化学修饰对组织型纤溶酶原激活剂的纤溶活性和体内半衰期的影响

用肝素、右旋糖酐和聚乙二醇对重组组织型纤溶酶原激活剂(rt-PA)进行化学修饰。不同的化学修饰物对rt-PA体外纤维蛋白溶解活性的影响不同。肝素修饰小PA的纤维蛋白溶解活性提高63％;右旋糖酐和聚乙二醇修饰rt-PA的纤维蛋白溶解活性则分别降低了36％和63％。rt-PA经化学修饰后半衰期均有所延长,其中聚乙二醇修饰的小PA的半衰期由修饰前的3．4min延长至6．3mim。     

Control of pharmaceutical properties of soybean trypsin inhibitor by conjugation with dextran. I: Synthesis and characterization

The Kunitz-type soybean trypsin inhibitor (STI), a model protein, was conjugated with dextran (Mw, approximately 9900; STI-D), and its physicochemical and biochemical properties were studied to develop a novel delivery system for a protein drug. Conjugation was carried out using periodate oxidation, and cyanogen bromide, carbodiimide, cyanuric chloride, epichlorhydrin, and N-succiniimidyl-3-(2-pyridyldithio)propionate (SPDP) reagent methods. Dextran was conjugated to STI at a molar ratio of 1.5 to 4.6, but the degree of modification, as well as yield and contamination extent of unreacted STI and dextran, varied with the method of synthesis. Gel filtration and electrophoresis confirmed the covalent attachment of dextran to STI but also demonstrated the broad molecular weight distribution of the conjugates. The STI-D conjugate retained satisfactory activity, although the attachment partially reduced its inhibitory activity against trypsin. The periodate oxidation method seemed to be the best for the preparation of STI-D since it gave the conjugate with a high modification ratio (4.6 molecules per STI), high yield (95%), and satisfactory activity recovery (63%). Chemical modification of STI was also carried out with activated polyethylene glycol (PEG) for comparison. The STI-PEG conjugate was obtained in a satisfactory yield (96%) and modification degree (5.8 molecules per STI), but the remaining activity was considerably lower (34%). Thus, conjugation of protein with dextran by the periodate oxidation method is suggested to be preferable for preparing a protein-carrier system without significant diminution of its biological activity.     

Studies on possible mechanisms for the interaction between cyanamide and aldehyde dehydrogenase

Cyanamide is known to interfere with the metabolism of alcohol by decreasing the activity of aldehyde dehydrogenase in vivo, thereby leading to an accumulation of acetaldehyde following the ingestion of ethanol. We have studied various mechanisms for the chemical interaction between aldehyde dehydrogenase and cyanamide (or some of its possible metabolites). Cyanamide was shown to react under physiological conditions with amino and thiol groups, forming guanidino and isothiouronium compounds respectively. However, it was found that the enzyme is not appreciably affected in vitro by high concentrations of cyanamide. Dicyandiamide and the aminoethylisothiouronium ion (AET) also have no effect in vitro. It was postulated that the pathway of in vivo enzyme modification by cyanamide may involve thiourea (formed by breakdown of isothiouronium compounds) and formamidine disulphide (an oxidation product of thiourea). However, although the disulphide is a moderately-effective inactivator of aldehyde dehydrogenase in vitro, administration of thiourea or AET to rats does not result in any significant loss of aldehyde dehydrogenase activity in either the cytoplasm or the mitochondria     

Purification and partial characterization of hyaluronidase from five pace snake (Agkistrodon acutus) venom

—A hyaluronidase (EC 3.2.1. 35) was isolated and purified from Agkistrodon acutus venom. The purification procedure included CM-Sephadex C-50 chromatography, gel-filtration on Sephadex G-75 and CM-Sephadex C-25 chromatography. The purified preparation of the enzyme was homogeneous on polyacrylamide gel electrophoresis at pH 4.3, a 45-fold purification being obtained. The hyaluronidase was a glycoprotein (positive PAS staining) with a molecular weight of 33,000 and a pI of 10.3. No hemorrhagic activity was found. The hyaluronidase had an optimum pH of 3.5–5.0 and an optimum temperature of 37°C. It was heat sensitive, was more stable in the acidic than in the neutral region, and lost its activity in the alkaline region. Fe²⁺, Cu²⁺ and heparin inhibited the venom hyaluronidase. The K_m value for hyaluronic acid was 6.2 × 10⁻³ mg/ml.     

