Degradation of human apolipoprotein B-100 by apolipoprotein(a).

Human plasma low density lipoproteins (LDL) contain a very high molecular weight protein termed apoB-100 (M(r) = 550,000). In many samples of LDL, minor components designated as apoB-74 (M(r) = 407,000) and apoB-26 (M(r) = 145,000) are present. It has been shown that they can arise as a result of proteolytic degradation of apoB-100. Our earlier studies demonstrated that the active forms of lipoprotein(a) (LP(a)) and apolipoprotein(a) (apo(a)) possess proteolytic activity. In the present study we investigated the possibility of apoB-100 degradation in the presence of activated apo(a). LDL were incubated with the purified apo(a) and analyzed by SDS-polyacrylamide gel electrophoresis. It was found that apoB is cleaved by apo(a) with formation of proteolytic fragments including B-74 and B-26. The physiological significance of apoB degradation under the action of apo(a) is considered.

The preparation and properties of factor V from bovine plasma.

1. A procedure for the purification of factor V from bovine plasma with a good yield and in a high state of purity is described. The isolation procedure involves BaSO4, adsorption, chromatography of the absorbed plasma on DEAE-Sephadex A-50, activation with thrombin of the A-50 eluate, chromatography on Amberlite CG-50 and precipitation with (NH4)2SO4. Factor V is obtained with a 160% yield and 5000-fold purification. 2. Some of the physico-chemical properties of Ac-globulin have been measured. The molecular weight was determined by electrophoresis is SDS-polyacrylamide gel and found to be 30 000. The studies employing disc electrophoresis and immunoelectrophoresis suggest the existence of several active isoforms of factor V.

The influence of the antidepressant pirlindole and its dehydro-derivative on the activity of monoamine oxidase A and GABAA receptor binding.

The influence of pirlindole and dehydro-pirlindole on GABAA receptor binding and MAO-A activity was investigated in vitro. Inhibition of rat brain and human placenta MAO-A by both compounds was much more potent (with IC50 range 0.3-0.005 microM) than that of GABAA receptors. Pirlindole was inactive as a GABA antagonist. Dehydro-pirlindole exhibited selective blockade of GABA-A receptors with EC50 12 microM. Effects of both compounds on MAO-A activity were partially reversible. Data obtained suggest that in contrast to pirlindole dehydro-pirlindole may act not only as a MAO-A inhibitor but also as a potent GABAA receptor blocker.

[Atherogenic modifications of low density lipoproteins during fibrin formation and fibrinolysis]. / Aterogennye modifikatsii lipoproteidov nizkoi plotnosti v protsesse obrazovaniia fibrina i fibrinoliza.

Low density lipoproteins (LDL) were modified after incubation with fibrinogen and fibronectin at physiological concentrations in presence of thrombin and, following the fibrin formation, in presence of plasmin. The modified LDL (LDL-F) isolated from plasmin digested fibrin by means of gel permeation chromatography on Sepharose 6B, were associated with fibrinogen and fibronectin degradation products. The LDL-F differed from control LDL in their physico-chemical properties: LDL-F contained the degraded apoprotein B, its electrophoretic mobility was increased, cholesterol/protein ratio as well as flotation coefficient at d = 1.063 were decreased. The effect of LDL-F on lipid accumulation was studied. Content of cholesterol esters in macrophages incubated with LDL-F was higher 3.8-fold as compared with that of the cells incubated with control LDL. Thus, after incubation of LDL with fibrinogen and thrombin, 20% of the lipoprotein was bound to fibrin. The data obtained suggest that thrombosis may promote both LDL deposition in the vascular intercellular matrix and cellular lipid accumulation.

[Effect of the modification of low density lipoproteins by thrombin on their interaction with fibronectin]. / Vliianie modifikatsii lipoproteidov nizkoi plotnosti trombinom na ikh vzaimodeistvie s fibronektinom.

Interaction of native and thrombin-modified human low density lipoproteins (LDL) with immobilized homologous fibronectin (either covalently bound to Sepharose or adsorbed from blood serum on collagen-Sepharose) was studied. Treatment of LDL with thrombin at pH 7.5 and 37 degrees within 60 min (thrombin/apo B ratio 1:20 w/w) led to formation in LDL preparations of 3 new fragments of apoprotein B which were detected by polyacrylamide gel electrophoresis in presence of sodium dodecylsulfate. Chromatography of native and thrombinmodified LDL on fibronectin-Sepharose showed that 30% of the modified LDL and 2% of native LDL were bound to fibronectin-Sepharose at physiological pH values and NaCl concentrations. Study of the interaction of LDL with fibronectin adsorbed on collagen-Sepharose showed that thrombin-treated LDL partially released fibronectin from the sorbent due to the formation of a modified LDL-fibronectin complex. Native LDL did not act in a similar manner. Complexes of modified, LDL with fibronectin were detected under conditions of both electrophoresis in 3% polyacrylamide gel and immunoelectrophoresis. Interaction of LDL with fibronectin may promote accumulation of lipoproteins in the vascular wall and thus may serve as a model system for evaluation of the extent of atherogeneity of LDL and detection of the modified LDL in vivo.

