Paul X. Callahan

A specific radioimmunoassay for osteocalcin with advantageous species crossreactivity

The specificities of immunoassays to rat and bovine osteocalcin are examined. Extracts of noncalcified tissues and tissue fractions are unreactive to the antibody, with the exception of the kidney, in which the reactive component appears to be identical with osteocalcin by gel filtration and dose dilution analysis. The assays, developed against protein isolated from bone, are also demonstrated to be reactive to the native protein (bone in situ) and to osteocalcin in serum. The assays are sensitive to less than 50 pg osteocalcin. Intra- and interassay coefficients of variation are less than 6.8%. The bovine antibody crossreacts with human, horse, monkey, baboon, and cat osteocalcin, while the rat antibody crossreacts with dog and mouse.

[22] Preparation and specificity of dipeptidyl aminopeptidase I.

Publisher Summary The dipeptidyl aminopeptidases (DAP) are enzymes that catalyze the consecutive removal of dipeptide moieties from the unsubstituted NH 2 termini of polypeptide chains. Four such enzymes are characterized and shown to have distinctive substrate specificities. Within this group, dipeptidyl aminopeptidase I (DAP I) exhibits the broadest substrate specificity. When DAP I is observed in bovine pituitary extracts, it is characterized as a sulfhydryl-dependent Ser-Tyr-β-naphthylamidase with an absolute or near-absolute chloride requirement and a pH 4 optimum. The purification serves as the basis for the preparation of DAP I. The practicability of using DAP I as a sequencing reagent is greatly enhanced by the relative ease with which a suitable preparation can be made. The broad substrate specificity of DAP I is first observed with β-corticotropin used as a substrate. DAP I activity has also been measured on Gly-Phe-NH 2 . An analysis of the specific bonds cleaved in polypeptide substrates revealed that DAP I can remove dipeptides having penultimate residues with both hydrophilic and hydrophobic side chains.

Separation and identification of dipeptides by paper and column chromatography

Abstract The behavior of approximately 140 dipeptides on column chromatography as well as in four different solvent systems on paper chromatography has been reported along with the ninhydrin color values of the dipeptides. These data are useful in predicting the elution times and Rf values of unknown dipeptides. The use of these systems for separation and identification of dipeptides coupled with N-terminal analysis in a few equivocal situations makes possible the separation and identification of practically all dipeptides.

ISOLATION AND PROPERTIES OF RAT AND RABBIT GROWTH HORMONES

The present studies were undertaken with the aim of developing a method for isolating highly purified rodent growth hormones that would be useful in studies on the comparative biochemistry of growth hormone as well as for immunological assays for plasma growth hormone in the rat. The isolation of a highly purified growth hormone from frozen rat pituitaries has been described recently by Reisfeld and colleagues,’ who by means of fractionation with ethanol and zone electrophoresis a t pH 8.6, isolated the hormone having a potency of 1 to 1.5 USP units per mg. Employing acetone-dried pituitaries, Wilhelmi2 has obtained rat growth hormone with a potency of 2 USP units per mg by fractionation with (NH4)2S04 and chromatography on DEAE-cellulose a t pH 8.0. To facilitate the recovery and isolation of other pituitary hormones as well as growth hormone, the sequential extraction procedure, previously employed with bovine and ovine gland^,^ has been applied to the extraction of rat and rabbit glands in our l a b ~ r a t o r y . ~

[23] Sequencing of peptides with dipeptidyl aminopeptidase I

Publisher Summary This chapter presents the methodology by which dipeptidyl aminopeptidase I (DAP I) can be utilized in a new approach to the sequencing of small amounts (0.5 μmole or less) of peptides. The ability of DAP I to remove dipeptides, in sequence, from the NH 2 termini of oligopeptides can be followed, for at least several dipeptide products, by time-course chromatography. Although often this is insufficient for establishing sequence, it is possible to modify the peptide substrate in such a way that DAP I generate the alternate, overlapping dipeptides––an advantage not possible with monoaminoacyl aminopeptidases. Certain advantages over chemical methods of sequencing are inherent in this approach. Asparagine and glutamine are recovered as such (not as aspartic or glutamic acid), and there is no tendency to form pyroglutamic acid, as occurs with chemical methods. The inability of DAP I to remove dipeptides with an NH 2 -terminal arginine or lysine is not a problem if the peptide substrates are derived from tryptic digests wherein most, if not all, lysines and arginines are COOH terminal. A limitation arises from the inability of DAP I to cleave the bond on either side of proline. The chapter focuses on the separation of the dipeptide products that result from the action of DAP I on oligopeptides, their recognition in mixtures when separation of the dipeptides is unnecessary, and the approaches necessary for the reordering of those dipeptide products.

Accelerated chromatographic method for determination of hydroxyproline.

Abstract An automated method is described for the determination of urinary hydroxyproline. The method utilizes accelerated column chromatography on spherical sulfonated polystyrene resin, PA-35, and a Beckman/Spinco model 116 amino acid analyzer equipped with sample injection and high sensitivity recording. The effluent is monitored by ninhydrin analysis. The method requires only 30 min per analysis, and allows better than 98% reproducibility and recovery. Moreover, compared to other methods, less handling is required for each sample, and less time is required of the analyst. Eight normal subjects, whose sole source of collagen was derived from a controlled diet containing 10 oz of meat per day, excreted 38.71 mg of hydroxyproline per 24 hr. After correcting for the urinary hydroxyproline excretion arising from dietary collagen, the same urinary excretion of hydroxyproline was obtained as that reported by others for subjects on collagen-free diets.

Ames Research Center Life Sciences Payload Project for Spacelab Mission 3

The Research Animal Holding Facility, developed to support rodent and squirrel monkey animal husbandry in the Spacelab environment, is to be tested during the Spacelab Mission 3 flight. The configuration and function of the payload hardware elements, the assembly and test program, the operational rationale, and the scientific approach of this mission are examined. Topics covered include animal life support systems, the squirrel monkey restraint, the camera-mirror system, the dynamic environment measurement system, the biotelemetry system, and the ground support equipment. Consideration is also given to animal pretests, loading the animals during their 12 hour light cycle, and animal early recovery after landing. This mission will be the first time that relatively large samples of monkeys and rats will be flown in space and also cared for and observed by man.