Simplified assay for lysyl oxidase activity.

A simplified method for the assay of lysyl oxidase activity was developed. The method is based on the measurement of tritiated water released by enzyme action from labeled protein-bound lysine and hydroxylysine. Trichloroacetic acid (TCA) supernates of the incubation mixtures are passed through small Dowex 50 (H⁺) columns and the effluents are counted. For rapid screening purposes an indication of the presence of enzyme activity in enzyme preparations can be obtained by measuring the radioactivity present in aliquots of the TCA supernates as such and by measuring the radioactivity after drying at 60°C, taking the difference between the two as a measurement of enzyme activity.     

trans-cinnamic acid 4-hydroxylase of Phaseolus mungo: A new assay method

A simple and sensitive method for the assay of trans-cinnamic acid 4-hydroxylase by use of tritiated substrate is described. This method is based on the migration of tritium during the enzyme-catalyzed hydroxylation. The hydroxylase activity is detected in microsomes from Phaseolus mungo. The tritium method can be used practically with sensitivity similar to that of the ¹⁴C method. In view of the time and labor required the tritium method is obviously more advantageous.     

Modification of the tritium-release assays for prolyl and lysyl hydroxylases using Dowex-50 columns.

The tritium release assay of Hutton et al. (Anal. Biochem.16, 384, 1966) for prolyl hydroxylase and of Miller (Anal. Biochem.45, 202, 1972) for lysyl hydroxylase have been modified. The reaction is carried out on a microscale, and tritiated water is collected after passage of the trichloroacetic acid-soluble reaction products through a small Dowex-50 (H⁺) column instead of using a vacuum distillation apparatus as described in the original procedures. When measured by the modified procedures, both the prolyl and lysyl hydroxlyase reactions showed regions of linearity with respect to enzyme concentration, time and substrate concentration and were almost completely dependent on ascorbate and α-ketoglutarate. In addition, both reactions were completely inhibited by the iron chelator, α,α′-dipyridyl. The results indicate that these assay procedures are valid means of measuring prolyl and lysyl hydroxylase activities.     

A simple and sensitive fluorometric assay for tyrosine hydroxylase.

A simple, rapid, and sensitive fluorometric assay for measuring the activity of tyrosine hydroxylase is described. The enzyme activity is detected by converting tyrosine to 3,4-dihydroxyphenylalanine (dopa), which is then subjected to conversion to the highly fluorescent product by the trihydroxyindole method. The assay method is very reproducible, more sensitive than a radiochemical method for the determination of tyrosine hydroxylase activity using the isolation of [³H]water commonly used, and linear from 0.2 to 12 nmol of dopa. The method should be applicable for the assay of the enzyme with a wide range of activity.     

Mimivirus collagen is modified by bifunctional lysyl hydroxylase and glycosyltransferase enzyme

Collagens, the most abundant proteins in animals, are modified by hydroxylation of proline and lysine residues and by glycosylation of hydroxylysine. Dedicated prolyl hydroxylase, lysyl hydroxylase, and collagen glycosyltransferase enzymes localized in the endoplasmic reticulum mediate these modifications prior to the formation of the collagen triple helix. Whereas collagen-like proteins have been described in some fungi, bacteria, and viruses, the post-translational machinery modifying collagens has never been described outside of animals. We demonstrate that the L230 open reading frame of the giant virus Acanthamoeba polyphaga mimivirus encodes an enzyme that has distinct lysyl hydroxylase and collagen glycosyltransferase domains. We show that mimivirus L230 is capable of hydroxylating lysine and glycosylating the resulting hydroxylysine residues in a native mimivirus collagen acceptor substrate. Whereas in animals from sponges to humans the transfer of galactose to hydroxylysine in collagen is conserved, the mimivirus L230 enzyme transfers glucose to hydroxylysine, thereby defining a novel type of collagen glycosylation in nature. The presence of hydroxylysine in mimivirus proteins was confirmed by amino acid analysis of mimivirus recovered from A. polyphaga cultures. This work shows for the first time that collagen post-translational modifications are not confined to the domains of life. The utilization of glucose instead of the galactose found throughout animals as well as a bifunctional enzyme rather than two separate enzymes may represent a parallel evolutionary track in collagen biology. These results suggest that giant viruses may have contributed to the evolution of collagen biology.     

Inhibition of lysyl hydroxylase by catechol analogs.

Catechol analogs inhibit the activity of lysyl hydroxylase (peptidyllysine, 2-oxyglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4), a microsomal enzyme which catalyzes the transformation of certain lysyl residues in collagen to hydroxylysine. Chick embryo lysyl hydroxylase activity was measured by specific tritium release as tritiated water from an L-[4,5-3H]lysine-labelled unhydroxylated collagen substrate prepared from chick calvaria. Catechol analogs did not bind irreversibly to either enzyme or substrate, as full activity was restored with dialysis. Addition of excess cofactor, Fe2+, ascorbic acid, or alpha-ketoglutarate, did not affect inhibition. Kinetic analysis revealed that with respect to collagen substrate, catechol demonstrated a noncompetitive type of inhibition with a Ki of 15 muM.     

