Comparison between avian and human prolyl 4‐hydroxylases: Studies on the holomeric enzymes and their constituent subunits

Abstract

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.

Improved solid‐phase microextraction device for use in on‐line immunoaffinity capillary electrophoresis

“…1), coupled covalently to Blue Carrier immunogenic protein, were raised in rabbits using a method similar to those described elsewhere [47,48]. The purification of the antibodies from rabbit antisera was performed by HPLC using r-Protein A affinity chromatography as previously described [48].…”

“…Immediately after collection of a pool of six urine samples, a cocktail of protease inhibitors was added including 0.1 mM of PMSF, soybean trypsin inhibitor, iodoacetate, p-aminobenzamidine, and 1 mM leupeptin [38,42,47]. A 0.20 mm porous diameter Nylon filter was used to filter the pooled samples, to remove particulate matter or cells.…”

“…The peptides were linked to CPG through a procedure described elsewhere [51]. The columns were individually eluted with 3 M sodium thiocyanate in 0.01 M sodium phosphate buffer, pH 7.0 [47]. The highly specific pure antibodies were dialyzed against 0.01 M sodium phosphate buffer, pH 7.0, aliquoted in small fractions, and stored at 2707C until use.…”

A Quantitative Assay for the Determination of Proline and Hydroxyproline by Capillary Electrophoresis

“…Measurements of hydroxyproline content in a particular tissue have not only been useful in assessing the quantity and quality of collagen in that tissue, but also in determining the rate at which that collagen is synthesized or degraded. The intense interest in imino acids is evident by the great number of methods and modifications of methods developed over the years (16)(17)(18)(19)(20)(21)(22)(23). It is also evident from these reports that a simple specific method for measuring proline and hydroxyproline is lacking.…”

“…A variety of procedures have been described, including colorimetric (paper chromatography, thinlayer chromatography, spectrophotometric), liquid chromatography (radioactive, spectrophotometric, fluorimetric), gas chromatography, and mass spectrometry (16)(17)(18)(19)(20)(21)(22)(23). In this report, we have evaluated capillary electrophoresis (CE) as a complementary or alternative technique to assess proline and hydroxyproline.…”