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Human peroxidasin 1 (hsPxd01) is a homotrimeric multidomain heme peroxidase embedded in the extracellular matrix. It catalyses the two-electron oxidation of bromide by hydrogen peroxide to hypobromous acid which mediates the formation of essential sulfilimine cross-links between methionine and hydroxylysine residues in collagen IV. This confers critical structural reinforcement to the extracellular matrix. This study presents for the first time transient kinetic measurements of the reactivity of hsPxd01 compound I and compound II with the endogenous one-electron donors nitrite, ascorbate, urate, tyrosine and serotonin using the sequential stopped-flow technique. At pH 7.4 and 25 °C compound I of hsPxd01 is reduced to compound II with apparent second-order rate constants ranging from (1.9 ± 0.1) × 104 M−1 s−1 (urate) to (4.8 ± 0.1) × 105 M−1 s−1 (serotonin). Reduction of compound II to the ferric state occurs with apparent second-order rate constants ranging from (4.3 ± 0.2) × 102 M−1 s−1 (tyrosine) to (7.7 ± 0.1) × 103 M−1 s−1 (serotonin). The relatively fast rates of compound I reduction suggest that these reactions may take place in vivo and modulate bromide oxidation due to formation of compound II. Urate is shown to inhibit the bromination activity of hsPxd01, whereas nitrite stimulates the formation of hypobromous acid. The results are discussed with respect to known kinetic data of homologous mammalian peroxidases and to the physiological role of human peroxidasin 1.

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine required for the intermolecular cross-linking of collagen, which is an essential step in collagen biosynthesis. Dermal fibroblasts from patients with Ehlers-Danlos Syndrome type VI (EDS VI), an inherited connective tissue disorder, express decreased levels of LH activity. In the present study we have shown that both the mRNA and the enzyme activity of LH in skin fibroblasts from one EDS VI patient (AT750), a compound heterozygote for the LH gene, are increased by administration of ascorbate and hydralazine, either individually or in combination. Although the AT750 cells express only 24% of the LH activity found in normal cells, a similar fold increase in activities in both EDS VI and normal cells was observed following treatment with ascorbate (1.5- to 2-fold) and hydralazine (2- to 4-fold), which paralleled the increase in their steady state LH mRNAs. Ascorbate increased total collagen production by 2-fold from a similar basal level of collagen synthesis in each cell type. This was confirmed by protein gel analysis which showed increases in pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen chains in both normal and EDS VI cells. This ascorbate-mediated increase of alpha 1(I) collagen resulted from increased mRNAs for alpha 1(I) collagen in both cell types. Hydralazine treatment, with or without ascorbate, severely decreased the alpha 1(I) collagen mRNAs in fibroblasts from both AT750 and the normal donor; total collagen synthesis was similarly reduced. This study shows that LH activity, which is severely deficient in fibroblasts from an EDS VI patient, can be upregulated by administration of ascorbate and hydralazine as a result of the increased mRNA for LH, suggesting that the mechanism for the regulation of the LH gene is functioning normally in this patient.

