Catalytic effect of gamma-thrombin on synthetic low molecular weight peptide substrates]

Hydrolysis and respective catalytic parameters of hydrolysis of ester peptide substrates that contain residues of hydrophobic and nonpolar amino acids in P2, P3 subsites have been studied. It is shown that efficiency of hydrolysis by thrombin is determined by the length of polypeptide chains and by the nature of the amino acids in P2, P3 subsites in the substrate. In spite of the fact that gamma-thrombin retains the conformation activity of the catalytic centre the local conformation changes of the second binding region of the enzyme have been discovered.     

Kinetics of low molecular weight substrate hydrolysis by immobilized trypsin]

The catalytic properties of trypsin immoblized on silochrome were studied in a flow reactor with replacement. The hydrolysis of methyl ester N-n-tosyl-L-arginine obeys the Michaelis--Menten kinetics. The apparent K'm value for the system with immobilized trypsin is considerably lower than for native trypsin. The K'm value was decreased with an increase in the rate of the substrate flow through the reactor or when smaller-sized silochrome granules were used. It is assumed that the apparent K'm value for the immobilized system is due to diffusion. The effects of diffusion on the catalytic properties of the immobilized enzyme were estimated.     

Inhibition of peptide initiation by a low molecular weight RNA from rabbit reticulocytes

A heat-stable inhibitor of protein synthesis has been isolated from the postribosomal supernatant of rabbit reticulocytes. Its activity is not susceptible to protease treatment but is destroyed by incubation with alkali. Inhibitory activity can be quantitatively recovered in the aqueous phase after phenol extraction and has the ultraviolet absorption spectrum of a nucleic acid. It is concluded that the inhibitor is RNA. The inhibitory activity sediments in the range of 3 S, but it has not been demonstrated whether the inhibitor RNA is a single molecular species. The inhibitory RNA does not affect peptide elongation but rather blocks a step of peptide initiation. It does not interfere with the formation of the ternary complex between initiation factor 2, GTP, and methionyl-tRNAMetf and does not activate a protein kinase phosphorylating initiation factor 2. The inhibitory RNA appears to be a novel type of RNA that inhibits polypeptide initiation at a step involving ribosomal subunits.     

Inhibition of platelet aggregation by dinitrosyl iron complexes with low molecular weight ligands

The dinitrosyl iron complexes (DNIC) with thiosulphate, cysteine or phosphate were shown to inhibit in vitro (in citrate plasma) the human platelet aggregation induced by ADP, collagen or adrenaline. This effect cannot be explained by the toxic action of DNIC on the platelet membrane, since DNIC-pretreated platelets are capable of aggregating under the action of 10(-8) M/ml of phorbol ester, which is known to cause direct activation of protein kinase C. The antiaggregatory activity of DNIC exceeds that of Na-nitroprusside and seems to be due to nitric oxide capable to activate guanylate cyclase of platelets. Using the EPR method, it was shown that addition of DNIC to platelet-enriched plasma results in a rapid transfer of Fe(NO)2 groups to the coupled RS(-)-groups proteins of plasma and, apparently, of platelet membrane proteins. These protein DNIC seem to be the source of NO which inhibits human platelet aggregation.     

A low molecular weight inhibitor of steroid receptor activation.

Dilution at 0 degrees of rat liver cytosol incubated with [3H]triamcinolone acetonide provoked an enhanced binding of steroid-receptor complexes to nuclei. The explanation of this phenomenon was found to be an "activation" of the complexes. Dilution acted by decreasing the concentration of a cytosol inhibitor. This reaction was irreversible at 0 degrees: once activated the complexes could not be reversed to the nonactivated state by the addition of inhibitor. The presence of hormone was necessary, since hormone-free receptor molecules could not be activated by dilution. Removal of the inhibitor did not lead to activation of all complexes: after 24 h a "plateau" was attained where 55 to 70% of the complexes were activated. The inhibitor was shown to be a low molecular weight molecule by dialysis, Sephadex G-25 chromatography, ammonium sulfate precipitation, and ultrafiltration. Thus [3H]triamcinolone acetonide-receptor complexes present in a cytosol from which the inhibitor had been removed by Sephadex G-25 chromatography became spontaneously activated at low ionic strength and at 0 degrees. The inhibitor is not a steroid (at least of usual polarity) since it cannot be extracted by methylene chloride or adsorbed by activated charcoal. It is thermostable (resists to 30 min at 100 degrees). Its removal by incubation with a cation exchange resin suggests that it may be positively charged, however it is not complexed by EDTA. This inhibitor must be distinguished from a previously described inhibitor of steroid-receptor complexes binding to nuclei. The latter compound has been shown in various systems to be responsible for an artifactual saturation of nuclear acceptor by steroid-receptor complexes. It inhibits the binding to nuclear acceptors of already activated complexes and is probably a macromolecule. It is thus different from the low molecular weight activation inhibitor described in the present paper.     

