Purification of Fully Active Human Encephalitogenic Basic Protein on Sulfoethyl-Sephadex

1972 ◽  
Vol 50 (6) ◽  
pp. 684-688 ◽  
Author(s):  
M. A. Barton ◽  
T. A. McPherson ◽  
J. K. Martin
Keyword(s):  
Guinea Pigs ◽  
Basic Protein ◽  
Serum Antibody ◽  
Gel Filtration ◽  
Cross Reactivity ◽  
High Yield ◽  
Two Samples ◽  
C 50 ◽  
Detectable Serum ◽  
Experimental Allergic

Human encephalitogenic basic protein (HEP) was obtained in high yield after chromatography on sulfoethyl-Sephadex C-50 (SE-Sephadex). The protein was shown to be homogeneous and was identical in physical properties with samples of human encephalitogenic protein obtained by other methods. It was also shown to induce typical clinical and histological experimental allergic encephalomyelitis (E.A.E.) in adult guinea pigs injected with 50 μg HEP in Freund's complete adjuvant (FCA). Gel-filtration radioimmunoassay was used to study immunogenicity, antigenicity, and antigenic cross-reactivity of HEP; seven of 11 guinea pigs injected with HEP in FCA had detectable serum antibody to 125I-HEP, indicating that the SE-Sephadex preparation was fully immunogenic and antigenic; HEP and another preparation of human encephalitogenic basic protein prepared by a different method were equally capable of inhibiting the binding between 125I-HEP and anti-HEP serum indicating that the two samples tested were antigenically identical. We believe that the new technique represents a significant technological advance because it provides a most effective and rapid method for the isolation of fully active encephalitogenic basic protein.

Experimental Allergic Encephalomyelitis (E.A.E.): Synthesis of the 7–11, 11–20, and 12–20 Peptides of Human Encephalitogenic Protein and Inhibition of Histological E.A.E. by the 12–20 Peptide

1972 ◽  
Vol 50 (6) ◽  
pp. 689-696 ◽  
Author(s):  
M. A. Barton ◽  
T. A. McPherson ◽  
R. U. Lemieux ◽  
G. O. Bain
Keyword(s):  
High Voltage ◽  
Guinea Pigs ◽  
Basic Protein ◽  
Solid Phase ◽  
Severe Disease ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Layer Chromatography ◽  
Homologous Peptide ◽  
Experimental Allergic

Peptides comprising the 7–11, 11–20, and 12–20 sequences of human encephalitogenic basic protein (HEP) were synthesized by the solid-phase procedure, purified by gel-filtration and high-voltage paper electrophoresis, and characterized by amino acid analysis, thin-layer chromatography, high-voltage paper electrophoresis, and paper chromatography.The peptides were studied for the capacity to induce E.A.E. when injected with Freund's complete adjuvant (FCA) into guinea pigs. The same peptides were also studied for the capacity to inhibit development of E.A.E. in guinea pigs injected with peptide/FCA 28 days before injection with encephalitogenic HEP in FCA. The results were as follows: (1) all three peptides induced mild clinical E.A.E. but the 12–20 peptide induced severe disease in five out of 40 guinea pigs; (2) none induced histological lesions typical of E.A.E.; (3) none induced antibody reactive with 125I-HEP; (4) none induced delayed sensitivity to the homologous peptide or HEP; (5) injection of the 12–20 peptide in FCA inhibited the development of histological E.A.E. in guinea pigs injected later with HEP/FCA, whereas the 7–11 and 11–20 sequences did not; (6) humoral immunity to injection of HEP/FCA was not prevented by prior injection of the synthetic peptides in FCA.

scholarly journals ANTIBODY FORMATION

1961 ◽  
Vol 113 (5) ◽  
pp. 959-970 ◽  
Author(s):  
Jonathan W. Uhr ◽  
Joyce B. Baumann
Keyword(s):  
Guinea Pigs ◽  
Antibody Formation ◽  
Serum Antibody ◽  
Active Immunization ◽  
Response Priming ◽  
Anamnestic Response ◽  
Partial Suppression ◽  
Free Antigen ◽  
Detectable Serum ◽  
Secondary Type

Diphtheria toxoid-antitoxin precipitates formed in antitoxin excess can prepare guinea pigs, rats, and rabbits for a secondary type of antitoxin response. Priming may occur without the development of detectable serum antibody. In rats, toxoid-antitoxin precipitates are more efficient than "free" toxoid in priming, whereas in guinea pigs, the magnitude of the anamnestic response varies with the precipitate employed. The possibility that priming is due to "free" antigen released from the specific precipitate rather than the precipitate itself is discussed. The anamnestic antitoxin response can be inhibited by passive antitoxin, but less efficiently than primary antitoxin formation. Partial suppression of the secondary antitoxin response was accomplished by injection of excess horse antitoxin as long as 4 days after reimmunization with toxoid. The importance of these findings for the understanding of passive-active immunization in the human is discussed.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

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