Secretion and stabilization of the layers of the egg capsule of the dogfish Scyuorhinus canicula

The egg capsule of the dogfish is a unique, collagcnous, layered structure secreted by the nidamental gland which has nine, remarkably discrete, transverse zones of tubular glands. The present paper traces the origin of the four layers of the capsule to particular zones within the gland. Evidence is presented for the existence of DOPA, DOPA oxidase. protein(s) rich in tyrosyl residues and a peroxidase within the same storage granules within the secretory cells of the C and E zones. It is suggested that these interact when secreted to cross-link the inner and outer surfaces of the egg capsule. Evidence is presented that the middle layer which forms the bulk of the thickness of the egg capsule and has the highest collagen content may be partly stabilized by the peroxidation of tyrosyl residues. The mechanical significance of crosslinking in this system and the possible cytological mechanisms involved in the secretion of the tanning agents and enzymes are discussed.     

Collagen type I and hyaluronic acid based hybrid scaffolds for heart valve tissue engineering

Tissue engineers have achieved limited success so far in designing an ideal scaffold for aortic valve; scaffolds lack in mechanical compatibility, appropriate degradation rate, and microstructural similarity. This paper, therefore, has demonstrated a carbodiimide-based sequential crosslinking technique to prepare aortic valve extracellular matrix mimicking (ECM) hybrid scaffolds from collagen type I and hyaluronic acid (HA), the building blocks of heart valve ECM, with tailorable crosslinking densities. Swelling studies revealed that crosslinking densities of parent networks increased with increasing the concentration of the crosslinking agents whereas crosslinking densities of hybrid scaffolds averaged from those of parent collagen and HA networks. Hybrid scaffolds also offered a wide range of pore size (66-126 μm) which fulfilled the criteria for valvular tissue regeneration. Scanning electron microscopy and images of Alcian blue-Periodic acid Schiff stained samples suggested that our crosslinking technique yielded an ECM mimicking microstructure with interlaced bands of collagen and HA in the hybrid scaffolds. The mutually reinforcing networks of collagen and HA also resulted in increased bending moduli up to 1660 kPa which spanned the range of natural aortic valves. Cardio sphere-derived cells (CDCs) from rat hearts showed that crosslinking density affected the available cell attachment sites on the surface of the scaffold. Increased bending moduli of CDCs seeded scaffolds up to two folds (2-6 kPa) as compared to the non-seeded scaffolds (1 kPa) suggested that an increase in crosslinking density of the scaffolds could not only increase the in vitro bending modulus but also prevented its disintegration in the cell culture medium.     

Long-term culture of functional hepatocytes on chemically modified collagen gels

For long-term maintenance of functional hepatocytes in primary culture, a new culture system with chemically modified type-I collagen gel was developed. Isolated hepatocytes spread as flat cells and rapidly lost their viability and functions when cultured on native collagen gel. In contrast, they survived for several weeks when cultured on collagen gels that had been modified by treatment with sodium-borohydride (NaBH₄) or by digestion with pepsin, which resulted in destruction of crosslinking of collagen fibers and marked decrease in meachanical strength of the gels. These long-lived cells were round and aggregated and maintained high levels of various differentiated liver functions including albumin secretion and activities of tyrosine aminotransferase and P450. Moreover on collagen gels modified by treatment with NaBH₄ or pepsin, the cell showed less DNA synthesis in response to mitogenic stimulation than cells cultures on gel containing native collagen. Interestingly, crosslinking of these chemically modified gels with D-ribose resulted in changes in various phenotypes of hepatocytes cultures on them including shape, longevity, and functions expressed when the cells were cultured on native collagen gel, suggesting that the effect of modification of the collagen gel is reversible. Thus the structure of collagen gels, probably due to the degree of crosslinking, seems to affect the morphology, maintenance of differentiated functions, and growth of primary cultured hepatocytes.     

Differentiation in brain and liver DOPA/5HTP decarboxylase activity after L-DOPA administration with or without pyridoxine in cats.

