A STUDY OF THE HETEROGENEITY OF PARATHYROID HORMONE PREPARATIONS

1969 ◽  
Vol 44 (2) ◽  
pp. 195-202
Author(s):  
D. J. LEA ◽  
J. M. ZANELLI
Keyword(s):  
Biological Activity ◽  
Parathyroid Hormone ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Parathyroid Glands ◽  
Biologically Active ◽  
Antigenic Determinants ◽  
High Potency

SUMMARY Bovine parathyroid glands, stored in several ways, have been extracted by published methods using urea and HCl, or phenol, and recent modifications of the latter method utilizing mercaptoethanol. The extracts were purified by gel filtration. Parathyroid hormone of high potency has been obtained only from rapidly frozen glands by the method using phenol and mercaptoethanol. Purified hormone preparations were extremely heterogeneous, were indistinguishable from material of much lower potency by gel electrophoresis, and shared common antigenic determinants with components of fractions having insignificant biological activity. It is possible that parathyroid hormone which has been modified irreversibly at the biologically active site is commonly present in gland extracts.

HETEROGENEITY OF INSULIN: I. ISOLATION OF A CHROMATOGRAPHICALLY PURIFIED, HIGH-POTENCY INSULIN AND SOME OF ITS PROPERTIES

1966 ◽  
Vol 44 (8) ◽  
pp. 1171-1181 ◽  
Author(s):  
W. W. Dillon ◽  
R. G. Romans
Keyword(s):  
Biological Activity ◽  
Gel Electrophoresis ◽  
Sodium Phosphate ◽  
Chemical Degradation ◽  
Minor Components ◽  
Slight Improvement ◽  
High Potency ◽  
Sodium Phosphate Buffer ◽  
A Value ◽  
Main Component

A slight improvement in the chromatography of insulin on Amberlite IRC-50 resin in 7 M urea −0.13 M sodium phosphate buffer, pH 6.05, at 3 °C revealed the presence of at least four components.The major component was found to have a potency greater than that of any other purified insulin hitherto reported and substantiated. The biological activity of the minor components varied from a value close to that of the starting insulin down to very low or negligible values, indicating a loss of biological activity with increasing chemical degradation. The potency studies were quantitative.Starch-gel electrophoresis showed that the main component was not completely homogeneous, traces of other components being present.A highly fluorescent, colored material was separated from among the minor components and may be of interest in relation to allergy produced by some insulins.The separation reported here is discussed in relation to other fractionation procedures.

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.

Characterization of Immunoreactive Angiotensin in Canine Cerebrospinal Fluid as Des-Asp1-Angiotensin II

1978 ◽  
Vol 54 (2) ◽  
pp. 147-151 ◽  
Author(s):  
J. S. Hutchinson ◽  
J. Csicsmann ◽  
P. I. Korner ◽  
C. I. Johnston
Keyword(s):  
Cerebrospinal Fluid ◽  
Biological Activity ◽  
Angiotensin Ii ◽  
Gel Electrophoresis ◽  
Biologically Active ◽  
Pressor Activity

1. Immunoreactive angiotensin II was measured in cerebrospinal fluid of four normal dogs. 2. The migration of this immunoreactive angiotensin II on polyacrylamide-slab gel electrophoresis was identical with the migration of the heptapeptide, Des-Asp1-angiotensin II, in each case. 3. The biological activity of the material from canine cerebrospinal fluid in a pressor bioassay was similar to that of Des-Asp1-angiotensin II. 4. The pressor activity of the canine material was abolished by treating the pressor bioassay rat with a competitive antagonistic analogue, Sar1-Ala8-angiotensin II. 5. The results suggest that the biologically active immunoreactive angiotensin II present in normal canine cerebrospinal fluid is composed mainly of the heptapeptide fragment of angiotensin II, Des-Asp1-angiotensin II.

scholarly journals Effects of Plasmin on Human Factor VIII (AHF)

Blood ◽  
1973 ◽  
Vol 41 (1) ◽  
pp. 105-111 ◽  
Author(s):  
R. Pasquini ◽  
E. J. Hershgold
Keyword(s):  
Factor Viii ◽  
Gel Electrophoresis ◽  
Human Factor ◽  
Gel Filtration ◽  
Antigenic Determinants ◽  
Hemorrhagic Diathesis ◽  
Activity Assay ◽  
Factor Viii Activity ◽  
Immunologic Reactivity ◽  
Human Factor Viii