Cationic derivatives of dextran and hydroxypropylcellulose as novel potential heparin antagonists

Cationic derivatives of dextran (Dex) and hydroxypropylcellulose (HPC) were studied as potential alternatives of protamine sulfate (PS) used in the reversal of anticoagulant activity of heparin. The modification was performed by the attachment of cationic groups to the Dex main chain or by grafting short side chains of a polycation onto HPC. The cationic derivatives of these polysaccharides were found to bind heparin with the efficiency increasing with growing degree of cationic modification. The degree of cationic modification and consequently the ζ potential of the polymers do not have to be high to achieve effective heparin binding. The size of the complexes of cationic Dex with unfractionated heparin (UFH) is a few micrometers. For complexes of cationic HPC and UFH the size is much below 1 μm, both below and above the lower critical solution temperature of HPC. None of the cationic polysaccharides studied caused hemolysis. The concentrations of the polymers inducing the aggregation of human erythrocytes in vitro were determined.     

几种因素在L-天冬酰胺酶化学修饰中对酶活力及抗原性的影响

为了寻求在较好地保持酶活力的同时解除L-天冬酰胺酶抗原性的方法,采用不同分子量的乙酸酐、右旋糖酐和单甲氧基聚乙二醇,作为修饰剂和不同的修饰方法对该酶进行了化学修饰。结果表明在保持酶活性和降低抗原性方面,大分子修饰剂右旋糖酐、单甲氧基聚乙二醇优于小分子乙酸酐,底物保护修饰优于直接修饰;活化PEG,优于活化PEG₁。在底物保护下的PEG,修饰酶其抗原性完全解除的同时,酶活力保持在30%以上。     

Characterization of human erythrocyte aldehyde dehydrogenase

Human erythrocyte aldehyde dehydrogenase was purified to homogeneity. The enzyme exhibited a single band of activity on starch gel electrophoresis and on isoelectric focusing. It was a tetramer with an estimated molecular weight of 230,000 daltons and an isoelectric point of 5.0. Its pH optimum of 8.5, Michaelis-Menten constant for acetaldehyde of 46 microM, and high sensitivity to noncompetitive inhibition by disulfiram resembled human liver cytosolic aldehyde dehydrogenase. Low concentrations of magnesium (5-10 microM) resulted in enhancement of erythrocyte aldehyde dehydrogenase activity, whereas higher physiological concentrations of magnesium resulted in uncompetitive inhibition of enzyme activity. Magnesium inhibited the enzyme activity by increasing the binding of NADH to the enzyme as had been found to be the case for the inhibitory effect of magnesium on the human liver cytosolic enzyme. Erythrocyte aldehyde dehydrogenase may metabolize small amounts of acetaldehyde escaping the liver during ethanol metabolism and protect extrahepatic tissues from acetaldehyde toxicity.     

The functional role of histidine and sulfhydryl groups in rat liver thiamine pyrophosphokinase

The functional role of histidine and sulfhydryl groups of thiamine pyrophosphokinase (ATP: thiamine pyrophosphotransferase, E.C.2.7.6.2) has been studied by the methods of photooxidation and chemical modification by diethylpyrocarbonate (DEPC) and Ellman's reagent, 5,5' - dithiobis (2 - nitrobenzoic acid) (DTNB). Histidine amino acid residues have been shown to be destroyed during photoinactivation. Titration of the protein with DEPC has established that modification of two histidine groups decreases the catalytic activity of thiamine pyrophosphokinase. Interaction of the subsequent three groups with the reagent does not affect residual activity. Substrates protect thiamine pyrophosphokinase from inactivation. Two -SH groups have been identified in a molecule of thiamine pyrophosphokinase with Ellman's reagent. Modification of only one of them results in complete loss of the enzymatic activity. Treatment of the enzyme with 8M urea has shown no differences in the amount of thiol groups of the native and denatured enzymes. Mg2+ + ATP and somewhat more weakly Mg2+ partially protect thiamine pyrophosphokinase from inhibition by DTNB. In their presence a high degree of enzyme reactivation is observed. Both histidine and sulfhydryl groups are suggested to play an important role in the catalytic mechanism of thiamine pyrophosphokinase.     

