Elijah Adams

Fluorometric determination of pyridoxal phosphate in enzymes.

Abstract The pyridoxal phosphate contents of several enzymes have been compared both by fluorometry and by absorption spectrophotometry of the phenylhydrazone, and give results in good agreement. The fluorometric method is as rapid and simple as the spectrophotometric method and in addition is about as sensitive as the best biological assay methods. The characteristic excitation and emission spectra also permit qualitative identification of the coenzyme.

Intraspecies composition difference in collagen from cuticle and body of Ascaris and Lumbricus

Collagens of diverse origin have a generally similar amino acid composition, the extreme differences recorded being for Lumbricus cuticle, in which there are 12–20 hydroxyproline residues per proline (Watson, 1958;Maser and Rice, 1962; Josse and Harrington, in press∗∗ and for Ascaris cuticle, in which this ratio is reversed, with about 15 proline residues per hydroxyproline (Watson and Silvester, 1959; Josse and Harrington). In connection with studies on the synthesis and turnover of Lumbricus cuticle collagen, to be described elsewhere, we have made observations comparing the amino acid composition of cuticle collagen with that of collagen extracted from cuticle-stripped bodies in each species of worm. For both Ascaris and Lumbricus, these measurements indicate marked differences when body and cuticle collagen are compared with respect to hydroxyproline/proline ratios. To our knowledge, such large intraspecies tissue difference in collagen composition have not been reported, although only limited date are available comparing different tissues in the same species (Tristram and Smith, 1963).

Coenzyme content of purified alanine racemase from Pseudomonas

Abstract Alanine racemase, induced in Pseudomonas putida by growth on DL -alanine, was purified about 1000-fold to homogeneity. The purified enzyme contains approximately one molar equivalent of pyridoxal phosphate by absorbance, fluorescence, and microbiological assay. No evidence for the presence of a flavin coenzyme was found.

Basement membrane and interstitial collagen content of whole animals and tissues

Summary A method for estimating total basement membrane collagen in whole animals or tissues was based on measuring 3-hydroxyproline and 4-hydroxyproline of samples. Basement membrane collagen is 1% or less of total mouse collagen, but is a substantial fraction of kidney cortex collagen. In agreement with earlier analysis by a different method, total mouse collagen is 2–3% of wet weight.

Induction of separate catabolic pathways for L- and D-lysine in Pseudomonas putida

Abstract A strain of Pseudomonas putida was found to oxidize L-lysine by an inducible pathway through δ-aminovalerate, and D-lysine via Δ 1 -piperideine-2-carboxylate and pipecolic acid. Each pathway is selectively induced by L- or D-lysine and the appropriate intermediates. Lysine racemase is inadequate to permit growth on L-lysine after a block in the L-pathway, but is sufficient to permit cross induction of D-lysine-related enzymes.

Proline incorporation into the proline and hydroxyproline of earthworm-cuticle collagen

Abstract Studies in vivo of the incorporation of proline into earthworm cuticle have shown that, as with vertebrate collagens, proline and not free hydroxyproline is the source of both proline and hydroxyproline in cuticle collagen. Purified salt-soluble cuticle collagen preparations contain a component highly labeled in proline and resistant to collagenase (EC 3.4.4.19), but sensitive to trypsin (EC 3.4.4.4). The labeled proline in crude collagen preparations, although much higher in specific activity than hydroxyline, decays with about the same time-constant as the hydroxyproline; this and related observations suggest that injection-stimulated synthesis rather than normal turnover is being measured in these experiments. Digestion of cuticle collagen with collagenase releases most of the hydroxyproline in dialyzable form, implying extensive degradation to small fragments.

Alpha-Ketoglutaric Semialdehyde: A Metabolic Intermediate

α-Ketoglutaric semialdehyde has been obtained as a product of hydroxyproline through reactions catalyzed by purified enzymes from Pseudomonas. It has been characterized both by chemical and enzymatic derivatives and by comparison with the chemically synthesized compound. This reactive compound, not previously known as a product of biological reactions, may participate in other metabolic pathways.

Collagen helix stabilization by hydroxyproline in (Ala-Hyp-Gly)n

Abstract (Ala-Hyp-Gly)n was synthesized and fractionated to yield three fractions in the molecular weight range 10,000 to 2,500. Optical rotatory and circular dichroism measurements indicated the collagen-like properties of this polymer in contrast to those reported for (Ala-Pro-Gly)n. We conclude that hydroxyproline contributes to collagen-helix stability in general, independent of the presence of an adjacent proline residue as in previously studied synthetic polypeptides. (Pro-Hyp-Gly)n showed identical circular dichroism spectra whether polymerized via the nitrophenyl ester or tetraethylpyrophosphite.

Observations on the gel chromatography of collagen-like peptides and use of a simple calibration method for molecular weight determination

Abstract Elution characteristics of collagen-derived polypeptides and of globular proteins were compared under identical experimental conditions with agarose gels. This comparison permitted calculation of the hydrodynamic radii of collagen polypeptide chains of different molecular weight, and these radii were shown to be in reasonable agreement with estimates made from intrinsic viscosity data. Two distinet linear relationships were observed for collagen polypeptide chains, relating logarithm of molecular weight to elution parameters. Peptide chains of MW 3300 and lower fell on a line of a steeper slope than did larger polypeptide chains. A simple procedure for molecular weight estimation of an unknown polypeptide chain of the collagen class is described, using only three commercially available standards for calibration: reduced, carboxymethylated Ascaris cuticle collagen, and the synthetic peptides ( l -Pro- l -Pro-Gly) 10 and ( l -Pro-L-Pro-Gly) 5 .

Formation and excretion of pyrrole-2-carboxylate in man.

A detailed investigation of the purification of pyrrole-2-carboxylate (PCA) from human and rat urine indicates that previously reported mean values overestimate the correct quantity of free PCA by a factor of approximately three for rat urine and approximately five for human urine. Although several criteria of purity were satisfied by a previous method, pyrrole-reactive impurities were still present in the final fractions. These impurities are separated from PCA by chromatography through an amino acid analyzer ion-exchange resin. With the corrected method, normal human values for endogenous urinary PCA in 16 individuals averaged 0.51 mumol/day, with a range of 0.20-1.3 mumol and a SD of 0.31 mumol. The probable source of human PCA is free hydroxy-L-proline, as inferred from the high value for PCA in the urine of a subject with hereditary hydroxyprolinemia, and from the threeto eightfold elevation in PCA excretion by two normal subjects after a large oral load of hydroxyl-L-proline. Subcutaneous administration of [2-(14)C]PCA to a single human subject indicated almost complete conversion of the exogenous compound to derivatives, which are largely excreted in the urine. Data are discussed suggesting that much or all of the PCA in human urine may be formed in urine from a labile precursor, presumably Delta(1)-pyrroline-4-hydroxy-2-carboxylate.