EXISTENCE OF BIG AND LITTLE FORMS OF LUTEINIZING HORMONE IN HUMAN SERUM

1976 ◽  
Vol 83 (3) ◽  
pp. 466-482 ◽  
Author(s):  
D. Graesslin ◽  
F. A. Leidenberger ◽  
V. Lichtenberg ◽  
D. Glismann ◽  
N. Hess ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Molecular Size ◽  
Normal Subjects ◽  
Exchange Chromatography ◽  
Pituitary Hormone ◽  
Gel Chromatography ◽  
Molecular Weight Range ◽  
Radioligand Receptor Assay ◽  
Gel Isoelectric Focusing

ABSTRACT Serum fractions from normal subjects obtained by gel chromatography have been investigated using three different assay systems: radioimmunoassay (RIA), radioligand receptor assay (RRA), and testosterone production assay (TPA). The bulk of immunoassayable and "bioassayable" LH-activity was found in two fractions differing widely in their molecular size. The slower moving component, designated as "little" LH, migrated identical to the radioiodinated pituitary hormone (LER 960) with a molecular weight of about 30 000, while "big" LH appeared in an elution volume consistent with a molecular weight range between 140 000 and 180 000. Concordance was seen between the LH-activities measured in all three assay systems. The RRA/RIA ratio varied between 1.6 and 8.9, the RRA/TPA ratio was close to unity. Treatment with 6 m urea and 0.1 % mercaptoethanol and also, exposure to different pH values and salt concentrations did not change the elution position of the two LH components. Also, "big" and "little" LH appeared unaltered after re-filtration and no conversion each other could be found. In another experiment injection of gonadotrophin releasing hormone (Gn-RH) into a male induced a profound shift of LH towards the low molecular weight species. Kinetic uptake studies with "big" and "little" LH using RRA showed identical affinities to the receptor preparation. Ion exchange chromatography of serum, however, did not give two LH components, indicating no major differences in charge properties. This finding could be confirmed by preparative gel isoelectric focusing. The RRA potencies following gel filtration were in good agreement with that applied to the column, however, the immunological activities exceeded that of loaded by a factor 3–4. A new aspect of serum LH heterogeneity is the finding of a low molecular substance (mol. weight approximately 1000) in the outer dialysate of serum, which has LH like activity in all three assay systems.

scholarly journals The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Colin H. Self ◽  
P. David J. Weitzman
Keyword(s):  
Molecular Weight ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Concentrations ◽  
Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

scholarly journals Purification and partial characterization of superoxide dismutase from the thermophilic bacteria Thermothrix sp

2004 ◽  
Vol 69 (1) ◽  
pp. 9-16 ◽  
Author(s):  
Svetlana Seatovic ◽  
Ljubinka Gligic ◽  
Zeljka Radulovic ◽  
Ratko Jankov
Keyword(s):  
Molecular Weight ◽  
Superoxide Dismutase ◽  
Specific Activity ◽  
Thermophilic Bacteria ◽  
Noncovalent Interactions ◽  
Gel Filtration ◽  
Antioxidant Defense System ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Sod Activity

Superoxide dismutase (SOD; EC 1.15.1.1), a high molecular weight component of the antioxidant defense system, provided promising results in the treatment of oxidative damage. Thermothrix sp., isolated from thermal spa water in Serbia, showed high superoxide dismutase activity. The SOD, from cell free extract, was purified to homogenity by ammonium sulfate precipitation, Sephadex G 75 gel filtration chromatography and QAE Sephadex ion exchange chromatography. The specific activity of the purified enzyme was 9191 U/mg. The purified enzyme was analyzed and partially characterized. SOD was localized in polyacrylamide gel by activity staining, based on the reduction of nitroblue tetrazolium (NBT) by superoxide. The enzyme molecular weight determined by gel chromatography is 37 kD. According to SDS PAGE it is composed of two subunits of equal size, joined by noncovalent interactions. The isoelectric point, assessed by isoelectric focusing is 5.3. The optimum pH for enzyme activity was in the range of 8 to 10. The optimum temperature for SOD activity was 60 ?C. After one hour of incubation at 40, 50 and 60 ?C the SOD activity increases, but at 80 ?C, the SOD is denaturated. After 24 hours of incubation at 25 ?C SO Dactivity only slightly decreases.

scholarly journals The isolation and partial characterization of low-molecular-weight phosphorylated component of the non-histone proteins of mouse nuclei

1978 ◽  
Vol 175 (1) ◽  
pp. 35-46 ◽  
Author(s):  
A J MacGillivray ◽  
C Johnston ◽  
R MacFarlane ◽  
D Rickwood
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Low Molecular Weight ◽  
Gel Chromatography ◽  
Liver Nuclei ◽  
Acidic Proteins

After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.