Peptidyl fluoromethyl ketones as inhibitors of cathepsin B. Implication for treatment of rheumatoid arthritis.

Peptidyl fluoromethyl ketones (FMKs), with the amino acid sequence Phe-Ala held constant but with variable N-terminal groups, were synthesized and tested for inhibition of the cysteine proteinase cathepsin B. The FMKs were effective in inhibiting cathepsin B activity in vitro. The inhibition was time dependent and was not reversed by dialysis, suggesting covalent modification of the enzyme. Cathepsin B activity present in livers and kidneys of rats treated with FMKs was reduced by 22-91% 4 hr after a single oral dose of 25 mg/kg. The FMKs inhibited the severity of inflammation and the extent of cartilage and bone damage in adjuvant-induced arthritis. These effects were seen during the late-stage of the disease with no effect on onset or incidence of disease. This is consistent with inhibition of protease-mediated damage. These FMKs or derivatives may be of clinical value in the treatment of arthritis.     

Lamotrigine�Cdextran conjugates-synthesis, characterization, and biological evaluation

Synthesis of lamotrigine–dextran conjugates is done by oxidation of dextran using sodium periodate (NaIO₄), where the aldehyde groups formed were coupled with the amino (–NH₂) group of lamotrigine and reduced to secondary imine groups. Characterization of synthesized conjugates was done by using ultraviolet, infrared, nuclear magnetic resonance spectroscopy. Viscometer was used to determine molecular weight, and degree of substitution was estimated by complete hydrolysis of conjugates in borate buffer, which was found to be 8%. Buffer solutions with different pH, i.e., 1.2, 7.4, and 9.0 were used to perform the in vitro hydrolysis study and the amount of conjugates released was estimated by high performance liquid chromatography. The study showed that the rate of hydrolysis increases with increase in pH. The anticonvulsant and hepatotoxicity screening of the synthesized conjugates in different rat models showed that lamotrigine–dextran conjugates proved to be potentially safer than parent lamotrigine formulations.     

Effects of iridoids on lipoxygenase and hyaluronidase activities and their activation by beta-glucosidase in the presence of amino acids

Enzyme inhibitory activities of 14 iridoids previously obtained from two Malaysian medicinal plants, Saprosma scortechinii and Rothmannia macrophylla, were evaluated in vitro using soybean lipoxygenase and bovine testis hyaluronidase. Most of the iridoids, including asperulosidic acid, paederosidic acid, and an epimeric mixture of gardenogenins A and B, did not show any effect on the enzyme activities, except for the bis-iridoids, which inhibited the lipoxygenase activity with their IC(50) values of approximately 1.3 times that of a known inhibitor, fisetin. Structural modification of asperulosidic acid and paederosidic acid through enzymatic hydrolysis by beta-glucosidase resulted in their inhibition towards the enzyme activities, and these activities were enhanced by the presence of some amino acids (lysine, leucine or glutamic acid) or ammonium acetate. Mixtures of gardenogenins A and B; isomers of non-glucosidic iridoids, incubated with amino acid or ammonium acetate did not show any inhibitory effect on the enzyme activities during the 6 h incubation period, except for lysine where spontaneous reaction between the iridoids and amino acid resulted in the inhibition of lipoxygenase activity. The results from these biomimetic reactions suggested that the iridoid aglycons and the intermediates formed by these reactive species could inhibit the enzyme activities, and thus substantiate previous reports that the formation of iridoidal aglycons is a prerequisite for the iridoid glycosides to demonstrate some of the biological activities. In addition, the results also indicated that it is worthwhile to further explore these intermediates as potential anti-inflammatory agents.     

