scholarly journals Carboxymethylation of methionine residues in bovine pituitary luteinizing hormone and its subunits. Effects on the binding activity with receptor sites and interactions between subunits

1976 ◽  
Vol 159 (1) ◽  
pp. 71-77 ◽  
Author(s):  
K W Cheng
Keyword(s):  
Biological Activity ◽  
Luteinizing Hormone ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Receptor Site ◽  
Iodoacetic Acid ◽  
Cross Reactivity ◽  
Receptor Interaction ◽  
Beta Subunits ◽  
Methionine Residues

The reaction of iodoacetic acid with bovine lutropin (luteinizing hormone) at pH 3.0 was specific for methionine residues; it was slow and reached its equilibrium after 12 h at 37 degrees C. The number of modified methionine residues increased proportionately with the amount of the alkylating reagent in the reaction mixture. In the presence of a 20-fold molar excess of iodoacetic acid with respect to methionine, essentially all methionine residues in both subunits of bovine lutropin were carboxymethylated. Studies of various recombinations of modified and native alpha and beta subunits showed that methionine residues in bovine lutropin were not essential for interactions between subunits. Various recombinants were characterized by polyacrylamide-gel electrophoresis and gel filtration of Sephadex G-100. Immunological cross-reactivity by radioimmunoassay of the recombinants of modified alpha and beta subunits was relatively similar to that of the native subunits. However, the biological activity measured by receptor-site binding of the recombinants of alpha and beta chains with a total of three alkylated methionine residues was less than 5% of the activity of native lutropin. It is noteworthy that recombinants of a modified subunit and a native counterpart subunit regenerated 20-30 % of biological activity. These findings suggested that at least 1-2 methionine residues in each subunit are involved in the hormone-receptor interaction for bovine lutropin.

scholarly journals Carboxymethylation of methionine residues in bovine pituitary luteinizing hormone and its subunits. Location of specifically modified methionine residues

1976 ◽  
Vol 159 (1) ◽  
pp. 79-87 ◽  
Author(s):  
K W Cheng
Keyword(s):  
Luteinizing Hormone ◽  
Gel Filtration ◽  
Receptor Site ◽  
Iodoacetic Acid ◽  
Binding Activity ◽  
Beta Subunits ◽  
Beta Chain ◽  
Tryptic Peptides ◽  
Alpha Chain ◽  
Methionine Residues

Bovine lutropin (luteinizing hormone) was carboxymethylated at pH3.0 for 12 h at 37 degrees C with iodoacetic acid for specific modification of methionine residues. To facilitate the location of preferentially modified methionine residues, iodoE114C]acetic acid was added as tracer. The alpha and beta subunits of bovine lutropin were carboxymethylated with a 2- or 5-fold molar excess of iodoacetic acid either in the presence or absence of their counterpart subunits. The modified subunits were separated and isolated by counter-current distribution followed by gel filtration on Sephadex G-100. To locate the modified methiones, the isolated alpha or beta chain was reduced. S-carboxymethylated and subjected to tryptic hydrolysis. The tryptic peptides were fractionated by gel filtration on Bio-Gel P-10. From analyses of the purified 14C-labelled tryptic peptides, it was observed that methionine-8 and -33 in bovine lutropin alpha chain and methionine-52 in the beta chain were preferentially modified. Similar results were obtained when isolated alpha and beta subunits were individually carboxymethylated in the absence of their counterpart subunit under identical conditions. The fact that a recombinant of native human lutropin alpha chain, in which a valine residue is present in the position corresponding to methionine-8 of bovine lutropin alpha chain, and carboxymethylated bovine lutropin beta chain regenerated a substantial amount of receptor-site-binding activity indicated that methionine-8 in bovine alpha chain was biologically not essential. These studies showed clearly that both methionine-33 in the alpha chain and methionine-52 in the beta subunit were involved for optimum binding between bovine lutropin and its receptors for expression of hormonal activity.