The structural features of minoxidil contributing to its inhibitory effect on lysyl hydroxylase in cultured fibroblasts were investigated. Minoxidil analogs in which the pyrimidine ring (two nitrogens) was replaced with a pyridine ring (one nitrogen) or a sym-triazine ring (three nitrogens) suppressed lysyl hydroxylase activity without affecting prolyl hydroxylase activity, as did an analog in which the piperidine ring was replaced with an N,N-diethyl group. By contrast, minoxidil analogs bearing an N-monoalkyl (ethyl or butyl) group in place of the piperidine substituent failed to suppress lysyl hydroxylase activity. The results indicate that nitroxides of pyridine and triazine, in addition to pyrimidine, having an ortho amino group can act as specific inhibitors of lysyl hydroxylase in the cell. The minimum structural requirement for inhibitory activity appears to be an organic moiety containing a tertiary nitrogen para to the nitroxide oxygen, a condition that is best fulfilled by the piperidine ring in minoxidil. Hydroxylation of minoxidil at the 3 or 4 position of the piperidine ring had no impact on its ability to inhibit the post-translational hydroxylation of lysine during collagen biosynthesis. Fibroblasts treated with minoxidil, 3'-hydroxyminoxidil, or 4'-hydroxyminoxidil synthesized a collagen specifically deficient in hydroxylysine by approximately 70%, which completely accounted for the diminished lysyl hydroxylase activity. The percentage of total proteins synthesized as collagen was reduced minimally by minoxidil but not by 3'- or 4'-hydroxyminoxidil. The studies offer a potential means for therapeutic intervention of excessive collagen deposition during fibrosis, using minoxidil or preferably its hydroxy derivatives to limit the supply of hydroxylysine for collagen crosslink formation.

Calf skin acid-soluble collagen, containing about 34 residues of lysine plus hydroxylysine per 100,000 dalton polypeptide chain, was treated with [14C]glucose in the presence or absence of NaCNBH3. In 144 h, under the conditions employed, the presence of NaCNBH3 increased the extent of glycosylation from 8 to 15% of the total residues of lysine plus hydroxylysine. The extent of glycosylation was estimated, using acid hydrolysates of the protein, by isolation and determination of reduced adducts (1-lysinohexitols) employing a system of paper chromatography followed by chromatography on an amino acid analyzer. By those means the difficulties of using specific color reactions such as that with thiobarbituric acid were obviated. Identification of the reduced adducts as forms of 1-lysinohexitol was made by comparison of that substance prepared by treatment of polylysine with [14C]glucose in the presence of NaCNBH3. Of interest is the fact that treatment of the polymer with glucose for 144 h under conditions similar to those used for the collagen, resulted in an increase of extent of glycosylation from 3 to about 50% of the total lysine residues when NaCNBH3 was present in the incubation medium. The greater degree of glycosylation of lysine residues in polylysine as compared with collagen (15 versus about 50%) may be ascribed to the different orders of macromolecular structure in the protein that could sequester certain of the residues from reaction with glucose. 1-Lysinohexitol was also identified in hydrolysates of neutral salt-soluble guinea pig skin collagen that had been reacted with glucose and then treated with NaB3H4. The glycosylated collagens were fragmented by treatment with CNBr, and modified lysine residues were found to occur along the entire length of the collagen chains. The use of NaCNBH3 in the manner described above permits measurement of both aldimine and ketoamine forms of the adducts made with glucose. The possible physiological significance of the reversibility of the ketoamine form of adduct is discussed briefly.

Matrix-free cells from chick embryo tendons were incubated for 2 h with [ 14 C]proline or [ 14 C]lysine to achieve steady-state labeling of intracellular proteins. Cell homogenates were then examined by velocity sedimentation under conditions in which nonhelical proγ chains of procollagen sediment more rapidly than other procollagen polypeptides. With these procedures it was shown that the cells contained some proγ chains which were either not triple helical or were in an unstable triple-helical conformation. These proγ chains were shown to contain less hydroxyproline than the triple-helical procollagen secreted by the cells. Their content of glycosylated hydroxylysine, however, was greater than that of the procollagen secreted by the cells. The results indicate that some of the procollagen synthesized by chick embryo tendon is assembled by the sequence in which disulfide bonding precedes complete hydroxylation. The proγ chains synthesized by this alternate route may reflect errors in protein assembly or may serve as a source of microheterogeneity in collagen.