A hyaluronidase from Potamotrygon motoro (freshwater stingrays) venom: Isolation and characterization

Freshwater stingrays (Potamotrygon motoro) are known to cause human accidents through a sting located in its tail. In the State of Goiás, this accident happens especially during the fishing season of the Araguaia River. The P. motoro venom extracted from the sting presented hyaluronidase activity. The enzyme was purified by gel filtration on Sephacryl S-100 and ion-exchange chromatography on SP-Sepharose. A typical procedure provided 376.4-fold purification with a 2.94% yield. The molecular weight of the purified enzyme was 79 kDa as estimated by gel filtration on Sephacryl S-100. The K_m and V_max values for hyaluronidase, using hyaluronic acid as substrate, were 4.91 μg/ml and 2.02 U/min, respectively. The pH optimum for the enzyme was pH 4.2 and maximum activity was obtained at 40 °C. The hyaluronidase from P. motoro was shown to be heat instable, being stabilized by bovine albumin and DTT, and inhibited by Fe²⁺, Mn²⁺, Cu²⁺ and heparin.     

Pentosan polysulfate, a sulfated oligosaccharide, is a potent and selective anti-HIV agent in vitro

Several sulfated oligo- or polysaccharides such as pentosan polysulfate, fucoidan, dextran sulfate, heparin and iota-, kappa- and lambda-carrageenans proved to be potent and selective inhibitors of human immunodeficiency virus type 1 (HIV-1) in vitro. The most potent anti-HIV-1 activity was recorded for the oligosaccharide pentosan polysulfate, its 50% antiviral effective dose (ED50) being 0.19 microgram/ml in MT-4 cells. It inhibited HIV-1 antigen expression in HUT-78 cells at an ED50 of 0.02 microgram/ml, and complete inhibition of HIV-1 antigen expression was obtained at a concentration of 4.0 micrograms/ml. No toxicity for MT-4 cells was observed with pentosan polysulfate at a concentration of 2500 micrograms/ml. The anticoagulant activity of pentosan polysulfate was more than ten-fold lower than that of heparin (14.4 and 177 U/mg, respectively). In fact, pentosan polysulfate achieved its anti-HIV-1 activity at a concentration that is 370-fold below its anticoagulant threshold (1 U). Pentosan polysulfate inhibits virus adsorption to the cells, as was demonstrated by monitoring the association of radiolabeled HIV-1 virions with MT-4 cells.     

Daboia russelli venom hyaluronidase: purification, characterization and inhibition by β-3-(3-hydroxy-4-oxopyridyl) α-amino-propionic Acid

The present study describes the purification and characterization of a hyaluronidase (DRHyal-II) from Daboia/Vipera russelli venom and its inhibition by β-3-(3-hydroxy-4-oxopyridyl) α-amino-propionic acid, the mimosine. Gel permeation and ion exchange chromatography were employed to isolate DRHyal-II. The molecular mass by MALDITOF mass spectrometry was found to be 28.3 kDa. Single band in reduced SDS-PAGE suggested the monomeric nature. It was optimally active at pH 5.5 and at 37C and require 150 mM NaCl in the reaction mixture. It was specific to hyaluronan substrate and belongs to class-I or the neutral active enzymes. DRHyal-II was non-toxic by itself but, it potentiated the myotoxicity of VRV-PL-VIII myotoxin and hemorrhagic activity of hemorrhagic complex (HC). In in vitro experiments, mimosine inhibited the activity of DRHyal-II and the hyaluronidase activity of whole venom dose dependently. In in vivo experiments, mimosine inhibited the DRHyal-II potentiated myotoxicity of VRV-PL-VIII myotoxin and hemorrhagic activity of HC. The inhibition was due to the formation of DRHyal-II-mimosine inhibitory complex that resulted in significant structural changes at secondary and tertiary levels as evidenced by fluorescence emission and CD spectral studies. Hence, in this study an attempt was made to establish the possible role of hyaluronidase activity in the pathology of Daboia/Vipera russelli venom and the beneficial effects of its inhibition with special emphasis on the management of local toxicity.     

