scholarly journals Characterization of amino acids and low-molecular-weight peptides bound to cytoplasmic granules from the posterior pituitary

1970 ◽  
Vol 48 (4) ◽  
pp. 455-462 ◽  
Author(s):  
Seon Shin ◽  
Frank LaBella ◽  
Gary Queen
Keyword(s):  
Amino Acids ◽  
Gel Filtration ◽  
Cation Exchange Resin ◽  
Exchange Chromatography ◽  
Posterior Pituitary ◽  
Total Acid ◽  
Cytoplasmic Granules ◽  
Molecular Weights ◽  
Positive Material ◽  
Fraction I

A number of peptides and amino acids, representing 30–40% of the total acid-extractable, ninhydrin-positive material of the tissue, were associated with cytoplasmic granules (sedimenting at 3 000 000 g-min after preliminary removal of "nuclei and debris") isolated from bovine posterior pituitary glands. Acetic acid (0.2 N) extracts of a purified neurosecretory granule fraction showed only slight differences in the pattern of peptides and amino acids from extracts of the total cell particulate fraction. Gel filtration of extracts on Sephadex G-25 yielded three major fractions: fraction I consisting of peptide material of molecular weights > 4000, fraction II of molecular weights averaging about 3000, and fraction III of molecular weights < 2000. Fraction III was further resolved by anion-exchange chromatography into 12 subfractions. Vasopressin and oxytocin were contained in subfractions 2 and 3, respectively. Each of these subfractions was in turn chromatographed on a cation-exchange resin and resolved into a total for fraction III of 22 major components: lysine, arginine, phenylalanine, ammonia, and 18 peptides. Three of the peptides contained only aspartic and glutamic acids in the ratios 8:1, 5:1, and 4:1. The sequences of four dipeptides were ascertained. Another peptide was not retarded by Dowex 50 and yielded glutamic acid upon acid hydrolysis. Still another peptide yielded tyrosine plus an unknown ninhydrin-positive component after hydrolysis. The amino acid compositions were determined for nine other peptides containing three to nine residues. Additional peptides in fraction III were detected in lesser or trace amounts. Isolated granule fractions from both bovine posterior pituitary and rat liver were dialyzed against isotonic sucrose or distilled water. The rate of loss of ninhydrin-positive material from the sample dialyzed against water indicated that a large proportion of the "free" amino acids and peptides of these tissues were contained within intracellular organelles.

Isolation of specific protease inhibitors from Neurospora crassa

1976 ◽  
Vol 54 (8) ◽  
pp. 699-703 ◽  
Author(s):  
Peter H. Yu ◽  
Maria R. Kula ◽  
Hsin Tsai
Keyword(s):  
Neurospora Crassa ◽  
Protease Inhibitors ◽  
Gel Filtration ◽  
Physiological Role ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Tryptophan Synthase ◽  
Molecular Weights ◽  
Specific Protease

Four natural protease inhibitors have been partially purified by heat treatment, ion-exchange chromatography and gel filtration from Neurospora crassa. The inhibitory activity has been estimated by measuring the inhibition of proteolysis of casein as well as by the protection of Neurospora tryptophan synthase from proteolytic inactivation. The inhibitors are all oligopeptides and possess molecular weights in the range 5000 – 24 000 and appear to be very specific to Neurospora proteases. They may be classified into two types. The first are specific to Neurospora alkaline protease and the second to acidic protease. None of them exhibited any effect on other proteases including trypsin, chymotrypsin, papain, pepsin, thermolysin, subtilisin and proteinase K. The possible physiological role of these inhibitors is discussed.

PURIFICATION AND CHARACTERISATION OF FIBRINOLYTIC ENZYMES FROM ENDOPHYTIC FUNGI AND LIGNOSUS RHINOCERUS

2016 ◽  
Vol 78 (6-5) ◽  
Author(s):  
Zainon Mohd Noor ◽  
Mohd Sidek Ahmad ◽  
Zaidah Zainal Ariffin
Keyword(s):  
Ion Exchange ◽  
Endophytic Fungi ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fibrinolytic Enzymes ◽  
Molecular Weights ◽  
Ammonium Sulphate Precipitation ◽  
Fusarium Sp ◽  
Different Sources

Three enzymes FH3, S13 and LR1 from three different sources showed fibrinolytic activities. Two were from endophytic fungal cultures and one from the sclerotium of Lignosus rhinocerus mushroom (LR1). FH3, S13 cultures and LR1, the crude extract of the sclerotium were concentrated and purified by ammonium sulphate precipitation, ion-exchange chromatography and gel-filtration. The molecular weights of the FH3, S13 and LR1 purified enzymes were estimated to be approximately 34kDa, 34kDa and 10kDa, respectively. Maximum fibrinolytic activities were observed for FH3 at pH 7 and 30°C, S13 at pH 8 and 40°C and LR1 at pH 6 and 40°C.  In our earlier paper we identified FH3 as Fusarium sp. and S13 as Penicilium citrinum. 