Abstract Highly purified, fibrinogen-free human factor VIII was incubated with plasmin, and the liberated split products of the factor VIII were analyzed by gel filtration, acrylamide gel electrophoresis, bioassay, and for immunologic reactivity. At least three fragments retaining different antigenic determinants are released from the factor VIII after prolonged digestion and at least three new fragments are seen in acrylamide gel electrophoresis. The split products were not anticoagulant in the factor VIII activity assay. In fact, the breakdown products in the hydrolysate increased the factor VIII activity of normal plasma mixed with it. Therefore, it is not likely that the factor VIII split products formed in fibrinolytic states contribute actively to the hemorrhagic diathesis.

scholarly journals STUDIES ON THE PATHOGENESIS OF FEVER

1968 ◽  
Vol 127 (2) ◽  
pp. 341-357 ◽  
Author(s):  
Marilyn Sue Kozak ◽  
Helmut H. Hahn ◽  
William J. Lennarz ◽  
W. Barry Wood
Keyword(s):  
Molecular Weight ◽  
Biological Activity ◽  
Active Material ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Sulfhydryl Groups ◽  
Biologically Active ◽  
Exhaustive Extraction

Small quantities of highly purified granulocytic pyrogen have been separated from contaminating proteins by disc electrophoresis in polyacrylamide gel. The biologically active material thus isolated was shown to be electrophoretically homogeneous at pH 9 and pH 3.8. Earlier work on the chemical properties of the pyrogen molecule has been extended to include: (a) estimation of its molecular weight by gel filtration; (b) demonstration of free sulfhydryl groups essential for its biological activity; and (c) evidence that it is not inactivated by exhaustive extraction with ethanolether or n-heptane.

DISCREPANCIES IN PLASMA LH ACTIVITIES AS MEASURED BY RADIOIMMUNOASSAY AND AN IN VITRO BIOASSAY

1974 ◽  
Vol 77 (4) ◽  
pp. 672-685 ◽  
Author(s):  
M. H. Qazi ◽  
P. Romaní ◽  
E. Diczfalusy
Keyword(s):  
Molecular Weight ◽  
Biological Activity ◽  
Gel Filtration ◽  
Biologically Active ◽  
Plasma Samples ◽  
Elution Volume ◽  
Immunological Activity ◽  
Molecular Weight Range ◽  
Weight Range

ABSTRACT Using a highly sensitive in vitro bioassay system, luteinizing hormone activity has been measured in parallel with radioimmunoassays in postmenopausal plasma and plasma obtained from women in various phases of the menstrual cycle (follicular, mid-cycle, luteal). Biological and immunological activities were measured directly in plasma samples without any chemical manipulation. The biological activity (B) was always higher than the immunological activity (I); the B/I ratio varied from 2.1 to 14.0. Gel filtration of pooled plasma samples through Sephadex G-100 revealed major discrepancies in each physiological state when immunological and biological activities were measured in each fraction. The biological activity was eluted as a single peak behind the elution volume of bovine serum albumin, but in front of the elution volume of chymotrypsinogen. It was invariably preceded by a small hump. The immunological activity was spread all over the chromatogram. Areas of immunological activity without any biological activity were located on either side of the biologically active fractions, both in the high molecular weight range (including the void volume) and in the low molecular weight range. The biological LH activity recovered following fractionation on Sephadex G-100 was in close agreement with that loaded, whereas the immunological activity recovered following gel filtration exceeded the loaded activity by a factor of 6–8. In the various physiological states, 11 to 44 % of the total immunological activity recovered was not associated with any biological activity. Furthermore, there was a marked variation in the ratio of biological to immunological activities of those fractions which contained biological activity. It is suggested that the specificity of current RIA methods could be improved significantly by preparing antisera which react only with biologically active LH species.

scholarly journals β-d-Glucosidases and related enzymic activities in pig kidney

1969 ◽  
Vol 111 (5) ◽  
pp. 749-755 ◽  
Author(s):  
Hope E. Abrahams ◽  
D. Robinson
Keyword(s):  
Molecular Weight ◽  
Isoelectric Point ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Small Difference ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Heat Denaturation ◽  
Pig Kidney ◽  
Approximate Molecular Weight