The biosynthesis in vivo of the two reducible aldimine crosslinks of immature rabbit articular collagen, hydroxylysinohydroxynorleucine and hydroxylysinonorleucine, is demonstrated. The peak amount of crosslink was detected 1–2 weeks following labeling of the cartilage with [ 14 C]lysine. The subsequent diminution which occurred was due primarily to a decrease in the amount of hydroxylysinohydroxynorleucine. Natural reduction of the aldimine crosslinks in vivo did not occur. Glucosylgalactosyl hydroxylysine and galactosylhydroxylysine, in a 1.45 1.00 ratio, were synthesized. Seventy-three percent of the hydroxylysine residues were glycosylated. [ 3 H]NaBH 4 reduction of non- 14 C-labeled cartilage showed diminished amounts of reducible crosslink with time and the presence of hexosyl lysines and hexosyl hydroxylysines in mature articular cartilage.

Human skin has previously been shown to contain at least two genetically distinct types of collagen, type I and III. Here the presence of an additional form of collagen, α1(I)-trimer, is demonstrated. Skin collagen was solubilized by limited pepsin digestion and then fractionated by sequential precipitation with 1.5, 2.5, and 4.0 m NaCl at pH 7.4. The α-chain subunits of collagen were isolated by gel filtration and carboxymethylcellulose chromatography under denaturing conditions. The 1.5 and 2.5 m NaCl precipitates contained predominantly type I collagen with a chain composition of [α1(I)] 2 α2. In the 1.5 m precipitate a small amount of type III collagen was also recovered. In contrast, the 4.0 m NaCl fraction consisted almost exclusively of α-chains which on the basis of cyanogen bromide peptide mapping were shown to be identical with α1(I). The amino acid composition of these chains was also similar to that of α1(I), except that hydroxylysine was increased and lysine was correspondingly decreased. The content of 3-hydroxyproline was also increased. These results suggest that the α-chains in α1(I)-trimer are the same gene products as α1 in type I collagen, but that the co-translational or post-translational hydroxylation of lysyl residues is more extensive in α1(I)-trimer. Estimation of the quantitative amounts of α1(I)-trimer indicated that this collagen accounts for less than 5% of the total collagen in adult human skin. It is speculated, however, that α1(I)-trimer collagen may play a role in the stability and tensile strength of normal human skin and other tissues, and defects in its biochemistry might be associated with diseases of connective tissue.

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, pC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 m m β-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, ϵ-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and pC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis.

A collagen fraction representing two-thirds of the collagenous sequences in bovine lens capsules has been isolated following limited pepsin digestion and purified by DEAE- and carboxymethyl-cellulose chromatography in native form. The denaturation products of this collagen contain two types of components. The more acidic components (C and 50K1) are, respectively an α-chain-sized collagenous polypeptide and a mixture of smaller molecular weight proteolytic cleavage products of the C chain. The more basic components (80K and 50K2) represent, respectively, a collagenous polypeptide with an apparent Mr = 80,000 and a mixture of smaller molecular weight components derived through proteolysis of the 80K component. The C chain and 80K components are unique with respect to chromatographic properties, amino acid composition, and cyanogen bromide cleavage products. These data are interpreted to indicate that lens capsule basement membrane collagen molecules collectively contain at least two genetically distinct collagen chains: the C chain representing the collagenous domain of one type of chain and the 80K component representing the major portion of the collagenous domain of a second type of chain, designated the D chain.

A new enzyme, δ- N -hydroxyornithine: acetyl CoA δ- N -transacetylase, was partially purified from Ustilago sphaerogena . The product of the reaction catalyzed by this enzyme is a hydroxamic acid derivative of ornithine, δ- N -acetyl-δ- N -hydroxyornithine, a precursor of the peptide hydroxamate, ferrichrome. Differential centrifugation and ammonium sulfate precipitation provided a 10–15 fold purification, but further purification was hampered by the extreme lability of the enzyme. The enzyme showed a maximum activity at pH 7 and is quite specific for δ- N -hydroxyornithine as the hydroxylamino substrate, although ϵ- N -hydroxylysine is also a good substrate. Neither hydroxylamine itself nor ornithine is acetylated by the enzyme in the presence of acetyl CoA. Michaelis constants of 3.2 × 10 −4 m and 2.2 × 10 −4 m were found for δ- N -hydroxyornithine and acetyl CoA, respectively. The enzyme is not inhibited by the iron chelates, ferrichrome, ferrichrome A, or heme at concentrations sufficient to suppress ferrichrome synthesis in vivo . The amount of enzyme obtained from different cultures of U. sphaerogena showed a direct correlation with the rate of in vivo desferriferrichrome synthesis by the cells measured at the time of harvesting. This observation, as well as the substrate specificity, is evidence that this enzyme is involved in desferriferrichrome synthesis in vivo .