Partial Primary Structure Of Human α2-Antiplasmin

1981 ◽  
Author(s):  
H R Lijnen ◽  
B Wiman ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Edman Degradation ◽  
Single Chain ◽  
Tryptic Digest

α2-Antiplasmin (α2AP), the main physiological inhibitor of plasmin in human plasma, is a single–chain glycoprotein with a molecular weight of 67,000 consisting of about 510 amino acids and containing 13 percent carbohydrate.A tryptic digest on 400 mg of reduced, carboxymethylated and citraconylated purified α2AP was performed. Peptides were separated by combinations of ion exchange chromatography, gel filtration and high performance liquid chromatography, and sequenced using the manual Edman degradation. Some peptides were further digested in order to establish overlaps. At the time of submission of this abstract we have sequenced 7 out of the approximately 21 arginyl peptides completely (each between 3 and 21 residues) and are working on the others. At present we have about 200 residues of sequence. Here we only report the stretches of 10 amino acids or more, which may be useful to compare the structure of α2AP with that of other serine protease inhibitors.

Isolation of a Crosslinked Peptide from Hitman Fibrin and Development of a Radioimmunoassay

1979 ◽  
Author(s):  
R. Canfield ◽  
B. Lahiri ◽  
R. D’Alisa ◽  
V. Butler ◽  
H. Nossel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Edman Degradation ◽  
Cnbr Cleavage ◽  
Normal Hemostasis

Factor XIIIa introduces up to six crossllnklng bonds per molecule of fibrin; the bonds between the γ chains on adjacent fibrin molecules form most rapidly. Since cross linking is essential for normal hemostasis and is likely to be important in tests to detect thrombosis, we have attempted to develop a radioimmunoassay that exhibits specificity for the γ chain crosslinks. The immunogen consisted of a 54 amino acid, crosslinked peptide, isolated from purified human γ-γ chains following CNBr cleavage, gel filtration on Sephadex G-50 and ion-exchange chromatography on SP-Sephadex. Amino acid analysis and Edman degradation through step 24 confirmed the sequence of Chen and Doolittle (Biochemistry 10: i486, 1971), and the two degradation steps that failed to liberate the expected PTH-amino acids matched the reported location of the Gin-Lys crosslinks. Antisera were obtained against this immunogen coupled either to bovine thyroglobulin or bovine serum albumin. All antisera elicited bound immunogen that was covalently coupled to ribonuclease radiolabeled with 125I as a tracer. The unlabeled γ-γ, crosslinked peptide effectively inhibited binding (0.03-0.08 picomoles for 50% inhibition), while with some antisera up to 500 times more of the 27 amino acid γ monomer peptide was required for the same degree of inhibition. Fibrinogen and fragment D also were poor Inhibitors. The results Indicate that it is possible by radioimmunoassay to distinguish the COOH-termlnal region of the γ-γ dlmer from that of uncrosslinked molecules.

scholarly journals The isolation of three neurophysins from porcine posterior pituitary lobes

1970 ◽  
Vol 116 (5) ◽  
pp. 899-909 ◽  
Author(s):  
L. O. Uttenthal ◽  
D. B. Hope
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Analysis ◽  
Gel Filtration ◽  
Biological Significance ◽  
Starch Gel Electrophoresis ◽  
Posterior Pituitary ◽  
Fresh Tissue ◽  
Molecular Weights ◽  
Molecular Dimensions

