Gelatin Zymography Using Leupeptin for the Detection of Various Cathepsin L Forms.

Zymography is a highly sensitive method to assess the activities as well as molecular weights of enzymes in crude biological fluids and tissue extracts. Cathepsin L is a lysosomal cysteine proteinase that is optimally active at slightly acidic pH and is highly unstable in alkaline solutions such as electrode buffer (pH 8.3). Large amounts of cathepsin L are secreted by various cancer cells, where it promotes invasion and metastasis. Leupeptin is a tight-binding inhibitor of cysteine proteinases, and its complex with cathepsin L is stable in alkaline solutions. Moreover, leupeptin can be easily removed from the complex because it is a reversibly binding inhibitor. In addition, leupeptin is too small to influence the electrode migration distance of the complex with cathepsin L on a sodium dodecyl sulfate-polyacrylamide gel. Here, a novel gelatin zymography technique that employs leupeptin to detect pro-, intermediate, and mature cathepsin L forms on the basis of their gelatinolytic activities is described. Further, the differences in the glycosylation, phosphorylation, and processing statuses of lysosomal and secreted cathepsin L forms isolated from cultured HT 1080 cells are demonstrated using this method.

High-temperature liquid chromatography coupled on-line to a continuous-flow biochemical screening assay with electrospray ionization mass spectrometric detection.

The potential of high-temperature liquid chromatography (HTLC) was investigated in an on-line combination with a screening system for bioactive compounds against the enzyme cathepsin B. Samples were separated by HTLC and subsequently analyzed by an on-line continuous-flow enzymatic assay. Detection was performed by electrospray ionization mass spectrometry, revealing both the bioactivity and the molecular mass of the bioactive compounds. Compared to conventional reversed-phase liquid chromatography, the amount of methanol necessary for separation could be decreased to only 10%, which improved the compatibility of LC with a biochemical assay. Sufficient preheating of the mobile phase prior to the separation and postcolumn cooling to prevent deactivation of the enzyme, even at column temperatures as high as 208 degrees C, was achieved as indicated by the reliable peak shapes obtained. The sensitivity was comparable with previously described systems operating at ambient temperatures as similar IC50 values were obtained. Exposing the inhibitors to high temperatures did not lead to thermal decomposition. The separation of inhibitors and the subsequent biochemical assay was performed either isothermally at various temperatures or by applying various temperature gradients as well as at various flow rates. The results obtained clearly show the compatibility of HTLC with an enzymatic screening assay.

Sustained-release delivery of leupeptin in the chinchilla: hearing results.

Transtympanic medical therapy is becoming an increasingly popular modality for the treatment of "inner-ear disorders." While investigators continue to examine the best dosing paradigms for gentamicin in the treatment of Ménière's disease and for steroids in the treatment of hearing loss, they have also begun to focus on the use of other agents. In particular, transtympanic therapy has been advocated as a plausible route for the treatment of tinnitus. Transtympanic therapy for tinnitus is not new, and a number of groups have reported success in the past. Despite this success, a number of laboratories have been focusing on newer agents that might yield higher success rates in the treatment of tinnitus and other inner-ear disorders. Many of these agents could have systemic side effects when delivered in high enough doses; therefore, they are ideal candidates for transtympanic administration. The goal of this study is to begin to define the effects of one of these agents--leupeptin, a calpain antagonist--on the normal inner ear of an animal model. In this investigation, we demonstrate the effects of sustained-release delivery of leupeptin (2.5 micrograms/ml) on the hearing of chinchillas. The medicine produced no hearing loss at the early time points but did produce some hearing loss at later time points. We discuss these results and begin to outline the next steps in the investigation of this agent.

The inhibition of the enzymic activity of blood coagulation and fibrinolytic serine proteases by a new leupeptin-like inhibitor, and its structural analogues, isolated from Streptomyces griseus.

A group of leupeptin analogues was found in Streptomyces griseus strain 254, isolated from a soil sample from Fujian Province, China. The inhibitors excreted in the culture filtrate were purified by adsorption on macroporous resin, followed by sequential ion exchange chromatography on DEAE-52 cellulose, CM-32 cellulose and affinity chromatography with immobilized trypsin. The preparation thus obtained was further purified by preparative HPLC. Several major components were found and characterized, which possessed different inhibitory properties toward trypsin. Based upon amino acid and mass spectrophotometric analysis, these peptides were placed in four major structural categories, viz., R-Val-Val-argininal, R-Leu-Leu-argininal, R-Ile-Ile-argininal and R-Thr-Thr-argininal, this latter component representing a newly identified leupeptin analogue. The structural variability of the R-group was partly responsible for the multiplicity of the peaks obtained with HPLC. All peptides displayed varying degrees of inhibitory activity toward proteases involved in blood coagulation and fibrinolysis, including plasmin, factor Xa, activated protein C and thrombin. Among these peptide inhibitors, the molecule containing threonine showed the strongest inhibitory activity.

High-performance liquid chromatographic method for monitoring leupeptin in mouse serum and muscle by pre-column fluorescence derivatization with benzoin.

A high-performance liquid chromatographic method is described for the determination of leupeptin, a possible therapeutic drug for muscular dystrophy, in mouse serum and muscle. Leupeptin is reduced with sodium borohydride to leupeptinol, and then converted to a fluorescent derivative with benzoin. The derivative is separated on a reversed-phase column (LiChrosorb RP-18) with isocratic elution and determined with fluorescence detection. The detection limits of leupeptin in serum and muscle are 250 pmol/ml (107 ng/ml) and 500 pmol/g (214 ng/g), respectively, corresponding to approximately 150 fmol each in a 100-microliters injection volume. This method is simple and sensitive enough to permit the quantification of leupeptin in biological samples from mice dosed with leupeptin.

High-performance affinity chromatography of plasmin and plasminogen on a hydrophilic vinyl-polymer gel coupled with p-aminobenzamidine.

p-Aminobenzamidine was covalently attached via a spacer moiety to a microparticulate hydrophilic vinyl-polymer gel (Toyopearl HW65S) and this affinity adsorbent was used for the separation of plasmin and plasminogen by high-performance affinity chromatography. Toyopearl HW65S was alkylated with chloroacetylglycylglycine in dimethyl sulphoxide using methylsulphinyl carbanion as a catalyst, then p-aminobenzamidine was coupled to the carboxyl group of glycylglycine to form an acid amide bond. A column packed with the adsorbent retained both plasmin and plasminogen. Plasminogen was eluted with 6-aminohexanoic acid, a haptenic compound for the lysine-binding sites of plasminogen. For the elution of plasmin, the coexistence of 6-aminohexanoic acid and leupeptin (a competitive inhibitor for plasmin) was necessary. The results indicate a two-site interaction of plasmin with the immobilized ligand, i.e., at the lysine-binding sites and the catalytic site. Fluorometric detection of eluted protein and on-line assay of plasmin activity using a fluorogenic substrate, peptidylmethylcoumarylamide, revealed that effective chromatographic separation of the enzyme could be achieved with high sensitivity (10 micrograms) within 1 h.