Simultaneous characterizations of 3-prolylhydroxylase and 4-prolylhydroxylase activities by ion exchange chromatography

3-prolyl hydroxylase activity measurements have already been described by Kivirikko and al, using specific methods. The aim of the present work was to show that the specific and rapid method used for 4-prolyl hydroxylase activity measurement, involving protocollagen [3H-4] proline (measuring of tritiated water enzymatically obtained), could be used for 3-prolyl hydroxylase activity estimation on the same sample: tritiated water enzymatically produced by 4-prolyl hydroxylase was collected by distillation, and the amino acids enzymatically modified were analysed after HCl 6 N hydrolysis of dried incubation medium, by cation exchange chromatography. The characterization of enzymatically obtained 3-hydroxyproline was performed using three means. The elution peaks reported were in the same position as the elution peak of pure 3-hydroxyproline and 4-hydroxyproline. Moreover, tritiated 3-hydroxyproline and 4-hydroxyproline were obtained only after incubation of labelled substrate with crude preparation of prolyl hydroxylases from chick embryos. Some possible artefacts such as dicetopiperazines and pyrrol-2-carboxylic acid have been shown to be distinguished chromatographically from 3-hydroxyproline and 4-hydroxyproline. The high ratio of measured (Formula: see text) activities, near 5.5 p. cent, is discussed.     

High resolution gel chromatography of proteins

An assay for the determination of l-glutaminase in extracts and intact cells is described. The method is based on the stereospecific release of ³H₂O from l-[2-³H]glutamine when l-glutaminase is coupled to l-aspartate:2-oxoglutarate amino transferase. The substrate, glutamine, and intermediate product, glutamate, are separated from the final reaction product, tritiated water, on a mixed-bed ion-exchange column which retains amino acids and organic acids but not water. The method has been adapted to determine the activity of l-glutaminase in cultured human diploid fibroblasts.     

A rapid assay for prolyl 3-hydroxylase activity.

An assay is reported for prolyl 3-hydroxylase activity. The method is based on the release of tritiated water (THO) during 3-hydroxylation of a 2,3-T-l-proline-labeled (T = tritium) polypeptide substrate in which all prolyl residues recognized by prolyl 4-hydroxylase have been converted to 4-hydroxyprolyl residues. The formation of THO was essentially linear with enzyme concentration and time, and the K_m for the polypeptide substrate was about 3.4 × 10^?8m. A linear correlation was found between THO release and the synthesis of 3-hydroxyproline, the latter being analyzed by amino acid analyzer. The assay is simple, rapid, sensitive, and reproducible, and it is specific even in tissue samples containing a large excess of prolyl 4-hydroxylase activity.     

Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor α-aminoadipic δ-semialdehyde was isolated in its reduced form, -hydroxynorleucine, together with its acid degradation product -chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the δ-semialdehyde derived from lysine and hydroxylysine.     

Characterization of collagen hydroxylysyl glycosyltransferases as mainly intramembranous microsomal enzymes

The localization of collagen hydroxylysine galactosyl- and galactosyl-hydroxylysine glucosyltransferases in purified chick embryo bone microsomes was studied by differential solubilization with nonionic detergents. Brij-35 (polyoxyethylene 25-lauryl ether) which selectively releases intracisternal proteins, and Triton X-100, whose specificity varies with its concentration, were used in the presence or absence of high ionic strength NaCl. These methods were used previously to characterize prolyl hydroxylase as intracisternal and lysyl hydroxylase as mainly intramembranous. The distribution of both glycosyltransferases within microsomes was similar to that of lysyl hydroxylase; approximately 70-80% of their activities are intramembranous with the remainder intracisternal. Collagen hydroxylysine glucosyltransferase differed from prolyl and lysyl hydroxylase and the galactosyltransferase in that its activity in vitro was apparently inhibited by membrane vesicles, even in the presence of detergents at concentrations which permeabilize the membrane. Accurate measurement of its activity could be achieved only by its separation from vesicles after detergent treatment. The common location of the major portion of lysyl hydroxylase and the glycosyltransferase activities suggests that they may act as a multienzyme complex to preferentially modify certain lysyl residues in nascent procollagen chains as they traverse the membrane of the endoplasmic reticulum. Since these enzymes do not act on helical collagen, their physical separation from prolyl hydroxylase may ensure that modifications of lysine residues occur prior to formation of hydroxyproline, which stabilizes the helical form.     

Electrochemical method for the determination of aniline hydroxylation in liver.

