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Hormonotoxins: the role of positive charge of lysine residue on the immunological, biological and cytotoxic properties of ovine lutropin-S-S-gelonin conjugates

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Abstract

Since the positive charge on the lysine residues plays an important role in the receptor recognition ability of oLH, the hormonotoxin has been synthesised with the use of 2-iminothiolane HC1 (2IT) and N-Succinimidyl-3-(2-pyridyldithio)-propionate (SPDP). The oLH activated with 2IT (oLH-10) was then mixed with SPDP activated gelonin (gelonin-30) in order to obtain a oLH-S-S-gelonin hormonotoxin. The conjugation mixture containing hormonotoxin was purified by gel-filtration chromatography according to the molecular weight and a complete physico-chemical, immunochemical and biochemical analysis were performed. The linkage occured through the ε-NH2 groups of α-subunit of oLH as judged from RP-HPLC analysis. A 1∶1 (oLH:gelonin) molar ratio was obtained when determined with the use of several techniques. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity. The competitive displacement analysis indicate that the binding occurs via the hormone part leaving the gelonin free which was probed with the gelonin antibodies. The presently described (C150A-02, C160A-02 and C170A-02) hormonotoxins exhibited higher receptor binding and toxicity to the target cells than the hormonotoxins prepared with the use of SPDP only. Therefore it is concluded that higher receptor binding and cytotoxicity may be due to the retention of positive charge on the lysine residues of oLH which was preserved during the conjugation process.

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Abbreviations

BSA:

Bovine Serum Albumin

CMC:

Carboxy methyl Cellulose

DTT:

Dithiothreitol

DMEM:

Dulbeco's Modified Eagle's Medium

DTNB:

Ellman's reagent [5,5′-dithio-bis-(2-nitrobenzoic acid)]

EDTA:

Ethylenediaminetetraacetic acid

FPLC:

Fast Protein Liquid Chromatography

FCA:

Freund's Complete Adjuvant

FCS:

Fetal Calf Serum

Gelonin-30:

Gelonin modified by SPDP

GnRH:

Gonadotropin-Releasing Hormone

Gelonin-SPDP:

SPDP modified derivative of gelonin

HEPES:

(N-[2-hydroxyethyl] piperazine-N′-[-2-ethanesulphonic acid])

IFA:

Incomplete Freund's Adjuvant

2IT:

2-Iminothiolane

IODOGEN:

1,3,4,6-tetrachloro 3α,6α-diphenylglycouril

oLH:

Ovine Luteinizing Hormone

oLH-SPDP:

SPDP modified derivative of oLH

oLH-10:

oLH modified by 2IT

oLH∶2IT:

Molar ratio of oLH and 2IT

PDP:

2-Pyridyl-dithiopropionate

PAP:

Pokeweed Antiviral Protein

RIP:

Ribosome Inactivating Protein

RP-HPLC:

Reverse-Phase High Performance Liquid Chromatography

RPMI:

Roswell Park Memorial Institute

RIA:

Radioimmunoassay

RRA:

Radioreceptor Assay

SPDP:

N-Succinimidyl-3(2-pyridyldithio)propionate

SDS-PAGE:

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

TCA:

Trichloroacetic acid

TFA:

Trifluroacetic acid

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Authors and Affiliations

  1. Institute of Self Organising Systems and Biophysics, North-Eastern Hill University, Permanent Campus, 793 022, Shillong, Meghalaya, India

    Vinod Singh

  2. Department of Biology, Washington University, 63130, St. Louis, Missouri, USA

    Roy Curtiss III

Authors
  1. Vinod Singh
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  2. Roy Curtiss III
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Singh, V., Curtiss, R. Hormonotoxins: the role of positive charge of lysine residue on the immunological, biological and cytotoxic properties of ovine lutropin-S-S-gelonin conjugates. Mol Cell Biochem 130, 91–101 (1994). https://doi.org/10.1007/BF01084272

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  • DOI: https://doi.org/10.1007/BF01084272

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