Size heterogeneity of circulating growth hormone in acromegaly. "Big-Big" GH forms are associated with inappropriately low IGF-I levels

1991 ◽  
Vol 125 (2) ◽  
pp. 150-159 ◽  
Author(s):  
Maura Arosio ◽  
Marina Nissim ◽  
Maria Ballabio ◽  
Rita Orefice ◽  
Nicoletta Bazzoni ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Molecular Size ◽  
Normal Subjects ◽  
Good Correspondence ◽  
Igf I ◽  
Size Heterogeneity ◽  
Serum Gh

Abstract. Circulating GH consists of several molecular size species with different biological activity. A reduced sensitivity of some monoclonal antibodies towards high-molecular weight GH variants has been reported. The aim of the present work was to evaluate the molecular size species of circulating GH using Sephadex G-100 gel filtration chromatography in acromegalic patients and in normal subjects employing both RIA and an immunoradiometric assay for all GH determinations. In 6 normal subjects, studied under GHRH stimulation, little GH was 69.8±6% (mean ±sd), big GH (44 kD) 26.4±6% and big-big GH (>80 kD) 2.8±4%, in IRMA, with a good correspondence with RIA results (70.8±8, 27.0±4, and 3.2±2%, respectively). In 13 untreated acromegalic patients, studied in basal conditions, the little form constituted 76.2±7%, the big form 18.3±4%, which is significantly lower than in normals (p<0.05), and the big-big form 5.5±7%. Similar results were obtained with RIA. A clear elevation of big-big GH (21% for both in IRMA, and 15.7 and 27.8% in RIA) was found in 2 patients with IGF-I levels lower than expected on the basis of mean GH concentrations. The study was extended to an additional acromegalic patient, previously operated and irradiated on, characterized by discrepant serum GH levels in RIA (4.6 μg/l), and in IRMA (1.4 μg/l), and by normal IGF-I levels. Serum GH showed a lack of parallelism to standard GH in RIA, but not in IRMA. RIA immunoreactivity was almost completely composed (92%) of a high molecular weight GH form (>90 kD), not recognized by IRMA. All IRMA immunoreactivity eluted with a Kav corresponding to 19–50 kD. In conclusion: a. the three main molecular size isomers of serum GH are similarly recognized by IRMA and RIA methods in normal subjects. b. in acromegaly, both quantitative and qualitative modifications of the GH chromatographic profile may be present. In particular, increased amounts of big-big forms, whether or not recognized by monoclonal antibodies, have been observed. Their lower bioactivity, suggested by the normal or lower than expected IGF-I levels, can account for the discrepancy between serum GH levels and the clinical picture or IGF-I levels sometimes observed in acromegaly.

Identification of N-Monoacetylcystine in Uraemic Plasma

1981 ◽  
Vol 60 (3) ◽  
pp. 331-334 ◽  
Author(s):  
F. Gejyo ◽  
G. Ito ◽  
Y. Kinoshita
Keyword(s):  
Plasma Levels ◽  
Gel Filtration ◽  
Normal Subjects ◽  
Exchange Chromatography ◽  
Cysteic Acid ◽  
Homocysteic Acid ◽  
Normal Limits ◽  
Acidic Nature ◽  
Sulphur Amino Acids

1. An unidentified ninhydrin-positive substance of an acidic nature was detected in the plasma of uraemic patients. This substance was isolated from haemodialysate by ion-exchange chromatography and gel filtration, and identified as a sulphur-containing amino acid: N-monoacetylcystine. 2. The quantitative determination of sulphur amino acids in plasma revealed that the plasma levels of cysteic acid, homocysteic acid, taurine, cystine and cystathionine as well as N-monoacetylcystine in uraemic patients were markedly higher than in normal subjects (P < 0.001 for each). However, the plasma levels of methionine in uraemic patients were within normal limits.