Activation of the matrix metalloproteinase inhibitor complex by a low molecular weight angiogenic factor

Tissue inhibitor of matrix metalloproteinases is the major inhibitor of the collagenolytic enzymes and the inhibitory complex has been thought to be irreversible. In this paper we show that a low molecular weight non-protein endothelial cell stimulating angiogenic factor is able to reactivate the enzyme from the inhibitor complex and liberate free inhibitor. The importance of an angiogenic factor able to initiate limited degradation of extra-cellular matrix such that space is created for new capillary growth is discussed.     

Inhibition of human collagenase activity by a small molecular weight serum protein.

Fractionation of human serum proteins by gel filtration in Sephadex G-200 revealed two regions of collagenase inhibition which corresponded to α₂-macroglobulin and a smaller serum component which eluted after α₁-antitrypsin. The smaller collagenase inhibitor, having a molecular weight of 40,000 was separated from α₁-antitrypsin by chromatography in Sephadex DEAE A.50. It was found to inhibit human collagenases derived from skin, rheumatoid synovium, gastric mucosa and granulocytes, but not the neutral proteases trypsin and papain. Purified preparations of α₁-antitrypsin inhibiting trypsin and papain had no effect on the collagenase activities. The small collagenase inhibitor may have importance as a regulatory factor in the control of collagenase activity in vivo.     

Anti-HIV-1 activity of low molecular weight sulfated chitooligosaccharides

Chitooligosaccharides are nontoxic and water-soluble compounds obtained by enzymatic degradation of chitosan, which is derived from chitin by a deacetylation process. Chitooligosaccharides possess broad range of activities such as antitumour, antifungal, antibacterial activities. Sulfated chitooligosaccharides (SCOSs) with different molecular weights were synthesized by a random sulfation reaction. In the present study, anti-HIV-1 properties of SCOSs and the impact of molecular weight on their inhibitory activity were investigated. SCOS III (MW 3-5 kDa) was found to be the most effective compound to inhibit HIV-1 replication. At nontoxic concentrations, SCOS III exhibited remarkable inhibitory activities on HIV-1-induced syncytia formation (EC₅₀ 2.19 μg/ml), lytic effect (EC₅₀ 1.43 μg/ml), and p24 antigen production (EC₅₀ 4.33 μg/ml and 7.76 μg/ml for HIV-1_RF and HIV-1_Ba-L, respectively). In contrast, unsulfated chitooligosaccharides showed no activity against HIV-1. Furthermore, it was found that SCOS III blocked viral entry and virus-cell fusion probably via disrupting the binding of HIV-1 gp120 to CD4 cell surface receptor. These results suggest that sulfated chitooligosaccharides represent novel candidates for the development of anti-HIV-1 agent.     

The isolation of a low molecular weight inhibitor of [H]TdR incorporation into hepatic DNA

An inhibitor of [³H]TdR incorporation into rat liver DNA has been partially purified from bovine and rat livers and from bovine serum. The material isolated does not contain TdR: it is species non-specific, tissue-specific, and non-cytotoxic, and may contain a hepatic chalone.     

Inhibition of the cathepsin B like proteinase by a low molecular weight cysteine-proteinase inhibitor from ascitic fluid and plasma alpha 2 macroglobulin

The cathepsin B like proteinase present in ascitic fluid of a patient with neoplasia has been purified and characterized after pepsin activation. From this fluid we have prepared the low molecular weight (LMW) cysteine-proteinase inhibitors. Three major inhibitor forms were found with isoelectric points of 7.4, 5.4, and 5.1, respectively. The interaction of the enzyme with the former inhibitor was studied because this inhibitor was the most abundant. The Ki value was found to be 4.3 X 10(-8) M. Two molecules of this proteinase were bound by one molecule of plasma alpha 2 macroglobulin (alpha 2M). The LMW inhibitor was able to bind to the enzyme entrapped in alpha 2M and reduced its endopeptidase activity on benzyloxycarbonyl-L-phenylalanyl-L-arginine-4-methyl-7-coumarylamide. These results may have a physiological significance in the regulation of the enzyme which, among other extracellular hydrolases, probably plays an important role in tumor invasion.     