A rapid electrochemical method for the measurement of aniline hydroxylase activity directly in liver homogenates is described. Metabolism is monitored by direct introduction of a three-electrode assembly into incubation mixtures containing liver suspensions. The assay is based on the anodic oxidation of the metabolite, p-aminophenol, at a carbon-paste electrode. Kinetic constants for aniline hydroxylase are determined electrochemically and compared with values derived from classical indophenol procedures. The electrochemical assay permits the detection of 1 × 10^?6mp-aminophenol in microsomal suspensions with 3–5% accuracy.     

Further studies on the effect of the collagen triple-helix formation on the hydroxylation of lysine and the glycosylations of hydroxylysine in chick-embryo tendon and cartilage cells

The hydroxylation of lysine and glycosylations of hydroxylysine were studied in isolated chick-embryo tendon and cartilage cells under conditions in which collagen triple-helix formation was either inhibited or accelerated. The former situation was obtained by incubating the tendon cells with 0.6mm-dithiothreitol, thus decreasing their proline hydroxylase activity by about 99%. After labelling with [(14)C]proline, the formation of hydroxy[(14)C]proline was found to have declined by about 95%. Since the hydroxylation of a relatively large number of proline residues is required for triple-helix formation at 37 degrees C, the pro-alpha-chains synthesized under these conditions apparently cannot form triple-helical molecules. Labelling experiments with [(14)C]lysine indicated that the degree of hydroxylation of the lysine residues in the collagen synthesized was slightly increased and the degree of the glycosylations of the hydroxylysine residues more than doubled, the largest increase being in the content of glucosylgalactosylhydroxylysine. Recovery of chick-embryo cartilage cells from temporary anoxia was used to obtain accelerated triple-helix formation. A marked decrease was found in the extent of hydroxylation of the lysine residues in the collagen synthesized under these conditions, and an even larger decrease occurred in the glycosylations of the hydroxylysine residues. The results support the previous suggestion that the triple-helix formation of the pro-alpha-chains prevents further hydroxylation of lysine residues and glycosylations of hydroxylysine residues during collagen biosynthesis.     

Neuraminidase assay utilizing sialyl-oligosaccharide substrates with tritium-labeled aglycone.

A new method for the radioisotopic assay of neuraminidase activity has been developed. The substrate utilized, α-d-N-acetylneuraminosyl-(2 → 3′)-lactit[³H]ol, was prepared by reduction of α-d-N-acetylneuraminosyl-(2 → 3′)-lactose with tritiated borohydride and purified by ion-exchange chromatography. After incubation with neuraminidase, the reaction mixtures were applied to small columns of AG 1-X2 (formate) in order to remove free sialic acid and unhydrolyzed substrate. The lactit[³H]ol released by neuraminidase action was then recovered by washing the columns with distilled water and quantitated by utilizing a liquid scintillation spectrometer. Studies with bacterial, avian, and mammalian neuraminidases are described.     

A rapid and simplified assay method for tyrosine hydroxylase

Tyrosine hydroxylase can be measured by release of tritiated water from labeled tyrosine, and the assay method has now been modified to allow recovery of 3H2O from the reaction mixture in a much more rapid and less tedious manner than previously possible. In the new method, the tyrosine hydroxylase reaction is stopped with sodium carbonate, pH 11.6. At this pH the tritium in 3H2O, but not other 3H species, is extracted into an organic scintillant containing 25% isoamyl alcohol, toluene, 2,5-diphenyloxazole, and p-bis-[2-(5-phenyloxazolyl)]benzene. The selective extraction occurs by means of exchange of tritium in 3H2O with the hydroxyl proton of isoamyl alcohol. It is the [3H]isoamyl alcohol that is then extracted into the scintillant and quantified by liquid scintillation spectrometry. Although the organic extraction method is somewhat less sensitive than the more frequently used ion-exchange method for isolating the 3H2O formed in the tyrosine hydroxylase reaction, it is much more rapid, as well as cost effective, since the enzyme reaction, extraction, and counting are carried out within the same vial.     

Studies on the phenylalanine hydroxylase system in vivo. An in vivo assay based on the liberation of deuterium or tritium into the body water from ring-labeled L-phenylalanine.

