High-efficiency cation-exchange chromatography of polypeptides and polyamines in the nanomale range

Radhakrishnan, A. N.; Stein, Stanley J.; Licht, A; Gruber, Kenneth A.; Udenfriend, Sidney

doi:10.1016/s0021-9673(00)82922-2

Cited by 47 publications

(3 citation statements)

References 7 publications

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“…The fraction eluting immediately after the salt peak (Fraction IV) was lyophilized and stored at -70" for high pressure liquid chromatography. Biogel Fraction IV was redissolved in 300 pl of .05 M HC1 and separated on a high pressure Partisil SCX (cation-exchange) column (Whatman Inc., Clifton, NJ) under a protocol previously described (7). Four minute fractions (-1 ml) were collected in a fraction collector.…”

mentioning

confidence: 99%

Further Characterization and Evidence for a Precursor in the Formation of Plasma Antinatriferic Factor

Gruber¹,

Buckalew²

1978

Experimental Biology and Medicine

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mentioning

confidence: 99%

Further Characterization and Evidence for a Precursor in the Formation of Plasma Antinatriferic Factor

Gruber¹,

Buckalew²

1978

Experimental Biology and Medicine

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“…Since human and bovine fl-lipotropins and fl-endorphins have already been shown to differ in structure (8), it appeared likely that the opioid peptides in rats would also have their own unique structures and would have to be characterized. Utilizing sensitive fluorometric procedures for protein, peptide, and amino acid assay developed in this laboratory (9)(10)(11) An automatic fluorescence detection system was used for column monitoring of peptides (9). In this system a discontinuous sampling valve allowed typically 2-10% of the column effluent to react with fluorescamine, while the remainder was directed to a fraction collector.…”

mentioning

confidence: 99%

Isolation and characterization of the opioid peptides from rat pituitary: beta-lipotropin.

Rubinstein¹,

Stein²,

Gerber³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACr Rat ,-lipotropin was isolated from 40 anterior pituitaries using high efficiency chromatography columns and sensitive fluorometric methods. The P-lipotropin was characterized as to molecular weight, amino acid composition, and generation of opioid activity by trypsinization. Other opioid peptides, as well as a precursor to opioid peptides with a molecular weight larger than that of P-lipotropin, were observed in this tissue. The series of peptides a-, fi-, and y-endorphin and methio- An automatic fluorescence detection system was used for column monitoring of peptides (9). In this system a discontinuous sampling valve allowed typically 2-10% of the column effluent to react with fluorescamine, while the remainder was directed to a fraction collector. An LKB (Hicksville, NY) gradient mixer, a Milton Roy (Riviera Beach, FL) minipump, and an Altex Scientific (Berkeley, CA) 7000 lbs./inch2 sample injection valve were the major components of the chromatography system. Aliquots of the collected fractions were hydrolyzed in constant boiling hydrochloric acid that had been redistilled over sodium dichromate (12). Thioglycolic acid (0.1% vol/vol) was added to prevent oxidation of cysteine (13). Amino acid analyses were performed on an instrument that used fluorescamine for detection at the picomole level as previously reported (10). Trypsin (twice crystallized) was obtained from Sigma (St. Louis, MO). Bioassay of opioid activity was carried out with neuroblastoma X glioma hybrid cells (NG 108CC15) (14), which were generously provided by M. Nirenberg, National Institutes of Health, Bethesda, MD. Opioid activity was measured by competitive binding with [3H]Leu-enkephalin (Amersham/Searle) to these cells, and activity is reported in terms of Leu-enkephalin equivalents. Details of the binding assay will be published elsewhere. Other experimental details are given in the figure legends and in Results. RESULTSAnterior pituitaries from 40 rats were extracted with acid/ acetone according to the procedure of Li et al. (15), with some modifications. The tissue was homogenized in 2.5 ml of cold 75% acetone/25% 0.2 M hydrochloric acid containing 0.01% thiodiglycol and 0.001% phenylmethylsulfonyl fluoride (a protease inhibitor). The following steps were performed at room temperature. Acetone was removed under a stream of nitrogen and the volume was brought to 2 ml by the addition of water. Lipids were extracted with three 1-ml portions of ethyl acetate/ether (3:1 vol/vol). The aqueous phase was fractionated on a Sephadex G-75 column and the effluent was monitored for fl-lipotropin by the following procedure. Aliquots (25 Ml) from each collected fraction were dried and then incubated with 10 Mg of trypsin in 50 ,l of 0.05 M Tris buffer (pH 8.5) at 370 for 18 hr. Opioid activity was then determined by the competitive binding assay. A minor peak of activity was found in the 30,000-to 60,000-dalton range (fractions 20-25) while the major activity was found in fractions 34-37, which corresponded to the sheep 03-lipotropin standard...

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“…The chromatographic technique used by these investigators (HPLC on Partisil SCX) also supported the concept that they were isolating a peptide, since the procedure used was ftrst developed for small peptides (76). Other groups have demonstrated enzymatic degradation of biologic activity, suggesting NH is a peptide (8).…”

Section: Biochemical Nature Of Nhmentioning

confidence: 90%

Natriuretic Hormone

Buckalew¹,

Gruber²

1984

Annu. Rev. Physiol.

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High-efficiency cation-exchange chromatography of polypeptides and polyamines in the nanomale range

Cited by 47 publications

References 7 publications

Further Characterization and Evidence for a Precursor in the Formation of Plasma Antinatriferic Factor

Further Characterization and Evidence for a Precursor in the Formation of Plasma Antinatriferic Factor

Isolation and characterization of the opioid peptides from rat pituitary: beta-lipotropin.

Natriuretic Hormone

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