Isolation and partial characterization of tammar wallaby luteinizing hormone and development of a radioimmunoassay

1997 ◽  
Vol 9 (4) ◽  
pp. 475 ◽  
Author(s):  
James R. McFarlane ◽  
Carl D. Rudd ◽  
Lynda M. Foulds ◽  
Terry P. Fletcher ◽  
Marilyn B. Renfree
Keyword(s):  
Luteinizing Hormone ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Tammar Wallaby ◽  
Exchange Chromatography ◽  
Significant Rise ◽  
Brushtail Possum ◽  
Response Curves ◽  
Antechinus Stuartii

Tammar wallaby (Macropus eugenii) luteinizing hormone (LH) was purified from pituitaries collected from wild and captive populations by salt sequential precipitation, ion exchange chromatography and gel filtration. Pituitary tissue (5 g) yielded 1·8 mg of purified wallaby luteinizing hormone (ME-14B), as verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A heterologous radioimmunoassay has been developed for measurement of LH in plasma of marsupials using a monoclonal antibody raised against bovine LH (518B7). This assay system was able to measure basal LH concentrations in male and female tammars and detected a significant rise in plasma LH in response to oestradiol benzoate in female tammars and luteinizing hormone-releasing hormone (LHRH) in males. Parallel dose–response curves were also obtained from pituitary extracts from four other species of marsupial (brushtail possum, Trichosurus vulpecula; brown antechinus,Antechinus stuartii; kowari, Dasyuroides byrnei; and Eastern pygmy possum,Cercartetus nanus) in this assay, which suggests its usefulness in the measurement of LH in other marsupial species.

SUBUNITS OF HUMAN CHORIONIC GONADOTROPHIN: IMMUNOLOGICAL AND BIOLOGICAL STUDIES

1975 ◽  
Vol 79 (4) ◽  
pp. 749-766 ◽  
Author(s):  
S. Donini ◽  
I. D'Alessio ◽  
P. Donini
Keyword(s):  
Biological Activity ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Biologically Active ◽  
Β Subunit ◽  
Reactive Material ◽  
Biological Studies ◽  
The Cross

ABSTRACT The α and β subunits of human chorionic gonadotrophin (hCG) were prepared by incubation in 8 m urea, pH 4.5. The separation of the two subunits was obtained by DEAE-Sephadex A-25 chromatography and purification was carried out by gel filtration on Sephadex G-100. The β subunit obtained was biologically active and was therefore further purified by affinity chromatography using as immuno-adsorbent the α antibodies coupled to Sepharose 4B. The β subunit so purified showed a biological activity less than 1 IU/mg. The immunological and biological properties of the hCG subunits have been studied. It was found that the anti hCG β serum can discriminate between hCG and hLH and that in the 125I-hCG + anti-β serum radioimmunoassay, the cross-reactivity of pituitary hLH was lower than that of urinary hLH. Moreover, it was observed that the less purified was the urinary LH preparation, the higher was the cross-reactivity. Therefore we considered the hypothesis that during the purification of human menopausal gonadotrophin (hMG) some LH subunits or smaller immunoreactive fragments could have been discarded with the waste fractions. In order to test the validity of this hypophysis, all the protein fractions obtained during the purification of the hMG were gel-filtered on Sephadex G-100. The immunoreactivity of the effluents from the gel filtration was tested by hCG, hCG-β, hCG-α and hLH radioimmunoassays. While the α reactive material was found in some fractions as a peak having the same Ve/Vo value as hCG-α, the β reactive material present in the crude hMG fractions was not observed in other fractions. The cross-reactivity with the anti β serum was very low and was found in the LH region of the gel chromatogram. Furthermore, the neutralization of the biological activity of hCG and of urinary and pituitary LH by the anti hCG β serum was studied by incubating a fixed amount of the three hormones with increasing volumes of antiserum and measuring the LH activity after incubation by the OAAD test. It was observed that the anti hCG β serum inhibits hCG more than urinary or pituitary LH.

