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Background The predictive significance of programmed death ligand 1 (PD-L1) for programmed death 1 (PD-1) inhibitors remains unclear in gastric cancer (GC) due to the dynamic alteration by treatments. We aimed to elucidate the effects of trastuzumab (Tmab) on PD-L1 expression in GC. Methods PD-L1 expression was evaluated by multicolour flow cytometry analysis after co-culturing GG cell lines and immune cells with Tmab. IFN-γ in the co-culture experiments was quantified. Immunohistochemistry (IHC) for PD-L1 expression using clinical samples was also performed to confirm PD-L1 alteration by Tmab. Results PD-L1 expression was significantly upregulated by Tmab in HER2-amplified GC cell lines co-cultured with peripheral blood mononuclear cells (PBMCs). PD-L1 upregulation by Tmab was also observed in the GC cells co-cultured with NK cells in time-dependent manner, but not with monocytes. IFN-γ concentration in conditioned media from co-cultured PBMCs and NK cells with Tmab was significantly higher and anti-IFN-γ significantly suppress the Tmab-induced PD-L1 upregulation. IHC also suggested PD-L1 upregulation after Tmab treatment. Conclusions Tmab can upregulate PD-L1 expression on GC cells through interaction with NK cells. These results suggest clinical implications in the assessment of the predictive significance of PD-L1 expression for PD-1 inhibitors.

PURPOSE To study the effect of the concomitant use of latanoprost and brinzolamide on the 24-hour variation in the intraocular pressure (IOP) in patients with normal-tension glaucoma (NTG). METHODS We studied a total of 44 eyes from 22 NTG patients. Mean 24-hour IOP variation was determined after a washout period of > or =4 weeks. Latanoprost monotherapy was continued in both eyes for 8 weeks. Thereafter, patients were randomized to continue latanoprost monotherapy in 1 eye whereas brinzolamide was added as an adjunct to latanoprost therapy in the other eye. Eight weeks after the initiation of brinzolamide treatment, the 24-hour IOP variation was remeasured. IOP was measured in the sitting position 8 times daily using a Goldmann applanation tonometer before and after treatment. RESULTS The eyes treated with latanoprost monotherapy and those treated with latanoprost and brinzolamide showed a significant decrease in IOP at all time points. Percent reductions in the diurnal mean IOP (mean IOP at 10 AM, 1 PM, and 4 PM) and in nocturnal mean IOP (mean IOP at 10 PM, 1 AM, and 3 AM) were significantly greater in the eyes treated with the combination of latanoprost and brinzolamide than those with latanoprost alone (diurnal mean IOP: latanoprost and brinzolamide=19.8%, latanoprost=14.1%, P

We have produced biodegradable or enteric-coated microspheres containing camostat mesylate, a protease inhibitor, using a water-oil-water emulsion solvent evaporation method. The characteristics of the microspheres were determined. When polylactic acid, a biodegradable polymer, was used as a wall material, the optimized microsphere obtained showed a loading efficiency of almost 95% and had a mean diameter of 30 pim. This microsphere showed a sustained-release profile, with nearly 25% of drug being released at seven days in a dissolution test. When hypromellose acetate succinate (AS-HG type, with a high content of succinyl group) was used as an enteric wall material, optimized microspheres showed a loading efficiency of almost 80%. In this case, pH 3.0 citrate buffer was used as an internal aqueous phase, and citrate buffer containing 0.5 % polyvinylalcohol was used as the external aqueous phase. These microspheres showed a rapid release profile in pH 6.8 buffer, whereas the release was extremely slow in pH 1.2 buffer. Hypromellose acetate succinate microspheres were also prepared containing 10% (w/w) N-benzoyl-dl-arg-4-nitroanilide as a model substrate for trypsin, with or without 5% (w/w) camostat. These microspheres were incubated in pH 6 or 7 buffer containing trypsin at 37°C. When camostat was included in the microspheres, the substrate was protected from attack by trypsin, while in the absence of camostat, the released substrate was immediately attacked by trypsin to produce the degradation product N-benzoyl-dl-arginine.