Abstract— Pyridoxine (50mg/kg, per os) given for 7 consecutive days did not modify the content of dopamine, noradrenaline, and serotonin in the neostriatum of the brain 3, 6 and 18 h after the last dose, but significantly increased DOPA/5HTP decarboxylase activity in both the neostriatum and liver. The administration of l-DOPA and pyridoxine (100 and 50mg/kg, per os, respectively) together for 7 days increased DOPA/5HTP decarboxylase activity in the brain to the same extent as did l-DOPA and pyridoxine given individually. Liver DOPA/5HTP decarboxylase activity remained normal when both drugs were administered together. However it decreased significantly after l-DOPA administration for 7 days but not after pyridoxine treatment. In cats under treatment with l-DOPA for 7 days, actinomycin D given for the final 3 days prevented the increased DOPA/5HTP decarboxylase activity induced by l-DOPA in the neostriatum and mesencephalon but had no effect on the enzymatic activity in the liver. These findings indicate that differences exist between brain and liver DOPA/SHTP decarboxylase activity in uivo. In addition, denatured supernatant from livers of animals treated with l-DOPA contained a dialysable compound which inhibits DOPA/SHTP decarboxylase activity in the supernatant from livers of untreated cats. In animals who received pyridoxine along with l-DOPA, no such inhibitor was found. These results may explain the mechanism by which l-DOPA exerts its beneficial effects and why pyridoxine administered with l-DOPA reduces the therapeutic effectiveness of l-DOPA in Parkinson's disease. These findings are consistent with the possibility that a tetrahydro-isoquinoline derivative formed in vivo in the liver after l-DOPA therapy for 7 days might affect DOPA/5HTP decarboxylase activity in the liver but not in brain. A tetrahydroisoquinoline derivative did not appear to be formed when l-DOPA and pyridoxine were administrated together suggesting that pyridoxine protected the enzyme and favored a more rapid degradation of l-DOPA peripherally with less l-DOPA available for the CNS.     

Biochemical complementation of the betalain biosynthetic pathway in Portulaca grandiflora by a fungal 3,4-dihydroxyphenylalanine dioxygenase

3,4-Dihydroxyphenylalanine (DOPA) dioxygenase from Amanitamuscaria catalyses the key reaction of betalain biosynthesis, namely the conversion of DOPA to betalamic acid by a 4,5-ring-opening reaction. In addition, it catalyses a 2,3 opening which yields the fungal pigment muscaflavin, a compound that has never been found in plants. In this work, a cDNA clone (DodA) encoding A. muscaria DOPA-dioxygenase was expressed in white Portulacagrandiflora petals, using the particle bombardment technique. Transformation resulted in the formation of yellow and violet spots that contained betalain pigments and muscaflavin, indicating that the fungal enzyme was expressed and active in plants, and could complement the plant betalain biosynthetic pathway. The presence of muscaflavin in transformed plants indicates a difference in the specificity of the plant and A.muscaria enzymes. Received: 24 February 1997 / Accepted: 20 May 1997     

The hydroxypyridinium crosslinks of skeletal collagens: Their measurement,properties and a proposed pathway of formation

A method is described for quantifying both reducible and mature crosslinking amino acids of collagen. The main crosslinking residue in cartilage, dentine and mature bone was the 3-hydroxypyridinium compound identified by Fujimoto et al. (1–3). Adult articular cartilage contained about one residue per collagen molecule, over forty times the content of the reducible crosslinks. We propose that hydroxypyridinium residues are formed by spontaneous interaction of two residues of hydroxylysino-5-ketonorleucine. This reaction explains the disappearance of reducible crosslinks at maturity and provides a novel mechanism for lateral crosslinking within and between fibrils which may account for some of the unique physical properties of hard tissue collagens.     

Characterisation of the slime gland secretion from the peripatus, Euperipatoides kanangrensis (Onychophora: Peripatopsidae)

The Onychophora display a distinctive mechanism of feeding that involves the entanglement of prey in a sticky secretion. This secretion is produced in the slime glands and ejected as adhesive threads from a pair of oral papillae on either side of the head. Biochemical analyses of the secretion reveals it to be a composite material containing protein, sugar, lipid and a surfactant, nonylphenol. The identification of nonylphenol in the secretion is significant in that this is the first report of this compound from a natural source. The proteins are the principal component of the slime and the amino acid composition of the crude secretion suggests the presence of collagen or a ‘collagen-like’ domain. One or more of the high molecular weight proteins are O-glycosylated where the predominant modification is a single N-acetylgalactosamine (GalNAc). This study adds to our understanding of the chemical and biochemical composition of the unusual onycophoran slime gland secretion.     