1. The β-glucosidase activity of pig kidney is located in the unsedimentable fraction of the cell and is not associated with the lysosomes. 2. The enzyme is active towards β-d-glucosides, β-d-galactosides, β-d-xylosides and α-l-arabinosides. 3. These activities could not be separated by gel electrophoresis, gel filtration or DEAE-cellulose chromatography. 4. Response to inhibitors, heat-denaturation and competitive substrates suggests that a single active site is responsible for all four activities. 5. Two forms of the enzyme were found to occur either separately or together in kidneys of pigs from several different breeds. 6. Electro-focusing experiments show these to have a small difference in isoelectric point (4·9 and 5·1), and gel filtration gives an approximate molecular weight of 50000 for both forms. 7. The characteristics of these two enzymes are compared.

scholarly journals Extraction and purification of recombinant intact human Parathyroid Hormone (hPTH) from bacterial cell

2017 ◽  
Vol 3 (2) ◽  
pp. 1 ◽  
Author(s):  
Adnan E. Al-badran ◽  
Rafeef Amer Abdul-jabbar
Keyword(s):  
Biological Activity ◽  
Parathyroid Hormone ◽  
Bacterial Cell ◽  
Biologically Active ◽  
Human Parathyroid Hormone ◽  
Hormone Production ◽  
Rp Hplc ◽  
Bioactivity Assay ◽  
Significant Difference ◽  
Mcf 7

Objective: The intact human Parathyroid hormone (hPTH) is one of the biopharmaceutical drug produced by biotechnology, this hormone can be provided in a good amount using simple batch culture, and therefore the main purpose of this study was the production of purified bioactive intact hPTH.Methods: A cloning BL21(DE3) strain (which already cloned in our Lab. by synthetic human Parathyroid hormone (shPTH) gene) was grown in LB medium and the cloning gene expression was induced by addition of IPTG to the medium. The recombinant fused protein was purified from the bacterial cell lysate by affinity chromatography and underwent proteolysis by Enterokinase enzyme to obtain the target hPTH, and it's further purified by DEAE and SP Sepharose ion exchange chromatography. RP-HPLC analysis and biological activity on the MCF-7 breast cancer cells were done for both the standard and produced hPTH. Furthermore, the haemolysis ability of the latter was tested on the human RBC.Results: 225ng/ml of hPTH was produced after SP chromatography which detected by specific PTH ELISA kit, standard and produced hPTH showed the same peaks in the same retention time when analyzed by HPLC. The produced hPTH haemolysis assay showed the ability of the produced hPTH to partially haemolyze the RBC in a concentration above 200µg/ml. The bioactivity assay for the produced and standard rhPTH demonstrated that both compounds had a biological activity on the MCF-7 cells with IC50 of about 84.4 and 75.7 µg/ml for standard and produced hPTH respectively, and no significant difference was detected between them.Conclusions: Via suitable purification processes, the biologically active hPTH with structure similar to the standard ones as detected by RP-HPLC and bioactivity assay, can be obtained by using already cloned isolate with shPTH gene for hormone production. The hormone showed haemolysis effect on the RBC similar to the normal secreted hPTH.

scholarly journals α-Lactalbumin and lactose concentrations in rat milk during lactation

1981 ◽  
Vol 194 (1) ◽  
pp. 149-154 ◽  
Author(s):  
K R Nicholas ◽  
P E Hartmann ◽  
B L McDonald
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Significant Negative Correlation ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
Alpha Lactalbumin

Homogeneous rat alpha-lactalbumin was prepared from whey by chromatography on DEAE-Sephadex A-50 and Ultrogel AcA 44. Two biologically active forms of alpha-lactalbumin were apparent after ion-exchange chromatography, but on gel filtration the combined forms were eluted as a single peak with a molecular weight of approx. 33000. The molecular weight when determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was 15100. Antiserum to alpha-lactalbumin was prepared from rabbits, and single radial immunodiffusion was used to measure the concentration of alpha-lactalbumin in milk expressed from rats during lactation and for 2 days after the cessation of lactation. A significant positive correlation (r = + 0.89) between the concentrations of alpha-lactalbumin and lactose was obtained for the first 20 days of lactation. This is consistent with the suggestion that alpha-lactalbumin may control the concentration of lactose in milk. However, a significant negative correlation (r = -0.91) between the concentration of alpha-lactalbumin and lactose was obtained for 2 days after the cessation of lactation on day 20.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

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