Modulation of lysyl oxidase by dietary copper in rats.

Lysyl oxidase levels were estimated in rat tissues using an enzyme-linked immunosorption assay (ELISA) and a functional assay standardized against known amounts of purified lysyl oxidase. High concentrations of lysyl oxidase (> or = 150 micrograms/g of tissue or packed cells) were detected in connective tissues, such as tendon and skin. Values for aorta, kidney, lung and liver ranged from 30 to 150 micrograms/g of tissue; values for skeletal muscle and diaphragm were < 30 micrograms/g tissue. Purified rat skin lysyl oxidase catalyzed the release of 50-100 Bq of tritium per micrograms enzyme in assays that used 3H-elastin-rich substrates. In dense connective tissues, good agreement was obtained for the values from ELISA and those derived from measurements of functional activity in aorta, lung, skin and tendon (r2 > 0.9). When egg white-based experimental diets containing 2 or 10 micrograms/g added copper were fed to weanling rats, values for skin lysyl oxidase functional activity in the group fed 2 micrograms/g added copper were one-third to one-half the values for skin lysyl oxidase functional activity in rats fed 10 micrograms/g copper. This reduction in lysyl oxidase activity, however, had minimal effect on indices of collagen maturation in rat skin, e.g., collagen solubility in neutral salt and dilute acid or the levels of acid stable cross-links. Moreover, copper deficiency did not influence the steady-state levels of lysyl oxidase specific mRNA in rat skin or the apparent amounts of lysyl oxidase in rat skin as determined by ELISA. These observations underscore that the concentration of lysyl oxidase is relatively high in dense corrective tissues, and although decreasing dietary copper influences functional activity, there is little apparent effect on the production of lysyl oxidase protein.

Purification, properties and influence of dietary copper on accumulation and functional activity of lysyl oxidase in rat skin.

Lysyl oxidase (protein-lysine 6-oxidase; EC 1.4.3.13) is a copper-containing enzyme that functions extracellularly and catalyses the oxidative deamination of peptidyl lysine. Lysyl oxidase was purified 150-175-fold from urea extracts of rat skin and uteri. Features of the enzyme were similar to those reported previously for lysyl oxidase obtained from rat aorta and bovine ligamenture. However, both approximately 40 and approximately 32 kDa polypeptide chains could be isolated from rat skin with apparent lysyl oxidase activity. Antibodies raised in chickens against the approximately 40 kDa form of lysyl oxidase detected the approximately 32 kDa form in immunoblots. Consequently it is inferred that the approximately 32 kDa form of lysyl oxidase is processed from the approximately 40 kDa form of the enzyme. The antibodies were also used to prepare anti(rat lysyl oxidase) affinity columns to facilitate the separation of lysyl oxidase from other proteins in studies to assess the extent to which lysyl oxidase serves as a reservoir for skin copper. At 16 h after an oral dose of copper, as 67Cu, about 6-8% of the total 67Cu incorporated into rat skin was found in association with lysyl oxidase. The lysyl oxidase concentration in rat skin was 2.5-7.5 nmol/g (determined by e.l.i.s.a.). Changing the copper status of rats by feeding a diet deficient in copper did not appear to influence lysyl oxidase accumulation in skin nor the percentage of incorporation of 67Cu in skin as lysyl oxidase. However, when rats were deprived of copper, the functional activity of lysyl oxidase in skin was one-third to one-half the normal values.

Elastin metabolism during recovery from impaired crosslink formation.

Accelerated proteolysis of tropoelastin and elastin occurs in the arteries of chicks rendered nutritionally copper-deficient. The process results in part from decreased elastin crosslinking. Repletion of copper-deficient chicks with copper causes a deposition of elastin that is proteinase resistant. Resistance to proteolysis is conferred within 48 h of dietary copper repletion. Deposition of aorta elastin to near normal values occurs after 3-4 days in copper-repleted chicks. Moreover, elastolysis was enhanced when the content of dehydrolysinonorleucine in elastin was abnormally low. The chemical modification of lysyl residue in elastin by citroconylation, however, did not influence the rate of elastolysis. We have shown previously that tropoelastin messenger RNA activity and synthesis are not influenced by dietary copper deprivation (1986, Biochem. J. 236, 17-23). Rather, as demonstrated herein, the decrease in elastin content in arteries of copper-deficient birds appears to be more the result of enhanced degradation. Restoration of normal crosslinking restores deposition and imparts resistance to elastolysis. Moreover, serum appears to be a good source of elastolytic proteinases when the elastin substrate is partially or abnormally crosslinked.

Nutritional importance of pyrroloquinoline quinone.

Mice fed a chemically defined diet devoid of pyrroloquinoline quinone (PQQ) grew poorly, failed to reproduce, and became osteolathyritic. Moreover, severely affected mice had friable skin, skin collagen that was readily extractable into neutral salt solutions, and decreased lysyl oxidase. The identification of functional defects in connective tissue and the growth retardation associated with PQQ deprivation suggest that PQQ plays a fundamental role as a growth factor or vitamin.