30 kDa basic protein) might have induced some steric hinderence for βoLH to recombine to the αoLH site which might have been masked in αoLH-S-S-gelonin conjugates. (Mol Cell Biochem120: 95–102, 1993)

Key Words

gonadotropin immunoreactivity receptor binding steroidogenesis 

Abbreviations

oLH

ovine Luteinizing Hormone

αoLH

alpha subunit of oLH

βoLH

beta subunit of oLH

BSA

Bovine Serum Albumin

DTT

Dithiothreitol

RP-HPLC

Reverse Phase High Performance Liquid Chromatography

TSH

Thyroid Stimulating Hormone

FSH

Follicle Stimulating Hormone

LH

Luteinizing Hormone

eCG

equine Chorionic Gonadotropin

DMEM

Dulbecco's Modified Eagles Medium

HEPES

4-(2-Hydroxyethyl)-1 Piperazine Ethane Sulfonic acid

PAP

Pokeweed Antiviral Protein

RIA

Radioimmunoassay

hCG

human Chorionic Gonadotropin

TRH

Thyrotropin Releasing Hormone

CRF

Corticotropin Releasing Factor

hPL

human Placental Lactogen

TFA

Trifluroacetic Acid

αoLH-SPDP

SPDP activated derivative of αoLH

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Copyright information

© Kluwer Academic Publishers 1993

Authors and Affiliations

  • Vinod Singh
    • 1
  • Roy CurtissIII
    • 2
  1. 1.Institute of Self Organising Systems and BiophysicsNorth-Eastern Hill UniversityShillongIndia
  2. 2.Department of BiologyWashington UniversitySt. LouisUSA

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