1. Three neurophysins, proteins that bind the polypeptide hormones oxytocin and vasopressin, have been isolated from acetone-dried porcine posterior pituitary lobes. The proteins have been named porcine neurophysins-I, -II and -III in order of their electrophoretic mobilities at pH8.1. 2. Electrophoretic comparison of the purified proteins, which are homogeneous on starch-gel electrophoresis, with the soluble proteins of fresh porcine posterior pituitary lobes extracted in 0.1m-HCl and in buffer pH8.1 suggests that the isolated proteins are native to the fresh tissue. 3. Neurophysins-I and -II are present in similar amounts in the tissue, whereas neurophysin-III is present only in small quantities. Acetone-dried tissue also contains traces of other hormone-binding neurophysin components. 4. All the neurophysins can bind both oxytocin and [8-lysine]-vasopressin. 5. The apparent molecular weights of the neurophysins increase with increasing protein concentration as measured by equilibrium sedimentation in the ultracentrifuge. 6. Neurophysins-I and -III are of similar molecular dimensions, contain one residue of methionine per molecule and lack histidine. The minimum molecular weight of neurophysin-I obtained by amino acid analysis is 9360. Neurophysin-II is of larger molecular dimensions than neurophysins-I and -III and can be separated from these by gel filtration on Sephadex G-75. It contains no histidine or methionine, and its minimum molecular weight has been estimated as 14020 by amino acid analysis. 7. Each of the three neurophysins possesses N-terminal alanine. 8. The possible biological significance of the existence of several neurophysins within one species is discussed.

Comparison of some properties of soil humic acids and synthetic phenolic polymers incorporating amino derivaties

Soil Research ◽  
1966 ◽  
Vol 4 (1) ◽  
pp. 41 ◽  
Author(s):  
JN Ladd ◽  
JHA Butler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Acid Hydrolysis ◽  
Humic Acids ◽  
Gel Filtration ◽  
Quinone Imine ◽  
Peptide Bonds ◽  
Molecular Weights ◽  
Soil Humic Acids ◽  
Properties Of Soil

Twenty-three model phenolic polymers, either nitrogen-free or incorporating amino acids, peptides, or proteins, have been prepared from p-benzoquinone and catechol under mild oxidative conditions. Two lines of experimentation have demonstrated properties of soil humic acids closely similar to those of polymers incorporating proteins, but different from those of polymers incorporating amino acids: (1) fractionation of humic acids and synthetic polymers by 'Sephadex' gel filtration showed that the percentage of components of molecular weights nominally greater than 100 000 ranged from 52-76 % for eight humic acids tested, 53-59 % for benzoquinone-protein polymers (excluding polymers containing protamine), but less than 20% for all other polymers; (2) acid hydrolysis with 6M HCl resulted in a partial release of polymer nitrogen. Amino acid nitrogen in the hydrolysates accounted for 32.4-51.9 % of humic acid nitrogen, 31.2-56.3 % of the nitrogen of polymers incorporating protein, but less than 10.8% of the nitrogen of polymers incorporating individual amino acids. Experiments with model monomeric N-phenylglycine derivatives and with polymers incorporating simple peptides showed that the bond between the carbon atom of an aromatic ring and the nitrogen atom of an a-amino acid is far more stable to acid hydrolysis than peptide bonds or bonds linking amino acids in humic acids. Glycine is, however, readily released from N-phenylglycine derivatives when conditions favour their oxidation to a quinone-imine intermediate. Incorporation of proteins into phenolic polymers prevented the detection of peptide bonds by the Folin reagent.

ISOLATION OF LH, FSH AND TSH FROM HUMAN PITUITARIES AFTER THE REMOVAL OF HGH

1974 ◽  
Vol 77 (3) ◽  
pp. 485-497 ◽  
Author(s):  
P. A. Torjesen ◽  
T. Sand ◽  
N. Norman ◽  
O. Trygstad ◽  
I. Foss
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Molecular Weights ◽  
Pituitary Glands

ABSTRACT Highly purified human LH, FSH and TSH were isolated from batches of 300 frozen pituitary glands (200 g) by pH, acetone and ethanol fractionation, Sephadex gel filtration, ion-exchange chromatography on DEAE-cellulose and CM-Sephadex, and preparative polyacrylamide-gel electrophoresis. Sodium dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis was used in order to check the purity, the identity and the molecular weight of the purified LH, FSH and TSH. This procedure showed that the hormone preparations consisted of two subunits with molecular weights of: LH: 21 300 and 17 900, FSH: 22 100 and 18 300 and TSH: 20 800 and 16 400. The purity of the hormone preparations was also evaluated by analytical disc electrophoresis at pH 8.9. The purified hormone preparations had radioimmunological activity as follows: LH: 20 000 IU/mg, FSH: 16 500 IU/mg and TSH: 5 IU/mg. All preparations had high biological potency.