INFLUENCE OF THE PURITY OF THE IODINATED TRACER ON THE SPECIFICITY OF THE RADIOIMMUNOASSAY OF HUMAN LUTEINIZING HORMONE

1978 ◽  
Vol 89 (3) ◽  
pp. 506-520 ◽  
Author(s):  
H. Suginami ◽  
D. M. Robertson ◽  
E. Diczfalusy
Keyword(s):  
High Resolution ◽  
Luteinizing Hormone ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Biologically Active ◽  
Immunological Activity ◽  
Subunit A ◽  
Adsorption Step ◽  
Reference Preparation

ABSTRACT Human luteinizing hormone (HLH) iodinated with 125I by the use of a lactoperoxidase method, was fractionated by either cellulose adsorption, gel filtration or by the combination of these methods. The products of iodination were characterized by their in vitro biological LH activity and by their binding profiles with antisera to HLH, HLHα and HLHβ subunits. Several radioactive components were obtained after gel filtration with or without an initial cellulose adsorption step. One of these fractions was identified as biologically active HLH and another as the HLHα subunit. Radioimmunoassay studies were conducted with different iodinated fractions as tracers, using three well defined and widely available antisera to HLH. The standard used was the HLH International Reference Preparation for immunoassay (68/40). Cross-reactivity was examined with several purified pituitary preparations, such as HFSH, HTSH, HLHα and HLHβ subunit. A significantly higher cross-reactivity with HLHα, HFSH and HTSH was obtained with the [125I]HLHα fraction as tracer than with biologically active [125I]HLH. Furthermore, in the radioimmunoassay of HLH preparations of varying purity, significantly higher estimates of immunological activity were obtained with the [125I]HLHα tracer than with the biologically active [125I]HLH. It is concluded that the presence of [125I]HLHα in the [125I]HLH tracer can result in serious overestimates of the immunological activity in the measurement of LH. Therefore [125I]HLHα should be separated from [125I]HLH prior to radioimmunoassay. Many of the fractionation methods commonly used (cellulose adsorption and short column gel filtration systems) are inadequate for this purpose. However, an adequate separation can be achieved by the use of high resolution gel filtration systems.

scholarly journals Structural comparison of two esterases from Drosophila mojavensis isolated by immunoaffinity chromatography

1986 ◽  
Vol 238 (3) ◽  
pp. 691-699 ◽  
Author(s):  
J Pen ◽  
J Van Beeumen ◽  
J J Beintema
Keyword(s):  
Gel Electrophoresis ◽  
High Efficiency ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Gel Filtration ◽  
Immunoaffinity Chromatography ◽  
Cross Reactivity ◽  
Drosophila Mojavensis ◽  
Structural Comparison ◽  
Final Purification

Antibodies raised against esterase-4 and esterase-5 from Drosophila mojavensis were coupled to Protein A-Sepharose CL-4B to prepare high-efficiency immunomatrices used for their purification. Final purification was achieved by anion-exchange h.p.l.c., in the case of esterase-5 followed by gel-filtration h.p.l.c. The resultant esterase preparations were homogeneous, as judged by gel-filtration h.p.l.c., SDS/polyacrylamide-gel electrophoresis and non-denaturing gel electrophoresis. Esterase-4 and esterase-5 are the products of a duplicated gene. They are differently localized in the insect's body and expressed in different periods during development. Although both enzymes exhibit little immunological cross-reactivity, their amino acid compositions show few significant differences and their N-terminal sequences are largely identical, which clearly show their common origin.

Isolation, characterization and radioimmunoassay of luteinizing hormone in the brushtail possum

1997 ◽  
Vol 9 (4) ◽  
pp. 419 ◽  
Author(s):  
L. G. Moore ◽  
W. Ng Chie ◽  
S. Lun, S. B. Lawrence ◽  
D. A. Heath ◽  
K. P. McNatty
Keyword(s):  
Amino Acid ◽  
Luteinizing Hormone ◽  
Plasma Concentrations ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Cross Reactivity ◽  
Brushtail Possum ◽  
Corpora Lutea ◽  
Rate Of Increase ◽  
Pituitary Glands