Localization of dopamine in the freshwater hydrozoanHydra attenuata

Summary High performance liquid chromatography (HPLC), with electrochemical detection, is an analytical method sensitive enough to permit quantification of dopamine, dihydroxyphenylalanine (DOPA) and 5-S-cysteinyl DOPA in single or hemisected specimens ofHydra attenuata. Dopamine and 5-S-cysteinylDOPA appear to be the quantitatively predominant catechol compounds inH. attenuata, whereas DOPA is present in minor amounts. The presence of DOPA and 5-S-cysteinylDOPA, and the quantitative correlation between dopamine and these compounds in many specimens, suggests that dopamine inH. attenuata, as in higher animals, is formed through decarboxylation of DOPA. Contrary to the dopaminergic nerves in higher animals, DOPA inHydra seems to be oxidized and 5-S-cysteinyl DOPA is formed as a by-product. The oxidation of DOPA indicates that the hydroxylation of tyrosine into DOPA in the tissues ofH. attenuata is mediated by a tyrosinase rather than a tyrosine hydroxylase. Immunocytochemical methods demonstrate a highly variable distribution of dopamine in the tissues of different specimens ofH. attenuata. Dopamine immunoreactivity is confined to ectodermal tissue and can be found in several different cell types including nerve cells, battery cells, nematocytes, epithelial cells and interstitial undifferentiated cells. The large amounts of dopamine found in some specimens ofH. attenuata indicate some biological function, although its sporadic occurrence in neurites makes it less plausible as a generally utilized neurotransmitter in this animal.     

Plasma membrane factor XIIIA transglutaminase activity regulates osteoblast matrix secretion and deposition by affecting microtubule dynamics

Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.     

Protective Effects of Soluble Collagen during Ultraviolet-A Crosslinking on Enzyme-Mediated Corneal Ectatic Models

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.     

Effects of products from macrophages, blood mononuclear cells and of retinol on collagenase secretion and collagen synthesis in chondrocyte culture

Collagenase secretion was studied in cultures of rabbit articular chondrocytes. Differentiation of the cells was assessed by characterizing the type of ³H-labelled collagen produced during treatment with (1) conditioned media from rabbit peritoneal macrophages and human blood mononuclear cells, and (2) with retinol, a potent cartilage resorbing agent in tissue culture. Conditioned media stimulated collagenase secretion. Total collagen synthesis was reduced due to a decrease of synthesis of α₁ chains; the amount of α₂ chains synthesized was unchanged. This is thought to be due to a reduction in type II synthesis. Retinol did not stimulate collagenase secretion. Total collagen synthesis was reduced by retinol. α₂ chain synthesis, however, was significantly increased, suggesting a switch of collagen synthesis in favor of type I collagen and, therefore, dedifferentiation. These results demonstrate that dedifferentiation of chondrocytes with respect to collagen synthesis is not necessarily associated with a stimulation of collagenase secretion.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

Surface-enhanced Raman spectroscopy of DOPA-containing peptides related to adhesive protein of marine mussel, Mytilus edulis

The common blue marine mussel adheres to underwater surfaces using an adhesive protein (Mefp-1) extruded from its foot. This highly hydroxylated protein contains a number of unusual amino acids, including 3,4-dihydroxyphenylalanine (DOPA), which is thought to contribute to the crosslinking of the extruded threads and adhesion to the substratum. Mefp-1 adheres to a wide variety of surfaces and is ultimately biodegradable. In this study we use surface-enhanced Raman spectroscopy (SERS) to characterize the adsorption of DOPA-containing peptides on colloidal gold. The peptides are simplified fragments of the Mefp-1 consensus decapeptide repeat, Ala-Lys-Pro-Ser-Tyr-DHP-Hyp-Thr-DOPA-Lys. Our results show that the peptides TDeltaKA, PTDeltaKA, and PPTDeltaKA (where Delta represents DOPA) coordinate to the gold surface through the catechol oxygens of the DOPA residue and through primary amine groups. The diproline sequence introduces conformational constraints that influence the conformations of the adsorbed peptides. These findings lay the groundwork for developing synthetic adhesives for underwater and medical applications.     

