No abstract

No abstract

Protocatechuate 2,3-dioxygenase (2,3-PCD) from Bacillus macerans JJlb has been purified to homogeneity for the first time. The enzyme catalyzes proximal extradiol ring cleavage of protocatechuate (PCA) with the attendant incorporation of both atoms of oxygen from 02. The holoenzyme has a mass of 143 7 kDa as determined by ultracentrifugation and other techniques. It is composed of four apparently identical subunits with Mrs of 35,500, each containing one iron atom. Mossbauer spectroscopy of "7Fe-enriched enzyme showed that the irons are indistinguishable and are high spin (S = 2) Fe2+ in both the uncomplexed and substrate-bound enzyme. However, the quadrupole splitting, AEQ, and isomer shift, 8, of the Mossbauer spectrum changed from AEQ = 2.57 mm/s and 8 = 1.29 mm/s to AEQ = 2.73 mm/s and 8 = 1.19 mm/s upon PCA binding to the enzyme, showing that the iron environment is altered when substrate is present. The enzyme was also found to bind variable and substoichiometric amounts of Mn2", but this metal could be removed without loss of activity or stability. The inherently electron paramagnetic resonance (EPR)-silent Fe2+ of the enzyme reversibly bound nitric oxide to produce an EPR-active species (g = 4.11, 3.95; S = 3/2). The specific activity of the enzyme was found to be correlated with the amount of the S = 3/2 species formed, showing that activity is dependent on Fe2'. Anaerobic addition of substrates to the enzyme-nitric oxide complex significantly altered the EPR spectrum, suggesting that substrates bind to or near the iron. The enzyme was inactivated by reagents that oxidize the Fe2 , such as H202 and K3Fe(CN)6; full activity was restored after reduction of the iron by ascorbate. Steady-state kinetic data were found to be consistent with an ordered bi-uni mechanism in which the organic substrate must add to 2,3-PCD before 02. The enzyme has the broadest substrate range of any of the well-studied catecholic dioxygenases. All substrates have vicinal hydroxyl groups on the aromatic ring except 4-NH2-3-hydroxybenzoate. This is the first substrate lacking vicinal hydroxyl groups reported for catecholic extradiol dioxygenases. 2,3-PCD is the final member of the PCA dioxygenase family to be purified. It is compared with other members of this family as well as other catecholic dioxygenases.Protocatechuate (PCA) is one of a relatively small number of single-ring aromatic compounds that are found at the points of confluence of bacterial pathways for metabolism of complex aromatics (12,17,28,37). The aromatic ring of PCA is opened in reactions catalyzed by dioxygenase enzymes that result in the incorporation of both atoms of 02 into the open chain products (14,19,25,46,65). Two of these enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) (10, 21, 25, 59, 65, 70) and 4,5-PCD (5,18,19,53) were among the first of this class of enzyme to be recognized. They continue to serve as prototypical enzymes for the two major subclasses of catecholic dioxygenases termed intradiol and extradiol on the basis of the site of ring clea...

Laminin, a basement membrane protein derived from the matrix of the Engelbreth-Holm-Swarm murine tumor, was nonenzymatically glycosylated in vitro in the presence of increasing glucose concentrations. The amount of glucose incorporated per laminin molecule was shown to be proportional to the molarity of glucose used. Nonenzymatic glycosylation resulted in formation of cross-links and alterations of the cruciform shape of laminin molecules; these alterations were dramatic when high concentrations of glucose were used. One of the functions of laminin, the process of self-assembly, was shown to be impaired after in vitro nonenzymatic glycosylation. Glucose incorporation resulted in a dramatic decrease of long-to-long laminin dimers, which normally form during the initial steps of assembly. Furthermore, nonenzymatic glycosylation of laminin reduced its ability to self-associate into complexes larger than dimers, as judged by turbidimetry. The observed decrease of maximal turbidity was proportional to the degree of nonenzymatic glycosylation. Aminoguanidine, which has been suggested to inhibit cross-link formation, was shown to restore to a large extent the shape of laminin, the percentage of long-to-long arm dimers, and the maximal turbidity when included in the mixtures of laminin and glucose. These data suggest that structural and functional alterations of laminin may be primarily due to formation of cross-links. Such modifications of laminin (along with our basement membrane components) may contribute to the morphological and physiological changes observed in basement membranes under diabetic conditions.

Abstract. Recent studies using solid-phase-binding assays and electron microscopy suggested the presence of a heparin-binding domain between the inner globule of a lateral short arm and the cross region of laminin. Using the information from the amino acid sequence of the B1 chain of laminin, several peptides were synthesized from areas with a low hydropathy index and a high density of lysines and/or arginines. One of these, peptide F-9 (RYVVLPRPVCFEKGMNYTVR), which is derived from the inner globular domain of the lateral short arm, demonstrated specific binding to heparin. This was tested in direct solid-phase binding assays by coating the peptide either on nitrocellulose or on polystyrene and in indirect competition assays where the peptide was in solution and either laminin or heparin was immobilized on a solid support. The binding of [3H]heparin to peptide F-9 was dramatically reduced when heparin but not other glycosaminoglycans other than heparin (dextran sulfate, dermatan sulfate) were used in competition assays. Modification of the free amino groups of peptide F-9 by acetylation abolished its ability to inhibit the binding of [3H]hepafin to laminin on polystyrene surfaces. Peptide F-9 promoted the adhesion of various cell lines (melanoma, fibrosarcoma, glioma, pheochromocytoma) and of aortic endothelial cells. Furthermore, when peptide F-9 was present in solution, it inhibited the adhesion of melanoma cells to laminin-coated substrates. These findings suggest that peptide F-9 defines a novel heparin-binding and cell adhesion-promoting site on laminin.