The objectives of this study were to prepare insulin-loaded acrylic hydrogel formulations containing various absorption enhancers, to perform in vitro and in vivo characterization of these formulations, and to evaluate the factors which affecting insulin availability on rectal delivery of insulin using this hydrogel system. The acrylic block copolymer of methacrylic acid and methacrylate, Eudispert, was used to make the hydrogel formulations. As absorption enhancers, 2,6-di-O-methyl-beta-cyclodextrin (DM-beta-CyD), lauric acid (C12), or the sodium salt of C12 (C12Na), were incorporated into the hydrogels. In an in vitro release test, the release rate of insulin from the hydrogels decreased as the polymer concentration of the hydrogel increased. The addition of C12Na to the hydrogel further increased the insulin release rate, which was greater at higher concentrations of the enhancer. A portion of the C12Na was found to remain bound to the acrylic polymer in dissolution medium. Serum insulin levels were determined at various time points after the administration of insulin solution or insulin-loaded (50 units/kg body weight) Eudispert hydrogels containing 5% (w/w) of C12, C12Na, or DM-beta-CyD to in situ loops in various regions of the rat intestine. The most effective enhancement of insulin release was observed with formulations containing C12Na. The bioavailability of insulin from the hydrogels was lower than that from the insulin solutions. Hydrogel formulations containing 7% or 10% Eudispert remained in the rectum for 5 h after rectal administration. However, the 5% (w/w) C12Na solution stained with Evan's-blue had diffused out and the dye had reached the upper intestinal tract within 2 h. Finally, the rectal administration of insulin-loaded hydrogels, containing 4%, 7%, or 10% (w/w) Eudispert and 5% (w/w) of enhancer (C12, C12Na, or DM-beta-CyD) to normal rats was shown to decrease serum glucose concentrations. The greatest effect was found with insulin-loaded 7% (Eudispert) hydrogel containing C12Na which having cosiderable large insulin release rate and bioadhesive characteristics.

The rectal delivery of insulin using a bioadhesive hydrogel preparation containing various absorption enhancers was evaluated in rats. Hydrogels were prepared from acrylic block copolymers of methacrylic acid and methacrylate (Eudispert hv). Sodium salts of various fatty acids were incorporated into the hydrogels as absorption enhancers. Sodium oleic acid and 2,6-di-O-methyl-β-cyclo-dextrin (DM-β-CyD) were used as control enhancers. Rectal administration of 50 units kg−1 insulin-loaded 7% (w/w) Eudispert hydrogels containing 5% (w/w) sodium lauric acid (C12) caused a significant (P < 0.01) decrease in plasma glucose levels compared with gels containing 5% DM-β-CyD or sodium oleic acid (43% vs 26 and 17%, respectively), in normal rats. The optimized gel formulation containing C12 was administered to non-insulin-dependent diabetes mellitus (NIDDM) rats. At a dose of 10 units kg−1 insulin, the decrease in plasma glucose levels was significantly (P < 0.05) greater in NIDDM rats compared with normal rats (35 vs 22%, respectively). Bioavailability was approximately 18% in both NIDDM and normal rats. As an index of local toxicity, the amount of protein released from the rectal membrane after administration of hydrogels was measured in normal rats. The amount of protein released for hydrogels containing C12 was significantly (P < 0.05) lower than that for C12 solution. Eudispert hydrogels containing lauric acid may be useful rectal preparations for the delivery of insulin.

The objective of this study was to prepare and evaluate the possibility of cylindrical xerogel preparations with zero-order release characteristics. As model polymers, polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC), and acrylic block copolymers of methacrylic acid and the methacrylate, Eudispert, were selected and formed into xerogel formulations. Tegafur, 5-fluorouracil (5-FU), aspirin, benzoic acid, p-methoxybenzoic acid, theophylline, and salicylamide were employed as model compounds and thereby incorporated into xerogel matrices.In a dissolution test (rotating basket method; pH 7.4), PVA xerogel did not erode nor swell in dissolution medium, and did not exhibit zero-order release, but rather exhibited Fickian's law diffusion followed by the initial burst release profile. In the case of HPMC xerogel, swelling of the polymer was observed to some extent, but the release profile was almost the same as PVA, suggesting the Fickian diffusion of drug from HPMC gel; in contrast, Eudispert gel showed zero-order release in every drug. The polymer was also gradually dissolved into the medium at a zero order release rate.Finally, the Eudispert xerogel containing theophylline was orally administered to beagle dogs, and plasma was withdrawn periodically. The sustained-release characteristics were observed and the possibility of the cylindrical xerogel preparation for oral usage was demonstrated.