Luteinizing hormone (LH) was purified from brushtail possum (Trichosurus vulpecula) pituitary glands. The purification procedure consisted of ammonium sulfate precipitation followed by triazinyl-dye chromatography, hydrophobic interaction chromatography and gel filtration. A yield of 10 µg LH g-1 pituitary with a recovery of 20% was obtained from 1400 pituitary glands (20·3 g). Contamination with possum follicle-stimulating hormone (FSH) was ≤0.05%. The amino acid analysis and the N-terminal sequencing for 10 cycles revealed close homology with LH from other mammals. Minor amounts of LH that had been truncated near the N-terminal were also detected. No contaminating proteins were found by amino acid sequencing. The potency of possum LH was 20% that of ovine LH in a receptor assay using possum testicular receptors and 4% that of ovine LH when bovine corpora lutea receptors were used. Possum LH was able to stimulate production of cyclic adenosine 3′ ,5′-monophosphate by bovine granulosa cells. A radioimmunoassay (RIA) for possum LH using 125I-possum LH and an antiserum raised against ovine LH was developed. The RIA has a sensitivity of 0·15 ng mL-1 , a 50% displacement of 1·9 ng mL-1 and a cross-reactivity of <0 · 02% against possum FSH. Plasma concentrations were 0·24 ± 0· 04 ng mL-1 (n = 8) and 0·39 ± 0·12 ng mL-1 (n = 8) in female and male possums respectively. Administration of mammalian gonadotrophin-releasing hormone (GnRH) and chicken GnRH II stimulated increases in plasma LH concentrations in male and female possums. When comparing LH responses with administration of mammalian GnRH or chicken GnRH II, plasma LH concentrations remained elevated for a longer period of time in males than in females (P < 0· 01); plasma LH concentrations also remained elevated for longer after mammalian GnRH than after chicken GnRH II (P < 0· 01). Gonadectomy stimulated an increase in plasma concentrations of LH in both male (P < 0· 01) and female (P < 0· 05) possums. The rate of increase in plasma LH concentrations in males was faster than that in females. In summary, we have purified, partially characterized, and developed a RIA for possum LH.

scholarly journals Purification of two hexosaminidases from human kidney

1977 ◽  
Vol 163 (1) ◽  
pp. 133-140 ◽  
Author(s):  
D V Marinkovic ◽  
J N Marinkovic
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Kidney ◽  
Protein Band ◽  
Iodoacetic Acid ◽  
Homogeneous State ◽  
Molecular Weights ◽  
Hexosaminidase A

Hexosaminidase forms A and B were isolated from human kidney in a homogeneous state as demonstrated by electrophoretic and enzymic criteria. The enzymes were stable for at least 18 months when stored at -20 degrees C in 0.025 M-phosphate buffer, pH 6.5. The molecular weights of forms A and B were estimated by gel filtration to be 111 000 +/- 1500 and 114 000 +/- 1600 respectively. The molecular weights of hexosamidase A and B subunits were determined by using polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Hexosaminidase A dissociated into one subunit with mol.wt. 68 000. Hexosaminidase B dissociated into three subunits with mol. wts. 100 000, 68 000 and 37000 respectively, and one protein band of mol.wt. 140 000. After treatment of hexosaminidases A and B with iodoacetic acid, the molecular weights of the carboxymethylated polypeptide subunits were also estimated. Carboxymethylated hexosaminidase A dissociated into one major subunit of mol.wt. 18 000 and two other protein bands of mol.wts. 65 000 and 100 000. Carboxymethylated hexosaminidase B dissociated into one major subunit for mol.wt. 19 000 and an additional band of mol.wt. 37 000. The Km of the enzymes for the synthetic substrate p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside was 0.8 mM. Both enzymes were inhibited or activated by various metal ions. Double pH optima for the enzymes were found at pH 4.5 and 4.8.

scholarly journals Isolation and characterization of the subunits of bovine follitropin

1978 ◽  
Vol 175 (1) ◽  
pp. 29-34 ◽  
Author(s):  
K W Cheng
Keyword(s):  
Glutamic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Methionine Residue ◽  
Isolation And Characterization ◽  
Receptor Assays