The total yield of salt-soluble and acetic acid-soluble collagens of human uterus, prepared in purified states, is approximately 0.06% by wet weight of tissue. After heat denaturation, the β- and α-components were separated on Sephadex G-200 columns. The separation of β11 and β12 and α1 and α2 was accomplished on carboxymethyl cellulose columns. The amino acid composition of each component, so separated, was determined. In comparison with salt-soluble collagen of skin, uterine salt-soluble collagen contains less α-component and more β-component. Amino acid analysis revealed that uterine collagen contained a somewhat higher level of hydroxyproline and hydroxylysine than dermal collagen. Cysteic acid, not seen in collagen from other mammalian tissues, was consistently present in all preparations at levels approximating 1 residue/1000 residues.

A protein resembling collagen was isolated from the giant acanthocephalan, Macracanthorhynchus hirudinaceus , by treatment with 0.1 m NaOH, solubilization with pepsin, and precipitation with ammonium sulfate. The protein resembled other collagens in amino acid composition; glycine accounted for a third of the total, and hydroxyproline and hydroxylysine were present. The carbohydrate content (5.5%) was intermediate between that of vertebrate and invertebrate collagens. The molecular weight (329,000) was similar to collagens of vertebrates, but lower than invertebrate collagens. Transition to gelatin was marked by a distinct reduction in viscosity and specific optical rotation, with denaturation temperatures ( T m ) of 45.8 and 46.5 °, respectively. The most distinctive characteristic of Macracanthorhynchus collagen is its apparent departure from the relationship between T m and total pyrrolidine content observed for other collagens.

Bovine glomeruli from which most cellular elements were removed contained a protein that shared several properties with collagen. These included the presence of hydroxylysine and hydroxyproline, digestion with collagenase, dissolution by heating, resistance to tryptic digestion, and an electron diffraction pattern characteristic of collagens. A distinctive feature of glomerular collagen was an unusually high content of hydroxylysine expressed as the hydroxylysine: hydroxyproline ratio. This collagen constituted about one half of the acellular glomerular mass.

Homocitrate synthase activity has been partially purified from yeast. Homocitrate synthase activity was sensitive to inhibition by lysine both in vivo and in vitro only at high concentrations of lysine. Two enzyme activities have been demonstrated in several strains of yeast by isoelectric focusing. The two enzymes were equally sensitive to inhibition by lysine. One enzyme activity was repressed to a greater extent when the culture medium was supplemented with excess lysine.

Collagen-galactosyltransferase and collagen-glucosyltransferase activities have been studied in cultured human fetal lung WI-38 and IMR-90 diploid fibroblasts. These enzymes functioned in concert to synthesize glucosylgalactosylhydroxylysine units as found naturally in collagens, basement membranes, and certain serum glycoproteins. The transferases used UDP-Gal and UDP-Glc as glycose donors, collagens and collagen-derived peptides or glycopeptides as glycose acceptors, and worked best in the presence of manganese as a required divalent cation. Two pH optima, between pH 6 and 6.5 and between pH 7.5 and 8, were noted for each type of transferase, and these optima, particularly in the case of glucosyltransferase, were evident regardless of size of acceptor employed in the assay. About 35% of the total activity of each enzyme was found in the soluble fractions of cell homogenates, and, of the particulate fraction activities, about 50% could be released by mild sonication or by treatment with Triton X-100. Assessment of transferase activities as a function of cellular aging in culture revealed that significant decreases in enzyme levels occurred as the cell approached senescence (late Phase II), and these effects were reversed when cells attained senescence (Phase III). Addition of ascorbic acid to young cultures, under conditions known to increase endogenous collagenpeptide hydroxylation, caused no effects on the activities of the glycosyltransferases toward exogenous acceptors. These results suggested that the activities of collagen-hydroxylases and glycosyltransferase might not be coordinately regulated, and that, regardless of the hydroxylation events, glycosylation of the peptide might be limited to a specific fraction of hydroxylysine residues during the post-translational modification of collagen.