The rate of release of deuterons into the body water from 2,3,4,5,6-pentadeutero-L-phenylalanine has been shown to be a valid measure of the activity of the phenylalanine hydroxylase system in vivo. At a dose of 0.5 g/kg, the rate of release of deuterons is linear for 60 to 90 min. Male rats, which had previously been shown to have 22 to 25% more phenylalanine hydroxylase activity in liver extracts than female rats, produced deuterons from deuterated phenylalanine at a rate 20 to 30% greater than female rats. p-Chlorophenylalanine, which irreversibly inhibits phenylalanine hydroxylase in vivo, caused a similar degree of inhibition of the rate of deuteron formation as was found when phenylalanine hydroxylase was measured in extracts from the same group of animals. Methotrexate, which inhibits the phenylalanine hydroxylase system by preventing regeneration of the tetrahydropteridine cofactor, caused parallel inhibition of the in vivo assay as well as when the conversion of phenylalanine to tyrosine was measured in liver slices. Randomly ring-tritiated phenylalanine can be used interchangeably with ring-deuterated phenylalanine if greater sensitivity is needed in the in vivo assay for phenylalanine hydroxylase. However, a dose of 20 to 30 muCi/kg is required. The in vivo deuterium release assay described in this paper should be useful in studying the physiological control of the phenylalanine hydroxylating system. It also may be of value in differentiating between individuals who are heterozygotes for phenylketonuria and those who are homozygotes for hyperphenylalaninemia.     

Recombinant human type II collagens with low and high levels of hydroxylysine and its glycosylated forms show marked differences in fibrillogenesis in vitro

Type II collagen is the main structural component of hyaline cartilages where it forms networks of thin fibrils that differ in morphology from the much thicker fibrils of type I collagen. We studied here in vitro the formation of fibrils of pepsin-treated recombinant human type II collagen produced in insect cells. Two kinds of type II collagen preparation were used: low hydroxylysine collagen having 2.0 hydroxylysine residues/1,000 amino acids, including 1.3 glycosylated hydroxylysines; and high hydroxylysine collagen having 19 hydroxylysines/1,000 amino acids, including 8.9 glycosylated hydroxylysines. A marked difference in fibril formation was found between these two kinds of collagen preparation, in that the maximal turbidity of the former was reached within 5 min under the standard assay conditions, whereas the absorbance of the latter increased until about 600 min. The critical concentration with the latter was about 10-fold, and the absorbance/microgram collagen incorporated into the fibrils was about one-sixth. The morphology of the fibrils was also different, in that the high hydroxylysine collagen formed thin fibrils with essentially no interfibril interaction or aggregation, whereas the low hydroxylysine collagen formed thick fibrils on a background of thin ones. The data thus indicate that regulation of the extents of lysine hydroxylation and hydroxylysine glycosylation may play a major role in the regulation of collagen fibril formation and the morphology of the fibrils.     

Regulation of the glycosylations of collagen hydroxylysine in chick embryo tendon and cartilage cells

The regulation of the glycosylations of hydroxylysine was studied in isolated chick-embryo cells by labelling with a [¹⁴C]lysine pulse. The course of the procollagen lysyl modifications was compared in tendon and cartilage cells, and the effect on the glycosylations of the degree of lysyl hydroxylation and the concentration of Mn²⁺ and Fe²⁺ were also studied, in tendon cells. Procollagen triple helix formation was inhibited in most experiments in order to eliminate the effect of this process on the continuation of the reactions.Both in the tendon and cartilage cells the intracellular lysyl modifications proceeded in a biphasic fashion. After an initial sharp linear increase, the reactions did not cease but were protracted at a slower but constant rate. Lysyl hydroxylation was followed by rapid galactosylation in both cell types and this was followed almost immediately by rapid glucosylation, suggesting a close association of the corresponding enzymes. The data further suggest that other factors must also exist, in addition to the differences in the timing of triple helix formation and the actual hydroxylysine content, which are responsible for the different amounts of galactose in the collagens synthesized by these cell types. The amount of glycosylgalactosylhydroxylysine nevertheless seemed to be determined by the available acceptor sites, i.e., the amount of galactosylhydroxylysine.In further experiments wiht tendon cells the oxygen participating in lysyl hydroxylation was displaced by nitrogen at various points in time. When the degree of lysyl hydroxylation was reduced to less than one-third of the original, the total amounts of glycosylated residues decreased correspondingly, but their proportion relative to total hydroxylysine remained unchanged.Extra Mn²⁺ increased the proportion of galactosylated hydroxylysine, suggesting that the activity of hydroxylysyl galactosyltransferase is not saturating in respect of the catalyzed reaction. Experiments on the addition of Fe²⁺ or its chelation by α, α′-dipyridyl gave indications that the presence of this co-factor is not required for either glycosylation reaction in isolated tendon cells.     

A convenient radiometric assay for flavin-containing monooxygenase activity

A rapid, convenient assay to determine the activity of the flavin-containing monooxygenase is described. The method is based on direct analysis of quenched incubation mixtures by thin-layer chromatography and utilizes tritiated dimethylaniline as the substrate. The synthesis of the radiolabeled substrate is described. The usefulness of dimethylaniline N-oxide formation as a measure of flavin-containing monooxygenase activity was assessed using the purified hog liver enzyme, hog liver microsomes, and liver microsomes from untreated and phenobarbital-pretreated rats.     

The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.