Interaction of enzymatically modified low-density lipoproteins with fibronectin

Interaction of human low-density lipoproteins (LDL) with homologous fibronectin fixed on collagen-Sepharose was studied. LDL were digested with pepsin, the degree of hydrolysis amounting to 10%. Upon passing modified LDL through a fibronectin-collagen-Sepharose column the desorption of fibronectin occurred. Addition of the increasing amount of fibronectin to the pepsin-treated LDL solution in the presence of Ca2+ ions led to the formation of LDL-fibronectin insoluble complexes. Interaction of native LDL with fibronectin was not observed. The data suggest that enzymatic modification of LDL increasing interaction of modified LDL with fibronectin, a component of extracellular matrix, could promote the accumulation of such LDL in arterial walls.     

Isolation and properties of bovine kidney aminopeptidase A]

Aminopeptidase A, which specifically hydrolyses N-terminal dicarbonic amino acid residues containing free alpha-amino groups, is isolated from bovine kidney. The enzyme is 500-fold purified and is homogenous under electrophoresis and ultracentrifugation. Aminopeptidase A has pH optimum of 7.5, it is activated with Ca2+ and inactivated with EDTA. Its molecular weight is 53000. The enzyme hydrolyses alpha-L-aspartyl-beta-naphtylamide and splits peptides having N-terminal glycine, lysine, arginine and alanine are hydrolyzed by the enzyme much slower. Aminopeptidase A does not attack alpha-L-alanyl-beta-naphtylamide, leucineamide, insulin, peptides with blocked N-terminal amino acid and peptides which have proline to be the second N-terminal amino acid.     

Cytoskeleton-induced alterations of the lectin activity in winter wheat under cold hardening and abscisic acid (ABA)

The roots and leaves of 7-day seedlings of three winter wheat cultivars differing in frost resistant were used to study changes in lectin activity under cytoskeleton modifiers (DMSO-7%; colchicine-1 m m; oryzalin-15 microm; cytochalasin B-15 microm) of non-hardened (23 degrees C) and hardened (2-3 degrees C, 3-7 day) plants. Plants were grown with ABA (30 microm) or without ABA. Pretreatment with colchicine, oryzalin [inhibitors of microtubules (MT) polymerization], cytochalasin B [inhibitor of microfilament (MF) polymerization] increased the activity of cell wall lectins, although pretreatment with DMSO (stabilizer of microtubules) decreased the activity. Both hardening and ABA decreased the effect of the cytoskeletal modifiers. These results could be explained by the appearance of tolerant MTs with less affinity. It is probable that increase in the activity of cell wall lectins may be the compensatory mechanism which stabilizes the cytoskeleton structure in conditions tending to disrupt it. The genotype with low resistance had higher sensitivity of lectin activity to cytoskeleton modifiers than the frost resistant genotype. The results suggest that leaves have more stable MTs and MFs and stronger MT-MF binding than roots.     

Effect of modification of cell calcium status on lectin activity

Effects of oryzalin (10 microM), an inhibitor of microtubule polymerization, on the activity of soluble and cell wall lectins were studied in 7 day-old seedlings of unhardened (23 degrees C) and cold acclimated (7 days at 2-3 degrees C) winter wheat (Triticum aestivum L.). Seedlings were grown in the presence of 25 microM and 1 mM Ca2+, 500 microM verapamil, 250 microM chlorpromazine or without modifiers of calcium status in the medium. Inhibitor of the microtubule polymerization inhibitor, likely as inhibitors of Ca(2+)-signal, decreased the activity of soluble lectins and increased that of cell wall lectins. Apparently, injury of microtubule phosphorylation results in a more considerable microtubule disorganization, than that observed after oryzalin effect. A low Ca2+ concentration (25 microM) depressed, while a high concentration (1 mM) prompted microtubule sensibility to oryzalin. Such an effect of high Ca2+ concentration may be related to destabilizative action of Ca(2+)-calmodulin in these conditions, because chlorpromazine decreased oryzalin-induced increase in the activity of cell wall lectins with 1 mM Ca2+. It is concluded that the activity of cell wall lectins depends on the microtubule status that is regulated by calcium signal.     

Lectin and mitotic activities in root meristems of winter wheat under oryzalin effect

The effect of oryzalin, a microtubule polymerization inhibitor (10 MM), on lectin and mitotic activities (mitotic index and duration of mitotic phases) was studied in unhardened (23 degrees C) and hardened (7 days, 2-3 degrees C) winter wheat seedlings. Three wheat cultivars differing in their frost tolerance were compared. Oryzalin treatment (3 h) decreased activity of soluble lectins, increased activity of cell wall lectin mitotic index. Under these conditions, prolongation of anaphases and disappearance of telophases were detected. Plant hardening reduced the sensitivity of cell wall lectins and mitotic activity to the cytoskeleton inhibitor due, presumably, to the appearance of cold-stable microtubules. Plant growing and hardening with oryzalin stopped mitoses and caused the appearance of polyploid cells and cells with micronuclei. These abnormalities were preserved after hardening. The results obtained demonstrate an important role of microtubules in adaptation of plants to low temperature.     

Effect of cartolin on oryzalin-induced changes in lectin activity during low-temperature plant hardening

The effect of cartolin (0.33 μM), an antistress regulator of cytokinin type, on the cytoskeleton-dependent changes in lectin activity in the roots of unhardened (23°C) and cold-hardened (3°C, 7 days) 7-day-old plants of three cultivars of winter wheat (Triticum aestivum L.) was studied. In unhardened plants, cartolin increased activity of soluble and cell wall-bound lectins in a cultivar-specific mode. This is evidently important for subsequent enhancement of adaptation processes in the cell. The inhibitor of microtubule polymerization, oryzalin, reduced the activity of soluble lectins and increased that of cell wall-bound lectins. A reduced sensitivity of lectin activity to oryzalin after cartolin treatment could result from its stabilizing action on the cytoskeletal structures and on the interaction between cell-wall lectins and microtubules. The most efficient cartolin action, the suppression of oryzalin effect on lectin activity in hardened plants, was observed in the frost-sensitive wheat cultivar. It is likely that cartolin treatment is more efficient in the activation of adaptation processes occurring with the involvement of cytoskeletal structures in the cultivars of lower tolerance.     

Enzymatic properties of active form of human apolipoprotein (a)

Lipoproteins (d = 1.05-1.12 g/ml) were obtained from pooled serum by density gradient ultracentrifugation and used as a source for isolation of apolipoprotein (a) (apo(a]. It was found that both these lipoproteins and purified apo(a) possess negligible amidolytic and proteolytic activity. After preincubation of lipoproteins and apo(a) with collagen-Sepharose, the increase in enzymatic activity was observed. The activation of purified apo(a) also occurred upon its storage in the cold. After two week storage at 7 degrees C, the amidase activity, as measured by splitting of the substrate D-Pro-Phe-Arg-pNA, was increased from 0.009 U/mg to 0.85 U/mg. The amidase activity was completely inhibited by phenylmethylsulfonyl fluoride (10(-3) M) and by soybean trypsin inhibitor (10(-5) M); it was not inhibited by aprotinin (10(-6) M). Activated apo(a) did not split azocasein but converted plasma prekallikrein to kallikrein and degraded apolipoprotein B-100.