A biomimetic chitosan composite with improved mechanical properties in wet conditions

Chitosan is one of the most widely used structural polymers for biomedical applications because it has many favorable properties. However, one of the most critical drawbacks regarding the use of chitosan as a biomedical material is its poor mechanical properties in wet conditions. Here, we designed a method to improve the mechanical properties of chitosan in wet conditions and minimized the swelling behavior of chitosan film due to water adsorption by mimicking the sclerotization of insect cuticles and squid beaks, that is, catechol‐meditated crosslinking. The biomimetic chitosan composite film was prepared by mixing chitosan with l ‐3,4‐dihydroxyphenylalanine (DOPA) as a catecholic crosslinker and sodium periodate as an oxidant. The catechol‐meditated crosslinking provided a sevenfold enhancement in the stiffness in wet conditions compared to pure chitosan films and reduced the swelling behavior of the chitosan film. This strategy expands the possible applications for the use of chitosan composites as load‐bearing biomaterials. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 505–512, 2013     

Enzymatic modification of vegetable protein: Immobilization of Penicillium duponti enzyme on reconstituted collagen and the use of the immobilized-enzyme complex for solubilizing vegetable protein in a recycle reactor

Penicillium duponti enzyme was immobilized on reconstituted collagen by macromolecular complication, impregnation, and covalent crosslinking techniques. The immobilization of the enzyme on collagen has a twofold purpose: (1) providing a protein microenvironment for the proteolytic enzyme; and (2) extending the useful life the enzyme once immobilized on the collagen matrix. Two types of collagen were used, one produced by the United States Department of Agriculture and the other produced by FMC. The USDA collagen contained unhydrolyzed telepeptide linkages and required pretreatment to reduce collagenaselike activity of the enzyme. Activity analysis of the immobilized enzyme complex showed that membranes with enzyme loading less than 10 mg enzyme/g of wet membrane in the reactor were dimensionally stable. The degree of crosslinking was an important parameter. Membranes with structural opening up to three times the initial dry thickness were found to be the maximum limit for controlled release of enzyme from the collagen membrane during enzymatic reaction. Higher activities and better stability of the enzyme in collagen membrane were found for covalent crosslinking of the enzyme to treated collagen films. The hydrolysis of soybean vegetable protein with the immobilized enzyme in a recycle reactor at enzyme loading of mg/g of wet membrane at 40°C, pH 3.4, produced 56.5% of soluble protein in 10h. The production is equivalent to 1.84 h total contact time between the substrate and the immobilized enzyme. The average productivity based on a stable enzyme activity and 20g of dry membrane was 329 mg of protein/g/mg of active enzyme immobilized. The productivity of the free enzyme in a batch reactor was 62.5 mg protein/h/mg enzyme.     

Analysis of the BadA stalk from Bartonella henselae reveals domain-specific and domain-overlapping functions in the host cell infection process

Human pathogenic Bartonella henselae cause cat scratch disease and vasculoproliferative disorders. An important pathogenicity factor of B. henselae is the trimeric autotransporter adhesin Bartonella adhesin A (BadA) which is modularly constructed and consists of a head, a long and repetitive neck‐stalk module with 22 repetitive neck/stalk repeats and a membrane anchor. The BadA head is crucial for bacterial adherence to host cells, binding to several extracellular matrix proteins and for the induction of vascular endothelial growth factor (VEGF) secretion. Here, we analysed the biological role of the BadA stalk in the infection process in greater detail. For this purpose, BadA head‐bearing and headless deletion mutants with different lengths (containing one or four neck/stalk repeats in the neck‐stalk module) were produced and functionally analysed for their ability to bind to fibronectin, collagen and endothelial cells and to induce VEGF secretion. Whereas a head‐bearing short version (one neck/stalk element) of BadA lacks exclusively fibronectin binding, a substantially truncated headless BadA mutant was deficient for all of these biological functions. The expression of a longer headless BadA mutant (four neck/stalk repeats) restored fibronectin and collagen binding, adherence to host cells and the induction of VEGF secretion. Our data suggest that (i) the stalk of BadA is exclusively responsible for fibronectin binding and that (ii) both the head and stalk of BadA mediate adherence to collagen and host cells and the induction of VEGF secretion. This indicates overlapping functions of the BadA head and stalk.     