Gradual inhibition of ammonium assimilation in Anacystis nidulans cells by increasing concentrations of 5-hydroxylysine resulted in a progressive enhancement of nitrate uptake. For 5-hydroxylysine-treated cells, the magnitude of the inhibition of nitrate uptake promoted by added ammonium was dependent on the ammonium assimilation capacity. In cells with a moderate ammonium assimilation activity, acceleration of CO2 fixation induced by bicarbonate addition antagonized the negative effect of ammonium, allowing full nitrate uptake activity. The results support the contention that nitrate utilization is under the feed-back control exerted by products of its own assimilation via ammonium, the inhibitory effect being potentiated by ammonium addition and alleviated by enhanced CO2 fixation. Results of amino acid analysis in cells exhibiting different capacities to utilize nitrate speak against these compounds as direct effectors of nitrate uptake.

To examine the effect of age on the glomerular basement membrane, compositional analyses were performed on membranes isolated in highly purified form from rats at different stages of their growth (35 to 200 days old). Substantial age-related changes were observed in the amino acid composition of the basement membranes. A significant correlation with age (P < 0.01) was evident in the contents of 3- and 4-hydroxyproline, threonine, serine, alanine, valine, half-cystine, hydroxylysine, and lysine. Of these amino acids, hydroxylysine and both isomers of hydroxyproline demonstrated a progressive increase with age, while the others were found to decline. The direct relationship of hydroxylysine content with age (P < 0.001) was associated with an inverse correlation of lysine with age (P < 0.001) so that the ratio of hydroxylysine to lysine increased in a highly significant manner from 0.92 at 35 days to 1.33 at 200 days. This elevation in the hydroxylysine content was accompanied by an augmentation in the number of saccharide units linked to it so that the percentage glycosylation of this amino acid was not significantly affected by age. The relative differences in the hydroxylysine and lysine levels between young and older rats were maintained in sodium dodecyl sulfateextracted membranes. These results suggest that the compositional changes observed during the aging process reflect an alteration in the subunit makeup of the basement membrane, possibly due to an increased synthesis or decreased degradation of the more collagen-like polypeptide components.

Resolution of the heavy microsomal fraction of lung tissue by Ficoll density gradient centrifugation yielded a rough endoplasmic reticulum microsomal fraction containing the highest specific activity of detergent-released lysyl hydroxylase. This same microsomal fraction was previously shown to contain the highest specific activity of detergent-released prolyl hydroxylase activity. When hydroxylation was inhibited during the biosynthesis of collagen, this microsomal fraction contained lysine-rich, hydroxylysine-deficient, collagenase-digestible substrate that could be hydroxylated in the absence of detergent. The results indicate coordinate localization of both prolyl and lysyl hydroxylation reactions within the cisternae of the rough endoplasmic reticulum.

