Gerald L. Mechanic

Location of the intermolecular cross-links in bovine dentin collagen, solubilization with trypsin and isolation of cross-link peptides containing dihydroxylysinonorleucine and pyridinoline

Abstract [3H]NaBH4 reduced bovine dentin collagen was denatured at 60°C for 1 hr and then digested with trypsin. The digest was still substantially insoluble suspension, but it was found that 99% of dentin collagen can be solubilized if the digest was heated again at 60°C for 15 min. Two cross-linked tryptic peptides were isolated from this digest by sequential chromatographies on Sephadex G50, phosphocellulose and DEAE-cellulose column. One isolated peptide was characterized as a 59 residue cross-linked peptide including one residue of dihydroxylysinonorleucine and the other was 103 residue including one residue of pyridinoline. The amino acid compositions were consistent with the identification of the 59 residue peptide as the sequence in α1-CB4-5 (76–90) linked to the sequence in α1-CB6 (990-23c), and the 103 residue peptide as the sequence 76–90 linked to two of the sequence 990-23c. These results strongly support the previously proposed precursor-product relationship between dihydroxylysinonorleucine and pyridinoline.

The sephadex gel filtration characteristics of the neutral soluble proteins of embryonic bovine enamel

Abstract 1. 1.|The behaviour of the neutral soluble proteins of embryonic enamel when filtered through columns of Sephadex G-75 and G-100 resin was investigated. 2. 2.|The relative proportion of the protein which was retarded and which was excluded from the resin was dependent on the initial concentration of the protein solution. 3. 3.|Each of the peaks eluted from the Sephadex columns was studied by ultracentrifugation, amino acid analysis, and by its elution profile when rerun through the Sephadex column. All but one of the peaks were found to contain both high and low molecular weight material in a state of labile equilibrium. 4. 4.|The composition of the fractions which were retarded on the Sephadex columns was found to be dependent on both a concentration effect and the elution of distinctive chemical moieties at particular void-volume to elution-volume ratios. Each of the fractions which was retarded on the Sephadex columns formed high molecular weight aggregates of variable composition. 5. 5.|High molecular weight material which did not dissociate on dilution, and which was apparently unrelated to the interacting system, dissociated in 6 M urea, and was therefore also composed of non-covalently bonded aggregates. The average molecular weight of the aggregates was found to be 2.9·10 6 , and the estimated molecular weight of the smallest aggregate, 1.86·10 6 . 6. 6.|The results of these experiments characterize the enamel proteins as a multicomponent system involving different aggregate complexes of approximately the same size.

Collagenolytic Activity During Active Bone Resorption in Tissue Culture.

Summary A tissue culture system has been used to study the release of collagenolytic activity from actively resorbing bone by measuring the degradation of purified, reconstituted H3-hydroxyproline- and H3-proline-labeled collagen fibrils. Under conditions where active bone resorption was observed morphologically, a collagenolytic factor was liberated from the bone which degraded reconstituted, undenatured collagen fibrils. This collagenolytic activity was increased by addition of parathyroid extract to the tissue culture medium, and still further enhanced when both parathyroid extract and heparin were added.

The ultracentrifugal and free zone electrophoretic characterization of the neutral soluble proteins of embryonic bovine enamel.

Abstract 1. 1. The neutral soluble proteins of embryonic bovine enamel exist in aqueous solution as a mixture of labile, reversibly equilibrating species. Sedimentation patterns show a number of peaks whose velocities are pH dependent. The relative proportions of the fast and slow moving peaks were found to de dependent on the total protein concentration, pH, salt concentration and solvent. Two peaks are present which have s ° 20, w values of 1 and 24 S at neutral pH, and another peak with an s ° 2-, w of 2 S is present in the alkaline pH region. In 0.1 M supporting electrolyte the sedimentation velocity of the fast-moving peak changes continuously with pH going through a minimum at pH 8.3 and a maximum at neutral pH. In 0.25 M supporting electrolyte the variation with pH is discrete on the alkaline side but remains continuous in the acid range. 2. 2. Electrophoresis patterns in the alkaline region show one major boundary representing at least 90% of the protein and three minor boundaries which appear to represent non-interacting species. In the acid range, the patterns show a greater number of peaks. 3. 3. In 2,4,6 or 8 M urea essentially all of the protein is represented by a single slow moving boundary. This dissociation, as well as that which occurs under relatively mild acid conditions, is reversible on neutralization or removal of the urea. 4. 4. It was concluded that the sedimentation and electrophoresis data are consistent either with a polymerizing multi-component system with rapid re-equilibration kinetics with respect to transport processes, or a polymerizing single-component system plus non-interacting species with intermediate reaction rate kinetics.