Effect of extractant and molecular size on the optical and chemical properties of soil humic acids

Soil Research ◽  
1969 ◽  
Vol 7 (3) ◽  
pp. 229 ◽  
Author(s):  
JHA Butler ◽  
JN Ladd
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Humic Acids ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Molecular Size ◽  
Carboxyl Content ◽  
Peptide Bonds ◽  
Molecular Weights ◽  
Amino Acid Contents

Humic acids extracted from soil with sodium pyrophosphate have greater proportions of lower molecular weight material, less acid-hydrolysable amino acid nitrogen contents, but greater carboxyl contents and extinction values (260 and 450 nm) than humic acids extracted subsequently from the same sample with alkali. Humic acids extracted with alkali from fresh soil samples have intermediate values. Extinction values at 260 nm are directly correlated with carboxyl contents for a given soil. Different crop histories have no significant effect on the measured properties of the extracted humic acids. An alkali-extracted humic acid has been fractionated by gel filtration into seven fractions of different nominal molecular weight ranges. As the molecular weights of the fractions increase, both aliphatic C-H (based on infrared absorption at 2900 cm-1) and acid-hydrolysable amino acid contents increase, whereas extinction values at 260 nm and carboxyl contents decrease. The infrared spectra of the high molecular weight fractions have peaks at 1650 and 1510 cm-1 which correlate with acid-hydrolysable amino acid contents and which correspond to amide I and II bands of peptide bonds. Alkaline hydrolysis to split peptide bonds eliminates both these peaks. The spectra also have peaks at 1720 and 1210 cm-1 which correlate with the carboxyl content.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

STRUCTURE ACTIVITY RELATIONSHIPS OF DERMATAN SULFATE : EFFECT OF MOLECULAR SIZE AND CARBOXYL GROUP ESTERIFICATION ON HEPARIN C0FACT0R-II ACTIVATION

1987 ◽  
Author(s):  
J Mardiguian ◽  
M Corgier ◽  
M Jouany
Keyword(s):  
Molecular Weight ◽  
Dermatan Sulfate ◽  
Methyl Esters ◽  
Molecular Size ◽  
Carboxyl Groups ◽  
Gel Chromatography ◽  
Carboxyl Group ◽  
Heparin Cofactor Ii ◽  
High Affinity

Dermatan is a high molecular weight glycosaminoglycan which has been shown to enhance the inhibition of thrombin by heparin-cofactor II. The aim of this study was to establish the influence of the molecular size and the role of the carboxyl group on the in vitro activity of Dermatan Sulfate. Pig skin Dermatan Sulfate was fractionated according to molecular size by gel-chromatography on Ultrogel Ac 44. Each fraction was characterized by its sulfur content and by its mean molecular weight measured on a TSK - 4000 column in reference to standard heparin fractions. Methyl esters of the unfractionated Dermatan Sulfate with varying degree of esterification, where prepared via activation of the carboxyl groups with a carbodiimide and reaction with methanol. The results of this study show that the heparin - cofactor II mediated anti-thrombin activity of Dermatan Sulfate is increasing with the molecular weight and is abolished by esterification of the carboxyl groups. Moreover, it can be speculated that each fraction contains the same amount of high affinity fraction and that, like heparin, the potency of the high affinity component is increasing with the molecular weight.

Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa

1980 ◽  
Vol 26 (1) ◽  
pp. 77-86 ◽  
Author(s):  
S. E. Jensen ◽  
L. Phillippe ◽  
J. Teng Tseng ◽  
G. W. Stemke ◽  
J. N. Campbell
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Aeruginosa ◽  
Clinical Isolate ◽  
Protease Activity ◽  
Gel Filtration ◽  
Laboratory Strain ◽  
Exchange Chromatography ◽  
The Other ◽  
Protease Production ◽  
Peptide Bonds

Exocellular protease production was examined in two separate strains of Pseudomonas aeruginosa, one a clinical isolate and the other a laboratory strain. Both strains produced two separate proteases (proteases 1 and 2) which were indistinguishable from one strain to the other. The two proteases were purified by a two-step procedure of gel filtration chromatography followed by ion-exchange chromatography. Proteases 1 and 2 were shown to be distinct serologically and unrelated by physicochemical parameters examined. Protease 1 was the major exocellular protein produced and contributed about 95% of the total protease activity of the culture. It was estimated to have a molecular weight of 34 850 and was also shown to contain 10% glucosamine by weight. Protease 2, in contrast, had an estimated molecular weight of 52750 and contained no detectable carbohydrate. Proteases 1 and 2 were both stimulated by Ca2+, and Mg2+ and inhibited by Co2+Zn2+, and 1,10-o-phenanthroline. Protease 1 was also inhibited by EDTA. In addition to protease activity, both proteases 1 and 2 demonstrated elastase activity as well as a limited collagenase activity. Specificity of the two proteases against synthetic peptides was, however, quite different. Protease 1, but not protease 2, showed a preference for peptide bonds in which the amino group was contributed by an amino acid with a hydrophobic R group.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

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