Partial purification of a low molecular weight inhibitor of epidermal DNA synthesis

Abstract. An attempt has been made to purify factors present in aqueous extracts of pig epidermis which inhibit epidermal cell proliferation. A lipophilic factor of low molecular weight (less than 10,000), has been shown to inhibit DNA synthesis as measured by the uptake of tritiated thymidine in mouse ear epidermis. Purification by alcohol precipitation, ethyl acetate extraction and silicic acid column chromatography produced a fifteen-fold increase in the specific activity of the inhibitory action. It seems likely that aggregation or absorption of this low molecular weight factor may explain the high molecular weight of epidermal cell proliferation inhibitors previously studied, as well as the difficulty in their characterization.     

Synthesis of low molecular weight compounds with complement inhibition activity

An attempt was made to synthesize a series of non-cytotoxic low molecular weight meta-substituted aromatic ethers (2-4, 5-7) and some of their bioisosteres (14-16) and to evaluate their activity on the activation of human complement (classical pathway) and their intrinsic hemolytic activity. The in vitro assay results of the inhibition of complement-mediated hemolysis by these analogues indicate that the aldehydic meta substituted aromatic ethers show inhibitory potency, while carboxylic acid meta substituted aromatic ethers show hemolytic activity. Some of the bioisosteres exhibit both inhibitory as well as hemolytic property.     

Analysis of low molecular weight compounds by MALDI-FTICR-MS

This review focuses on recent applications of matrix-assisted laser desorption ionization-Fourier-transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) in qualitative and quantitative analysis of low molecular weight compounds. The scope of the work includes amino acids, small peptides, mono and oligosaccharides, lipids, metabolic compounds, small molecule phytochemicals from medicinal herbs and even the volatile organic compounds from tobacco. We discuss both direct analysis and analysis following derivatization. In addition we review sample preparation strategies to reduce interferences in the low m/z range and to improve sensitivities by derivatization with charge tags. We also present coupling of head space techniques with MALDI-FTICR-MS. Furthermore, omics analyses based on MALDI-FTICR-MS were also discussed, including proteomics, metabolomics and lipidomics, as well as the relative MS imaging for bio-active low molecular weight compounds. Finally, we discussed the investigations on dissociation/rearrangement processes of low molecular weight compounds by MALDI-FTICR-MS.     

The isolation of a low molecular weight inhibitor of [3H]TdR incorporation into hepatic DNA.

An inhibitor of [³H]TdR incorporation into rat liver DNA has been partially purified from bovine and rat livers and from bovine serum. The material isolated does not contain TdR: it is species non-specific, tissue-specific, and non-cytotoxic, and may contain a hepatic chalone.     

Inhibition of cell adhesion by high molecular weight kininogen

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.     

Isolation of a low molecular weight inhibitor of lymphocyte proliferation from tumorous ascites

Intraperitoneal growth of P-815 mastocytoma cells in syngeneic DBA/2 mice produces ascites fluid which strongly inhibits mitogen-stimulated lymphocyte proliferation. The less than 10,000 m.w. fraction from gel filtration chromatography of tumorous ascites on Sephadex G-150 showed no inhibition of proliferation when eluted under physiologic conditions but was inhibitory when eluted with a high ionic strength, acidic buffer. The organic phase of a chloroform/methanol extract of the low m.w. fraction contained all the inhibitory activity. Purification of the inhibitor to relative homogeneity was achieved by reverse phase, HPLC with a gradient of acetonitrile in dilute acetate buffer. Inhibitory activity eluted between 30 and 35% acetonitrile. The active fraction contained less than 30 pg/ml PGE by RIA which was insufficient to inhibit proliferation and may actually have been stimulatory. Inhibition comparable to that produced by the ascites fraction required greater than 300 pg/ml of PGE. This low m.w. (less than 10,000), lipid-like inhibitor of lymphocyte proliferation is acid stable, not sensitive to proteolytic enzymes, soluble in both aqueous and organic solvents and occurs normally bound to a higher m.w. carrier molecule.     

Inhibition of the p53-hdm2 interaction with low molecular weight compounds

The hdm2 protein, upon binding to p53, inhibits its tumor suppressor activity. The inhibition of the p53-hdm2 interaction represents therefore a new therapeutic strategy to activate wild type p53 in tumors. Potent low molecular weight compounds inhibiting this protein-protein interaction, which are active in vivo, have just been identified. This offers new perspectives and hopes in this research area.