The molecular structure of the neutral-soluble proteins of embryonic bovine enamel in the solid state

Cross- β X-ray diffraction patterns have been obtained from fibers prepared from the cold, neutral-soluble protein fraction of decalcified, bovine, embryonic enamel matrix. Similar cross- β patterns have previously been reported from the intact organic matrix (8). The structural implications of the unusually high proline content of these proteins are discussed.

THE ELECTRON MICROSCOPIC LOCALIZATION OF THE NEUTRAL SOLUBLE PROTEINS OF DEVELOPING BOVINE ENAMEL.

Electron microscopy, coupled with amino acid analyses, has shown that the proline- and histidine-rich protein fractions of embryonic bovine enamel which are solubilized in cold, neutral buffer solutions, are derived primarily from the inter- and intraprismatic filaments. In contrast to the majority of the inter- and intraprismatic filament proteins, the prism sheath proteins were relatively insoluble in neutral buffer solutions. There were a small number of filaments in many of the prisms which were not solubilized after prolonged extraction with neutral buffer solution. Both these filaments and the prism sheaths were soluble in dilute acetic acid. There were significant differences in the compositions of the proteins of the inter- and intraprismatic filaments and those of the prism sheaths, which may reflect their different biological functions.

Rapid short-column chromatography of amino acids. A method for blood and urine specimens in the diagnosis and treatment of metabolic disease.

Abstract Methods are described for the rapid analysis of certain individual amino acids (e.g., homocystine) and of groups of amino acids (e.g., the branched-chain amino acids), which are relevant to particular metabolic diseases. All the methods use ion-exchange chromatography on a 20 × 0.9 cm column. These methods were developed especially for clinical application in the diagnosis and therapy of inborn errors of amino acid metabolism. All analyses are completed in less than 2.5 hr.

STUDIES OF COLLAGEN DEGRADATION DURING BONE RESORPTION IN TISSUE CULTURE.

Summary A tissue culture system has been used to study the removal of bone collagen from radioactively labeled, resorbing mouse calvaria, by measuring the amount of hydroxyproline and the radioactivity of H3-hydroxyproline released into the tissue culture medium. The amounts of hydroxyproline and H3-hydroxyproline released from the calvaria were enhanced by addition of parathyroid extract to the medium and correlated with the extent of bone resorption observed microscopically in the living cultures. Evidence is presented which indicates that bone collagen is partially degraded during active bone resorption in tissue culture to peptides similar in size and sequence to those produced by the action of bacterial collagenase on collagen.

A rapid quantitative estimation of tyrosine and phenylalanine by ion-exchange chromatography.

Abstract An automatic method for rapid quantitation of tyrosine and phenylalanine by ion-exchange chromatography is presented. The method may be adapted for use on any automatic amino acid analyzer. Complete quantitation of tyrosine and phenylalanine can be obtained in as little as 15 min, making it ideal for use in screening programs for phenylketonuria and other inborn errors of metabolism.

Collagen in the Spicule Organic Matrix of the Gorgonian Leptogorgia virgulata

Decalcification of the calcareous spicules from the gorgonian Leptogorgia virgulata reveals an organic matrix that may be divided into water insoluble and soluble fractions. The insoluble fraction displays characteristics typical of collagen, which is an unusual component of an invertebrate calcium carbonate structure. This matrix fraction exhibits a collagenous amino acid profile and behavior upon SDS-PAGE. Furthermore, the reducible crosslink, dihydroxylysinonorleucine (DHLNL), is detected in this fraction. The composition of the matrix varies seasonally; i.e., the collagenous composition is most prevalent in the summer. These results indicate that the insoluble matrix is a dynamic structure. Potential roles of this matrix in spicule calcification are discussed.