Effect of Oxygen Free Radicals on Corneal Collagen

Corneal collagen was labeled in vivo by injection of ¹⁴C-proline into the anterior chamber of rabbit eyes. The isolated corneal collagen was incubated in iron-free phosphate buffered saline (pH 7.4) containing I mM axorbate and 0.1 mM CuSO₄ for either 1 hour or 3 hours at 37°. Addition of 2 volumes of 8 M urea-I mM dithiothreitol and heating for 1 min at 100° solubilized virtually all of the collagen in the control incubations but left a significant amount of insoluble collagen in specimens exposed to the hydroxyl radical generating system. This residue amounted to 19% and 38% of the initial radioactivity in samples incubated for 1 h and 3 h, respectively. The chromatographic profiles (gel filtration on CL-4B) of the soluble fraction showed an increase in both aggregation and degradation products of collagen in the 1 h incubation mixture, whereas after 3 h there was an increase only in degradation products. These observations suggest that additional crosslinking of the soluble collagen aggregates observed at 1 h may be responsible for their subsequent disappearance at 3 h, with concomitant increase of the insoluble fraction. Collagen degradation by OH may play a role in corneal ulceration, whereas hydroxyl radical-mediated crosslinking is consistent with age-dependent increases in insoluble collagen.     

An in vitro study on the role of metal catalyzed oxidation in glyeation and crosslinking of collagen

The present investigation was carried out to understand the effect of metal catalyzed oxidation on glycation and crosslinking of collagen. Tail tendons obtained from rats weighing 200-225 g were incubated with glucose (250 mM) and increasing concentrations of copper ions (5, 25, 50 and 100 M) under physiological conditions of temperature and pH. Early glycation, crosslinking and late glycation (fluorescence) of collagen samples were analyzed periodically. Early glycation was estimated by phenol sulfuric acid method, and the crosslinking was assessed by pepsin and cyanogen bromide digestion. A concentrationdependent effect of metal ions on the rate of glycation and crosslinking of collagen was observed. Tendon collagen incubated with glucose and 100 M copper ions showed 80% reduction in pepsin digestion within seven days, indicating extensive crosslinking, whereas collagen incubated with glucose alone for the same period showed only 7% reduction. The presence of metal ions in the incubation medium accelerated the development of Maillard reaction fluorescence on collagen, and the increase was dependent on the concentration of metal ions used. The metal chelator Diethylene triamine penta-acetate significantly prevented the increase in collagen crosslinking by glucose and copper ions. Free radical scavengers benzoate and mannitol effectively prevented the increased crosslinking and browning of collagen by glucose. The results indicate that the metal catalyzed oxidation reactions play a major role in the crosslinking of collagen by glucose. It is also suggested that the prevention of increased oxidative stress in diabetes may prevent the accelerated advanced glycation and crosslinking of collagen.     

Collagen has a unique SEC24 preference for efficient export from the endoplasmic reticulum

SEC24 is mainly involved in cargo sorting during COPII vesicle assembly. There are four SEC24 paralogs (A–D) in vertebrates, which are classified into two subgroups (SEC24A/B and SEC24C/D). Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans. sec24d mutant fish also recapitulate the phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. Using Sec24d KO mice and cultured cells, we show that SEC24A and SEC24B also contribute to endoplasmic reticulum (ER) export of procollagen. In contrast, fibronectin 1 requires either SEC24C or SEC24D for ER export. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.     

A sensitive fluorometric assay for protein-bound DOPA and related products of radical-mediated protein oxidation

Oxidative attack on proteins results in the hydroxylation of tyrosyl residues to protein-bound DOPA (3,4-dihydroxyphenylalanine). Existing methods for assaying protein-bound DOPA have poor sensitivity and numerous possible interferences, such that accurate determination (especially of very low DOPA concentrations) has required time-consuming acid hydrolysis and HPLC analysis with fluorometric detection. This work presents a sensitive and selective assay for peptide or protein-bound o-benzoquinones derived from DOPA based on fluorometric detection of ethylenediamine derivatives. Detection limits for protein-bound DOPA are in tbe range 0.53–4.70 ng/mL for the assay mixture, corresponding to sample DOPA concentrations of 0.59–5.30 ng/mL (representing a minimum of 6–54 pmole detected), depending on the particular protein/peptide under study. The assay response increases linearly with DOPA concentration, and also with the extent of radical exposure of the protein. The assay is a simple and fast way to assess DOPA formation and thus oxidative damage in a protein.