The effect of hydralazine on several parameters of collagen biosynthesis has been studied in cultured human skin fibroblasts. Cells treated with hydralazine synthesized procollagen which was severely deficient in hydroxyproline and hydroxylysine, indicating inhibition of prolyl and lysyl hydroxylase reactions in the cell. Assays of prolyl and lysyl hydroxylase activities, however, revealed markedly increased levels in hydralazine-treated cells. The stimulatory effect of hydralazine could not be simulated in cell extracts, demonstrating its requirement for intact cells. The effect occurred slowly over a period of 96 h and was dependent on hydralazine concentration between 10 and 100 μ m . This phenomenon was also observed in lysyl hydroxylase-deficient mutants. In both normal and mutant cells the relative magnitude of the hydralazine effect could be modified by ascorbic acid in the culture medium. Ascorbic acid increased the response of prolyl hydroxylase to hydralazine from 1.5- to 2-fold to 3- to 7-fold, whereas it decreased the response of lysyl hydroxylase to hydralazine from 4- to 8-fold to 2- to 3-fold. Total collagen synthesis was substantially reduced in hydralazine-treated cells; the time course and the dose-response relationship were similar to those observed for the hydroxylases. α,α′-Dipyridyl, an iron chelator, mimicked these effects of hydralazine. The studies suggest the existence in cultured cells of a compensatory mechanism for overproduction of these crucial enzymes in collagen biosynthesis, a mechanism which remains functional in cells derived from patients afflicted with hydroxylysine-deficient collagen disease.

Tendons from 17-day-old chick embryos were incubated with α,α′-dipyridyl so that they synthesized type I protocollagen, the unhydroxylated form of type I procollagen. The incubation medium was cooled to 4 to 15 °C so that the protocollagen secreted into the medium folded into a triple-helical conformation, and the protein was purified by ion-exchange chromatography and velocity sedimentation. The isolated protocollagen had the expected amino acid composition in that it was similar to procollagen but it contained only 1 to 8 residues per 1000 of hydroxyproline and 1 residue per 1000 of hydroxylysine. At 15 °C, the circular dichroism spectrum of the protein was similar to that of triple-helical procollagen or collagen, and the protein underwent a thermal transition with a midpoint of about 25.5 °C. The folding and unfolding of the protein was reversible in that on recooling the denatured protein, essentially all the initial helicity was recovered. The folded form of the protein, however, did not consist of perfectly aligned triple-helical structures. Analysis of the circular dichroism spectrum demonstrated that the specific mean residue ellipticities of the peaks at 221 and 198 nm were less than the expected values of native procollagen. Also, the thermal transition for protocollagen was broader than that observed with procollagen. When examined by zonal centrifugation at 4 °C, most of the protein sedimented with the same apparent sedimentation coefficient as native procollagen but the peak of protocollagen was asymmetric and tailed toward the bottom of the gradient. When incubated with prolyl hydroxylase, some of the prolyl residues were hydroxylated. When incubated with a mixture of trypsin and chymotrypsin, about 40% of the protein was cleaved under conditions in which triple-helical procollagen and collagen resisted digestion. The results demonstrated that although type I protocollagen folds and refolds reversibly in vitro, about half of the molecules present in the folded form of the protein contain short regions which are not in a triple-helical conformation.

Sheep anterior lens capsule was isolated, and its amino acid and carbohydrate composition determined. Anterior lens capsule appears to be a complex polymer of a collagen-like glycoprotein in association with noncollagen-like glycoproteins. In this respect it resembles other basement membranes. It contains large amounts of glycine, hydroxyproline, and hydroxylysine (28, 12, and 4% of the amino acid residues, respectively). The total carbohydrate content of sheep anterior lens capsule is about 11.0 by weight, composed of glucose (4.2%), galactose (5.2%), mannose (0.5%), fucose (0.4%), glucosamine (0.46%), galactosamine (0.12%), and sialic acid (0.12%). The collagen component from sheep anterior lens capsule was isolated by limited digestion with pronase or pepsin at low temperatures. The amino acid and carbohydrate composition of the collagen from lens capsule resembles that of collagen obtained from glomerular and Descemet's membranes. In terms of residues per 1000 residues it contains glycine 338, proline 65, hydroxyproline 154, hydroxylysine 51, and half-cystine 5. It contains about 14% hexose composed of equimolar amounts of glucose and galactose. The collagen obtained after digestion with pepsin has an intrinsic viscosity of 13.1 dl/g and a molecular weight of 284,000. Anterior lens capsule contains a higher proportion of this collagen component compared to Descemet's membrane or glomerular basement membrane.

