Larry Brink

Fluorometric assay of vasopressin and oxytocin: a general approach to the assay of peptides in tissues

A fluorometric method for the quantitative assay of vasopressin and oxytocin in individual rat pituitaries has been developed. Acid extracts of pituitaries are freed of amino acids and polyamines by passage over a copper-Sephadex column, and the peptides fraction is then labeled by reaction with fluorescamine. The resulting peptide fluorophors are separated by chromatography on a reverse-phase bonded column. Specificity of the procedure was ascertained by several criteria, including bioassay and amino-acid analysis of the eluted peptide fluorophors. The procedure serves as a model system for the assay of tissue peptides in the picomole range.

New automated fluorometric peptide microassay for carnosine in mouse olfactory bulb

A procedure for assaying peptides at the picomole level in tissue extracts has been developed and used to measure the dipeptide carnosine in mouse olfactory bulb. In this procedure the tissue extract is reacted with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF), and the resultant fluorophors are resolved on a high performance reverse-phase column. Quantitation is performed in a filter fluorometer equipped with a flow cell. Carnosine was found to be present at a level of 1.93 ± 0.44 nmol/mg of tissue (mean + SD of 11 samples), in agreement with previous findings by other methods.

Characterization of multiple forms of folate-binding protein from human leukemia cells.

Folate-binding proteins were isolated from the particulate fraction (44,000 X g pellet) and the soluble fraction (44,000 X g supernate) of the homogenate of a spleen obtained from a patient who had an acute leukemic (blast) transformation of chronic myelogenous leukemia. The folate-binding activity which was obtained from the particulate fraction by solubilization with 1% Triton X-100 could be resolved into two binding proteins (Mr 310,000 and 28,000) by gel filtration through Sephadex G-200 after incubation with excess [3H]pteroylglutamic acid (PteGlu). The folate-binding protein in the solubilized particulate fraction and the soluble folate-binding protein in the 44,000 X g supernatant cytoplasm were purified by affinity chromatography. Only a 32 kDa protein was identified by SDS-polyacrylamide gel electrophoresis in the final preparation of the purified folate-binding protein from the particulate, whereas two protein bands (Mr 42,000 and 32,000) were identified by SDS-polyacrylamide gel electrophoresis in the purified preparation of the soluble folate-binding protein. Both of these species were immunologically crossreacting. Both the purified folate-binding protein from the particulate fraction and the purified soluble form had higher affinity for oxidized folate than for the reduced folate cofactors, and both proteins had very low affinity for the antifolate compound, methotrexate. The amino-acid composition of the soluble folate-binding protein was similar with regard to the content of apolar amino acids to that reported for the membrane-derived folate-binding protein purified from milk and human placenta.

Specific polyclonal antibodies to the carboxyl terminus of [Met]enkephalin-Arg6-Gly7-Leu8

The octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu was recently isolated from bovine adrenal chromaffin granules and serves as a marker for proenkephalin from which it is derived. Polyclonal antisera which are highly specific for the carboxyl terminus have been raised against the synthetic peptide. The only significant cross-reactivity was with the 18.2-k Da and 5.3-k Da enkephalin-containing peptides (EC peptides) which contain the octapeptide at their carboxyl termini and the [des-Tyr] and [des-Tyr-Gly] congeners of the octapeptide. Extracts of bovine adrenal medulla and rat spinal cord were shown to contain significant amounts of the octapeptide, the two larger EC peptides, and the two smaller congeners.

Amino acid analysis of proteins and peptides at the picomole level: the fluorescamine amino acid analyzer.

Publisher Summary This chapter describes an instrument that uses the reagent fluorescamine for post-column reaction. Preparation of the reagents required for analysis at the picomole level is also presented. A schematic diagram of the amino acid analyzer is given. Four eluents are selected in sequence using a rotary (six-way) valve. The eluents are kept under argon pressure. The column is preheated to 60˚ and then packed with a slurry of the resin in 0.2 N NaOH. The resin is added and packed in several steps, care being taken to avoid air bubbles, to a height of 33 cm (10% higher than the resin bed will be at the end of packing). The column is washed at 15 ml/hr with 0.2 N NaOH for 1.5 hr and then for 1 hr with buffer A. The complete set of buffers is cycled through the column before use. Chromatography is carried out at 18 ml/hr at 57 ° with the following program: A, 15 min; B, 17 rain; C, 24 min; D, 41 min, NaOH, 5 min; A, 18 min. Borate buffer and fluorescamine solution are pumped at 35 and 18 ml/hr, respectively. When used, the N-chlorosuccinimide is delivered at 6 ml/min by adjusting the pressure on the reservoir. Two chromatograms depicting hydrolyzates representing 17 pmol (0.3 /μg) of human leukocyte interferon are given.

Purification and partial sequencing of bovine liver alkaline phosphatase

Bovine liver alkaline phosphatase has been purified to homogeneity by procedures that include reverse-phase HPLC. The pure enzyme has an apparent Mr of 160,000 and is composed of what appears to be two identical monomers of Mr 82,000. About 80% of the material yielded the amino-terminal sequence Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln. The minor component was extended at the amino terminus by two residues that have not yet been identified, i.e., ?-?-Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln.

Site-directed antibodies for probing the structure and biogenesis of phosphatidylinositol glycan-linked membrane proteins: Application to placental alkaline phosphatase

An immunological approach to the study of the structure and biogenesis of the phosphatidylinositol glycan (PI-G) membrane anchor at the carboxyl terminus of human placental alkaline phosphatase (PLAP) is described. Based on the protein sequence predicted from full length PLAP cDNA, two epitopes were chosen in the region of the carboxyl terminus for the production of site-directed antibodies. The exo site represents the last nine residues of preproPLAP, (res. 505-513), which is part of the sequence that is expected to be cleaved from the nascent protein during processing and addition of the PI-G tail. A second site, the endo sequence, was selected close to the expected carboxyl terminus in mature PI-G-tailed PLAP (res. 474-484 of proPLAP). The two peptides were synthesized, polyclonal antibodies to the conjugated peptides were prepared, and the antisera were characterized. Analytical methods for both synthetic peptides and proteins are presented. Preliminary applications to the isolation and characterization of the PI-G-linked carboxyl terminus of mature PLAP and to the characterization of nascent PLAP are described. The application of both carboxyl terminal-directed antibodies, and a third antibody directed to the amino terminus of mature PLAP, in studies employing mutant forms of PLAP and to the PI-G tailing process itself are discussed. The immunological approach used here for PLAP should be applicable generally to the study of other PI-G-tailed proteins.

[5] Amino acid analysis of protein bands on stained polyacrylamide gels

Publisher Summary Amino acid analysis is carried out on proteins that are present as bands on stained polyacrylamide gels. After electrophoresis, the gel is stained with Coomassie Blue and destained extensively with 7% acetic acid-10% methanol in the usual manner. After hydrolysis, the hydrolyzate is removed from the gel residue, dried in vacuo, dissolved in the pH 2.2 diluent, and run on the fluorescamine amino acid analyzer. With this procedure, it is not necessary to extract the protein from the gel in order to carry out amino acid analysis. Extraction from the gel typically gives poor recovery of protein and introduces amino acid contamination. This procedure offers a unique opportunity to characterize a protein. In many instances gel electrophoresis is the simplest method for separating a protein from a mixture, and it is applicable at the low microgram level. In the studies on purified human fibroblast interferon, a minor contaminant of about 40,000 daltons was detected on gels. Amino acid analysis revealed it a dimer of interferon by its identity in composition with the 20,000-dalton band.