Changes in proteins of Domiati cheese during pickling

1972 ◽  
Vol 39 (2) ◽  
pp. 219-225 ◽  
Author(s):  
M. H. Abd El-Salam ◽  
Safinaz El-Shibiny
Keyword(s):  
Amino Acids ◽  
Room Temperature ◽  
Gel Filtration ◽  
Electrophoretic Pattern ◽  
Pasteurized Milk ◽  
Molecular Weights ◽  
Electrophoretic Patterns ◽  
Related Substances ◽  
Nitrogenous Substances

SummaryDomiati cheese from homogenized and unhomogenized pasteurized milk was pickled for 4 months at room temperature. During pickling, the soluble tyrosine and tryptophan contents of the cheese gradually increased, indicating progressive proteolysis.Gel filtration of cheese extract on Sephadex G-25 revealed the presence of nitrogenous substances of various molecular weights. The maximum formation of amino acids and related substances was observed after 15 days storage.The changes in the electrophoretic patterns of proteins in the cheese during pickling indicated that both αs - and β-caseins were attacked. β-Casein, however, was much less affected.Homogenization was found to affect the formation of soluble nitrogenous substances, but was without effect on the electrophoretic pattern of the protein in the cheese.

scholarly journals The chemistry of the collagen cross-links. Purification and characterization of cross-linked polymeric peptide material from mature collagen containing unknown amino acids

1980 ◽  
Vol 185 (2) ◽  
pp. 373-381 ◽  
Author(s):  
N D Light ◽  
A J Bailey
Keyword(s):  
Amino Acids ◽  
Type I Collagen ◽  
Gel Filtration ◽  
Amino Acid Content ◽  
Exchange Chromatography ◽  
Type I ◽  
Polymeric Form ◽  
Cross Links ◽  
Helical Region

A polymeric form of the alpha 1-chain C-terminal peptide alpha 1 CB6 (poly-alpha 1 CB6) was purified from CNBr digests of insoluble bovine tendon type-I-collagen by gel filtration and ion-exchage chromatography. The purified material had a molecular weight of 1.5 × 10(6)-5 × 10(6) on gel filtration and an amino acid content virtually identical with that of monomeric peptide alpha 1 CB6. The material could be adsorbed on affinity gels containing immobilized anti-(alpha 1 CB6-peptide non-helical region) antibodies and was an inhibitor of haemagglutination by the same antibodies of alpha 1 CB6-peptide-coated sheep erythrocytes. Periodate treatment of the material had no effect. Alkali hydrolysates were shown to contain two unknown amino acids, which were purified by gel filtration and ion-exchange chromatography in volatile buffers and are believed to be components of the mature cross-link of collagen.

Antifreeze glycoproteins in the plasma of Newfoundland Atlantic cod (Gadus morhua)

1981 ◽  
Vol 59 (11) ◽  
pp. 2186-2192 ◽  
Author(s):  
Choy L. Hew ◽  
Don Slaughter ◽  
Garth L. Fletcher ◽  
Shashikant B. Joshi
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Gadus Morhua ◽  
Atlantic Cod ◽  
Antifreeze Proteins ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Polar Cod ◽  
Antifreeze Glycoproteins ◽  
Molecular Weights

The plasma of the Atlantic cod, Gadus morhua, contained antifreeze glycoproteins which were present only during the winter months. The antifreeze proteins were isolated, using gel filtration and ion exchange chromatography, and characterized by high performance liquid chromatography. The antifreeze proteins appeared to consist of at least seven components with molecular weights ranging from 2 500 to 33 000. Chemical analysis of the larger components showed a predominance of alanine, threonine, and galactosamine in a ratio of 2:1:1. The smaller peptides contained proline, in addition to alanine and threonine. The amino acid sequence of the smallest glycopeptide (molecular weight 2500) was found to be Ala Ala Thr Pro Ala Thr Ala Ala Thr Pro Ala Thr Ala Ala.These glycoproteins are very similar, if not identical, in amino acid and carbohydrate composition to those isolated from Antaractic nototheniids and several northern gadoids. The sequence of the smallest glycopeptide from the Atlantic cod is identical to that reported for the polar cod, Boreogadus saida.

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