Some structural determinants of the antiproliferative effect of heparin-like molecules on human airway smooth muscle

Accumulation of airway smooth muscle (ASM) and its infiltration by mast cells are key pathological features of airway remodelling in asthma. Heparin, a major component of mast cell granules, inhibits ASM proliferation by an unknown mechanism. Here, unfractionated heparins and related glycosaminoglycans having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule that were required for its antiproliferative activity in cultured human ASM cells. Proliferation induced by 10% fetal bovine serum (FBS) was abrogated by two unfractionated commercial heparin preparations (Sigma and Multiparin) and this effect was reproduced with each of three low-molecular weight heparin preparations (3, 5 and 6 kDa, respectively), demonstrating that antiproliferative activity resided in at least a 3 kDa heparin fraction. N-desulphated 20% re-acetylated (N-de) heparin (anticoagulant) and O-desulphated heparin (O-de) (non-anticoagulant) fractions also inhibited FBS-dependent proliferation (rank potency: Sigma heparin > O-de > N-de) suggesting that the antiproliferative action of heparin involved N-sulphation but was independent of its anticoagulant activity. Other sulphated molecules with variable anionic charge (dextran sulphate, fucoidan, chondroitin sulphates A or B, heparan sulphate) inhibited proliferation to varying degrees, as did the non-sulphated molecules hyaluronic acid and poly-L-glutamic acid. However, nonsulphated dextran had no effect. In summary, attenuation of FBS-dependent proliferation of human ASM by heparin involves but does not depend upon sulphation, although loss of N-sulphation reduces antiproliferative activity. This antiproliferative effect is independent of anionic charge and the anticoagulant actions of heparin.     

The effects of cephem antibiotics and related compounds on the aldehyde dehydrogenase in rat liver mitochondria

The effects of cephem antibiotics and their related compounds on aldehyde dehydrogenase obtained from rat liver mitochondria were studied. A pH of 8.8 and reaction temperature 24 degrees were the conditions for measurement of enzyme activity. The apparent Michaelis constant Km values for NAD, acetaldehyde and propionaldehyde were 3.8 X 10(-5) M, 4.0 X 10(-5) M and 2.5 X 10(-5) M, respectively. Cefamandole, cefoperazone and cefmetazole, having a 1-methyl-5-thiotetrazol group at position 3 of the cephem ring, caused a relatively potent inhibition of aldehyde dehydrogenase. Cefmetazole and cefoperazone also showed a significant inhibition on highly purified yeast aldehyde dehydrogenase; the extent of inhibition on yeast enzyme was almost the same as that on rat mitochondrial aldehyde dehydrogenase. The decrease in enzyme activity effected by 1-methyl-1H-tetrazol-5-thiol (MTT) was greater than those of 1H-tetrazol (TZ), 1H-tetrazol-5-thiol and 1-(2-dimethylaminoethyl)-1H-tetrazol-5-thiol, but was, of course, less than that of disulfiram. Cefamandole, cefmetazole and MTT showed competitive inhibition with NAD, while TZ was uncompetitive inhibitor with respect to both NAD and acetaldehyde. Enzyme inhibition caused by disulfiram, cefmetazole and MTT increased time-dependently and the addition of 2-mercaptoethanol into the medium effectively and completely restored enzyme inhibition. These results suggest that thiol group at position 5 of 1H-tetrazol ring is responsible for the type of inhibition with NAD, and methyl group at position 1 of 1H-tetrazol ring is important to exhibit a potent inhibition on aldehyde dehydrogenase.     

A hyaluronidase from Tityus serrulatus scorpion venom: isolation, characterization and inhibition by flavonoids.

The purification procedure of a hyaluronidase from Tityus serrulatus scorpion venom is described. It involves basically an ion-exchange chromatography on CM-cellulose at pH 7.8 followed by a rechromatography of the active fraction on the same column at pH 4.7. The optima pH and temperature for maximum activity of the isolated enzyme was 6.0 and 40 degrees C, respectively. Its K(M) was 69.7 microg/ml at 37 degrees C and its specific activity was 19,900+/-1,730 turbidity reducing units (TRU)/mg against 845+/-88TRU/mg for the whole desiccated venom, representing a 23- to 24-fold purification range. The hyaluronidase activity of the purified protein (51kDa) was inhibited by some flavonoid compounds. This article also showed that T. serrulatus hyaluronidase affected on the activity of the venom's major toxin, tityustoxin-I (TsTX-I or Ts1), as reflected by alterations in the serum levels of creatine kinase (CK), lactate dehydrogenase (LD) and aspartate aminotransferase (AST) following injection of TsTX-I, in the presence or absence of hyaluronidase.     