[Interaction of enzymatically modified low-density lipoproteins with fibronectin]. / Vzaimodeistvie fermentativno modifitsirovannykh lipoproteidov nizkoi plotnosti s fibronektinom.

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.

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.

[Accelerin (factor V), the catobolic product of fibrinogen ]. / Aktselerin (faktor V)--katabolicheskii produkt fibrinogena

The authors describe a method of obtaining a highly active and well purified human Ac-globulin (factor Y) preparation. Factor Y proved to be a part of the fibrinogen molecule--its intermediate transformation product under the effect of thrombin or some other enzyme analogous by its action.

[Study of the interaction of low-density lipoproteins with immobilized fibrinogen and fibronectin using an immunoenzyme assay]. / Izuchenie vzaimodeistviia lipoproteidov nizkoi plotnosti s immobilizovannymi fibrinogenom i fibronektinom metodom immunofermentnogo analiza.

The interaction of serum low density lipoproteins (LDL) with fibrinogen, fibronectin and fibrinogen-fibronectin complex immobilized on polystyrene was studied. LDL binding by these proteins was assessed by enzyme-linked immunosorbent assay. It was shown that LDL interacts with both immobilized fibronectin and immobilized fibrinogen. The fibrinogen-fibronectin complex bound a greater amount of LDL than either component alone, in the same concentration as in the complex. The binding of LDL with immobilized fibronectin or fibrinogen increased in the presence of low concentrations of diluted fibrinogen or fibronectin, respectively, which suggests the formation of the three-component complex on the surface. The formation of fibrinogen-fibronectin-LDL complexes during homeostasis may promote the accumulation of LDL in the vascular wall.

[Isolation and properties of bovine kidney aminopeptidase A]. / Vydelenie i svoistva aminopeptidazy A iz pochek byka.

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.

[SH-dependent aminopeptidase from bovine kidney. Isolation, physico-chemical properties and substrate specificity]. / SH-zavisimaia aminopeptidaza pochek byka. Vydelenie, fiziko-khimicheskie svoistva i substratnaia spetsifichnost'.

The SH-dependent aminopeptidase was isolated from bovine kidney and purified 800-fold. The enzyme has a pH optimum of 7.0 and pI of 5.5 and is activated by dithiothreitol and inhibited by p-chloromercurybenzoate. The enzyme has a molecular weight of 56000 and is represented by four polypeptide chains with m. w. of 14000. Aminopeptidase hydrolyzes beta-naphthylamides of arginine, alanine, leucine and proline and peptides with free N-terminal neutral and basic amino acid residues having L-configuration as well as angiotensin III. The enzyme does not split proteins, angiotensins I and II, B-chains of insulin and peptides with a blocked N-terminal amino group and peptides with proline as a second amino acid from the NH2-end.

[Molecular structure of aminopeptidase A from bovine kidney]. / Molekuliarnaia struktura aminopeptideay A iz pochek byka.

Using gel-filtration through Sephadex G-100 and polyacrylamide gel electrophoresis in the presence of 0,5% sodium dodecyl sulfate, it was found that aminopeptidase A has a molecular weight of 65 000 +/- 2000 and is made up of two subunits with mol. weights of 33 000 +/- 2000. Each subunit consists of two polypeptide chains with mol. weights of 22 000 +/- 2000 and 12 000. During enzyme dissociation into subunits the aspartylnaphtylamidase activity is lost, while the glutamylnaphtylamidase activity is retained.

Effects of the antidepressant pirlindole and its dehydro-derivative on the activity of monoamine oxidase-A and on GABAA receptors.

The effects of pirlindole and dehydro-pirlindole on GABAA receptors and MAO-A activity were investigated in vitro. Pirlindole was inactive as a GABA antagonist. Dehydro-pirlindole exhibited partial and selective blockade of a subset of GABAA receptors with an EC50 of 12 microM and maximum reversal (delta Bopt) of 42%. Inhibition of rat brain and human placenta MAO-A by both compounds was much more potent (with IC50 range 0.3-0.005 microM). Their effects on MAO-A activity were partially reversible in vitro. In contrast to pirlindole, dehydro-pirlindole may act not only as MAO-A inhibitor but also as a clozapine-like selective GABAA receptor blocker, preferentially blocking a subset of GABAA receptors that are not sensitive to DMCM or Ro 5-4864.