Treatment of calf skin insoluble collagen with NaB 3 H 4 followed by acid hydrolysis and ion-exchange chromatography yields a new compound which is prominent in the chromatograms of several collagens. We now describe this compound as reduced N ϵ -hexosylhydroxylysine. It appears to arise by reduction of the Schiff base between the carbonyl moiety of a hexose and the ϵ-amino group of hydroxylysine; the postulated structure was derived from both high- and low-resolution mass spectrometry and by comparison with synthetic N ϵ -galactosylhydroxylysine. Conceivably, the unreduced compound may be a crosslink, uniting collagen and glycoproteins or proteoglycans in the connective tissue.

The fate of the four stereoisomers of 5-hydroxylysine has been determined by metabolic studies of the 6- 14 C-labeled racemates and d -isomers. Both l -isomers were rapidly degraded to 14 CO 2 via glutaryl-CoA in the intact rat and in isolated perfused liver. Approximately 10%, of the 14 C from hydroxy- d -lysine-6- 14 C and 1% from allohydroxy- d -lysine-6- 14 C was expired as 14 CO 2 in the intact animal. Neither d -isomer was taken up by the perfused liver. Hydroxy- d -lysine was excreted largely unchanged, together with small amounts of 5-hydroxypipecolate tentatively identified as the d -isomer. The 14 C from allo -hydroxy- d -lysine was excreted largely as 5-hydroxypipecolate, presumably the l -isomer. The urine contained no detectable allo -5-hydroxypipecolate. The urinary hydroxypipecolate derived from both hydroxy- and allohydroxy- d -lysine was taken up but not metabolized by perfused liver. 2-Amino-5-hydroxyadipate- 14 C was metabolized to a limited extent by the rat or perfused liver and did not label 2-ketoadipate or glutarate in overloading experiments. No evidence was found for the participation of hydroxypipecolate or 2-amino-5-hydroxyadipate in the metabolism of the l -isomers of hydroxylysine.

Post-γ-globulin has been purified from urine of kidney transplant patients by a procedure which includes ultrafiltration, gel filtration, and ion-exchange chromatography. The purified protein appears to be homogeneous by ultracentrifugal and gel electrophoretic criteria. The yield of the purified protein was 30–35% of the total amount of post-γ-globulin excreted in different urine samples. Electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol revealed a single band and established that the protein consists of one polypeptide unit with a molecular weight of approximately 11,500. Sedimentation velocity experiments indicated a s 20, w of 1.55 S. Amino acid analysis of post-γ-globulin revealed the presence of hydroxylysine and hydroxyproline. The protein is devoid of carbohydrate. Post-γ-globulin has been demonstrated in small quantities in normal urine, serum, cerebrospinal fluid, and mixed saliva; preparations derived from these fluids have been shown to be immunochemically identical.

The differences between lysine and δ-hydroxylysine were investigated with respect to acid dissociation and chelate formation with copper(II). Also, six divalent metals of the first transition series were chelated with a single ligand, δ-hydroxylysine hydrochloride. The hydroxy group adjacent to the ϵ-amino group of lysine creates a second chelation site in the hydroxylysine molecule. The molecule chelates first at the carboxyl and α-amino groups showing the following trend in stabilities of the metal chelates: Cu > Ni > Zn > Co > Fe > Mn. There is some evidence that chelation at the terminal end of the molecule occurs at high pH. However, competition with metal ion hydrolysis complicates interpretation of results. The presence of precipitate precludes calculation of formation constants in this region. Some spectral studies were made in an attempt to identify the different chelated species formed.