Activation of microsomal glutathione s-transferase by peroxynitrite

Peroxynitrite (ONOO-) toxicity is associated with protein oxidation and/or tyrosine nitration, usually resulting in inhibition of enzyme activity. We examined the effect of ONOO- on the activity of purified rat liver microsomal glutathione S-transferase (GST) and found that the activity of reduced glutathione (GSH)-free enzyme was increased 4- to 5-fold by 2 mM ONOO-; only 15% of this increased activity was reversed by dithiothreitol. Exposure of the microsomal GST to ONOO- resulted in concentration-dependent oxidation of protein sulfhydryl groups, dimer and trimer formation, protein fragmentation, and tyrosine nitration. With the exception of sulfhydryl oxidation, these modifications of the enzyme correlated well with the increase in enzyme activity. Nitration or acetylation of tyrosine residues of the enzyme using tetranitromethane and N-acetylimidazole, respectively, also resulted in increased enzyme activity, providing additional evidence that modification of tyrosine residues can alter catalytic activity. Addition of ONOO--treated microsomal GST to microsomal membrane preparations caused a marked reduction in iron-induced lipid peroxidation, which raises the possibility that this enzyme may act to lessen the degree of membrane damage that would otherwise occur under pathophysiological conditions of increased ONOO- formation.     

Interactions of low-molecular-weight semi-synthetic sulfated heparins with human leukocyte elastase and human Cathepsin G

Semi-synthetic low-molecular-weight heparin samples (LMWHs), having homogeneous degree of polymerization and saccharide backbone, but differing in the number and location of sulfate groups, were investigated in their ability to interfere with the pharmacologically relevant targets human leukocyte elastase (EL) and human Cathepsin G (CatG). Spectroscopic studies were performed for a quantitative evaluation of the enzyme-inhibitor dissociation constant, K(i), and of the IC(50) values for the inhibition of cleavage of target peptide sequences. Both proteases are inhibited by the tested polysaccharides through a mixed hyperbolic binding process. A non-linear relationship was found between degree of sulfation and binding affinity or enzyme inhibition properties, showing a composite correlation between heparin charge density and interference with EL/CatG activity.     

Increasing the plasma half-life of trichosanthin by coupling to dextran.

Trichosanthin (TCS) is a plant protein which has a wide spectrum of pharmacological activities. It was demonstrated recently that this compound suppressed the replication of human immunodeficiency virus (HIV-1) in vitro. The mechanism of action is believed to be inhibition of protein synthesis. Trichosanthin is a low molecular weight protein which is expected to be easily filtered and eliminated through the kidney. To minimize renal loss, the molecular size of trichosanthin can be increased by coupling to dextran. The larger complex will not undergo glomerular filtration and therefore renal loss can be prevented. This study investigates the kidney's role in trichosanthin elimination and the beneficial effect afforded by coupling to dextran in prolonging plasma half-life. For this purpose, a radioimmunoassay has been developed to determine the concentration of TCS in plasma and urine. The sensitivity of this assay is in the nanogram range. Trichosanthin was coupled to dextran T40 by a dialdehyde method and successful coupling was confirmed by gel filtration chromatography. The complex retained specific binding to trichosanthin antibodies with decreased affinity which can be partially reversed after incubation with dextranase; an enzyme that digested dextran. The pharmacokinetics of intravenously administered trichosanthin (0.75 mg/kg) was compared between two groups of rats with normal and impaired renal function (bilateral renal arterial ligation). Rats with ligation showed a decrease in plasma clearance from 4780 +/- 570 to 220 +/- 20 microL/min and an increase in the mean residence time from 9 +/- 1 to 145 +/- 16 min. Despite the several-fold difference in these parameters, recovery of trichosanthin from normal rat urine was only 0.38 +/- 0.05%. This value can be increased by using higher injection doses. The data indicate that the kidney is an important organ for the elimination of trichosanthin. When the dextran-trichosanthin complex was injected into normal rats trichosanthin activity was not detected in the urine. All the pharmacokinetic parameters suggest that the dextran-trichosanthin complex stayed longer in the body and maintained a much higher plasma concentration than trichosanthin.