The leucoyte surface antigen CD38 has been shown to be an ecto-enzyme with multiple catalytic activities. It is principally a NAD+ glycohydrolase that transforms NAD+ into ADP-ribose and nicotinamide. CD38 is also able to produce small amounts of cyclic ADP-ribose (ADP-ribosyl cyclase activity) and to hydrolyse this cyclic metabolite into ADP-ribose (cyclic ADP-ribose hydrolase activity). To classify CD38 among the enzymes that transfer the ADP-ribosyl moiety of NAD+ to a variety of acceptors, we have investigated its substrate specificity and some characteristics of its kinetic and molecular mechanisms. We find that CD38-catalysed cleavage of the nicotinamide-ribose bond results in the formation of an E.ADP-ribosyl intermediary complex, which is common to all reaction pathways; this intermediate reacts (1) with acceptors such as water (hydrolysis), methanol (methanolysis) or pyridine (transglycosidation), and (2) intramolecularly, yielding cyclic ADP-ribose with a low efficiency. This reaction scheme is also followed when using nicotinamide guanine dinucleotide as an alternative substrate; in this case, however, the cyclization process is highly favoured. The results obtained here are not compatible with the prevailing model for the mode of action of CD38, according to which this enzyme produces first cyclic ADP-ribose which is then immediately hydrolysed into ADP-ribose (i.e. sequential ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities). We show instead that the cyclic metabolite was a reaction product of CD38 rather than an obligatory reaction intermediate during the glycohydrolase activity. Altogether our results lead to the conclusion that CD38 is an authentic 'classical' NAD(P)+ glycohydrolase (EC 3.2.2.6).

Leucocyte proteinases, e.g. leucocyte elastase and cathepsin G, are inhibited by heparin. The activities of pig pancreatic and Pseudomonas aeruginosa elastases are unaffected by this polysaccharide. Heparin derivatives of known Mr and degree of sulphation were isolated. The inhibition of leucocyte elastase by these oligosaccharides can be classified as tight-binding hyperbolic non-competitive. K, values ranged from 40 nm to 100 /M and were found to be inversely correlated with the chain length of the oligosaccharides. Desulphated compounds lacked inhibitory potential towards leucocyte elastase. Over-O-sulphated di-and tetra-saccharides are more potent inhibitors than their over-N-sulphated counterparts. It is proposed that the therapeutic use of heparin and its derivatives could be extended to disease states such as emphysema and rheumatoid arthritis, where the role of leucocyte elastase has been clearly established.

The addition of highly purified elastic fibers to confluent human skin fibroblast or porcine aorta smooth muscle cell cultures resulted in a time-dependent, strong adhesion of the fibrils to the cell surface. The kinetics of adhesion was studied by video/time-lapse cinematography. After a 0.5-1 hr lag period, adhesion progressed to a maximum amount in 3-6 hr in the described conditions. Adhesion is strongly accelerated by the prior addition of soluble elastin peptides (K-elastin) to the cultures. Cycloheximide inhibits this induced adhesion. Adherent elastic fibers can be detached by treatment with elastase and trypsin but not with collagenase. The radioactive proteins adhering to elastic fibers, after a 6-hr incubation of the induced cultures in presence of [35S]methionine, were extracted and analyzed by NaDodSO4/PAGE. The proteins strongly adhering to the elastic fibers had apparent molecular sizes of about 120, 67, 60, and 45 kDa. Only the 120-kDa protein band showed a significant increase of its associated radioactivity in the induced cultures as compared to the noninduced cultures. We propose that the 120-kDa protein is responsible for the induced adhesion of mesenchymal cells to elastic fibers and designate it "elastonectin."In the past years, specific adhesive proteins that mediate cell attachment to the extracellular matrix have been identified and characterized. Fibronectin ensures the adhesion of mesenchymal cells to interstitial collagens and to some proteoglycans (1-4). Laminin is involved in the interaction between epithelial cells and basement membrane components, such as collagen type IV (5, 6). Elastin was originally considered to be an isotropic and inert constituent, but physicochemical studies have pointed to a more dynamic structure and to possible interactions with other connective tissue proteins (7,8). Elastin peptides were also shown to be able to interact with several other cell types (9, 10). Mecham et al. (11) studied the acquisition of chemotactic responsiveness to elastin peptides and the expression of the elastin phenotype by fetal bovine ligamentum fibroblasts during cell differentiation and showed that they were closely related events. Therefore, the investigation of whether specific protein(s) could mediate the adhesion of mesenchymal cells to elastic fibers was undertaken.In the present report, we have investigated the interactions between purified bovine ligamentum nuchae elastic fibers and human skin fibroblasts or pig aorta smooth muscle cells in culture and show that elastic fibers adhere strongly to both cell types. Fibronectin has been shown not to be involved in this process. Biosynthetic experiments carried out with human skin fibroblasts cultured in presence or absence of elastic fibers enabled us to carry out preliminary identification of an inducible protein closely associated with the added elastic fibers. We have designated this adhesive protein "elastonectin."MATERIAL AND METHODS Fibroblasts and smooth muscle cells were obtained from human dermis and porci...