Highly purified bovine follitropin was dissociated into its alpha- and beta-subunits after treatment with 1 M-propionic acid. The dissociated subunits were fractionated by chromatography on DEAE-cellulose and further purified by gel filtration on Sephadex G-100. The isolated alpha- and beta-subunits were biologically inactive, but their recombinants regenerated 80% of the follitropin activity. The alpha-subunit of bovine follitropin recombined with the beta-subunits of bovine lutropin and thyrotropin to regenerate 70% of lutropin and 50% of thyrotropin activities respectively. The beta-subunit of bovine follitropin recombined with the alpha-subunit of either bovine lutropin or thyrotropin to regenerate about 75% of follitropin activity. Recombinations were monitored by specific radioligand-receptor assays and polyacrylamide-gel electrophoresis. The elution volumes of the alpha- and beta-subunits of bovine follitropin after gel filtration on Sephadex G-100 were almost identical. The amino acid composition of bovine follitropin-alpha was low in histidine, arginine, isoleucine and leucine, but relatively high in lysine, threonine and glutamic acid. The bovine follitropin-beta contained one methionine residue and low amounts of histidine and phenylalanine, but relatively high in aspartic acid, threonine and glutamic acid. The N-terminal residues of the alpha- and beta-subunits of bovine follitropin were identified to be phenylalanine and glycine respectively.

scholarly journals Isolation of material displaying insulin-like immunological biological activity from the brain of the blowfly Calliphora vomitoria

1979 ◽  
Vol 184 (2) ◽  
pp. 221-227 ◽  
Author(s):  
H Duve ◽  
A Thorpe ◽  
N R Lazarus
Keyword(s):  
Biological Activity ◽  
Neurosecretory Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Insulin Receptors ◽  
Neurosecretory Cells ◽  
Bovine Insulin ◽  
Fat Cell ◽  
Median Neurosecretory Cells ◽  
The Brain

An insulin-like material from the brain of the blowfly Calliphora vomitoria was partially purified by acid alcohol extraction, gel filtration and ion-exchange cellulose chromatography. In addition, the RF value on polyacrylamide-gel electrophoresis was determined. The material was characterized by its ability to cross-react with bovine insulin antibody and by displaying diminished immunoreactivity on dilution. It displaced specifically bound 125I-labelled insulin from rat liver plasma membrane insulin receptors and displayed insulin-like biological activity on the isolated rat fat-cell. Within 30 min of injection into Calliphora, made hypertrehalocaemic and hyperglucaemic as a result of median neurosecretory cell removal, it caused the concentrations of both sugars to return to normal. The hypothesis is put forward that the median neurosecretory cells are the source of the material.

Purification and properties of extracellular endoxylanases from alkalophilic thermophilic Bacillus sp.

1992 ◽  
Vol 38 (5) ◽  
pp. 436-442 ◽  
Author(s):  
Devyani Dey ◽  
Jyoti Hinge ◽  
Abhay Shendye ◽  
Mala Rao
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
Bacillus Sp ◽  
Thermophilic Bacillus ◽  
Molecular Weights ◽  
Purification And Properties ◽  
Similar Temperature

An alkalophilic thermophilic Bacillus sp. (NCIM 59) isolated from soil produced two types of cellulase-free xylanase at pH 10 and 50 °C. The two enzymes (xylanase I and II) were purified to homogeneity by ethanol precipitation followed by Bio-Gel P-10 gel filtration and preparative polyacrylamide gel electrophoresis. The molecular weights of xylanase I and II were estimated to be 35 000 and 15 800, respectively, by sodium dodecyl sulfate gel electrophoresis. The enzymes exhibited immunological cross-reactivity and were glycoproteins. They had similar temperature (50–60 °C) and pH (6) optima. Both xylanases were stable at 50 °C at pH 7 for 4 days. However, xylanase I was comparatively more stable than xylanase II at 60 °C. The isoelectric points of xylanase I and II were 4 and 8, respectively. The apparent Km values, using xylan as substrate, were 1.58 and 3.5 mg/mL, and Vmax values were 0.0172 and 0.742 μmol∙min−1∙mg−1, respectively. Both xylanases were inhibited by N-bromosuccinimide, suggesting the involvement of tryptophan in the active site. The hydrolysis patterns demonstrated that the xylanases were endoenzymes. Xylanase I and II yielded mainly xylobiose, xylotriose, and higher xylooligosaccharides, with traces of xylose from xylan. Key words: cellulase-free xylanase, alkalophilic thermophilic Bacillus sp., enzyme purification, characterization.

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