The hypoglycemic effect and the alpha-glucosidase activity inhibition of acarbose (AC:alpha-glucosidase inhibitor) were investigated in normal and KK-Ay mice, an animal model of noninsulin-dependent diabetes mellitus (NIDDM). AC improved hyperglycemia after an oral administration of maltose or sucrose, dose dependently in normal mice (1, 10, and 50mg/kg body weight) and in KK-Ay mice (50mg/kg). Furthermore, AC (50mg/kg) significantly inhibited maltase and sucrase activities in the small intestines of normal and KK-Ay mice (inhibitory efficacy: sucrasemaltase). The enzymatic inhibition in KK-Ay mice is stronger than in normal mice. However, AC (50 mg/kg) did not suppress the blood glucose in oral lactose tolerance and did not inhibit the lactase activity in either normal or KK-Ay mice. These findings indicate that the AC effect on the inhibition of alpha-glucosidase activity is selective for sucrase and maltase in normal and NIDDM mice.

This chapter describes the structural chemistry and the biological aspects of carboxypeptidase G 3 . Carboxypeptidase G 3 consists of four subunits of identical molecular mass of 15 kDa. The pI is estimated to be 7.2. The enzyme is suspected to be a zinc-dependent metallopeptidase. The amino acid sequence and gene for carboxypeptidase G 3 are not known yet. The enzyme exists on the cell membrane of Pseudomonas sp. bacteria and seems to be a constitutive enzyme. The enzyme shows in vitro antitumor activity. Carboxypeptidase G 3 very slightly inhibits the growth of murine leukemia, but shows remarkable antitumor activities against human carcinoma KB and PC-3 cells among ten kinds of human carcinoma cell lines. Its antitumor activity mechanism seems to be based on the degradation of folic acid. Unlike carboxypeptidases G, G 1 and G 2 , the carboxypeptidase G 3 acts not only on the L-form of acidic amino acids, but also on the D-form and slightly hydrolyzes Bz-DL-Glu. However, some properties such as C-terminal glutamate-releasing activity and the requirement for Zn 2+ are quite similar to those of the other enzymes.

Determination of volatile components in essential oils from Atractylodis plants was studied by headspace gas chromatography. The crude drug of 0.20 g with 1.0 ml of water in a capped vial was heated at 130 degrees C for 45 min. Then 0.5 ml of vaporized components were collected by gas tightsillinge, and were applied into the injection port of GC or GC/MS. Consequently we could analyze and confirm the following components; hinesol and beta-eudesmol were found to be contained in Atractylodis Lanceae Rhizoma and atractylon in Atractylodis Rhizoma. beta-Eudesmol was analyzed by headspace gas chromatography, and the obtained calibration curve showed good linearity over the range from 2.5 micrograms to 10.0 mg. The result agreed with those obtained using numerical analyses by the steam distillation method. Atractylodis was found to have a wide variety of components depending on the available sources and on the stored conditions. This method was, therefore, more rapid and simpler determination of essential oils in crude drugs using headspace gas chromatography than those used previously. The method was useful means of the analyses of components in crude drugs such as Atractylodis plants and quality control.

A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60, 000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (Mr : 15, 000). The enzyme hydrolyzed predominately acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles resembles carboxypeptidase G1 or G2 in its glutamate-releasing activity. However, it acts not noly on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G1 or G2, it was named carboxypeptidase G3.

Internal-surface reversed-phase (ISRP) silica supports having N-octanoylaminopropyl phases bound to the internal surfaces of the porous silica and N-(2,3-dihydroxypropyl)aminopropyl phases bound to the external surfaces were synthesized from silica particles differing in nominal pore diameters and specific surface areas. These ISRP supports were characterized with regard to physical and chromatographic properties. The support with an N-octanoylaminopropyl phase coverage of 485 mumol/g and an average pore diameter of 65 A was the most suitable for the direct-injection determination of hydrophilic or hydrophobic drugs in serum or plasma. Non-steroidal anti-inflammatory (acetylsalicylic acid and salicylic acid) and tricyclic antidepressant drugs (desipramine and nortriptyline) in serum were successfully determined with this support and an acidic eluent.

1. In the present study, we evaluated fibrate-mediated potentiation of statin-induced apoptosis in IM-9 human lymphoblasts. 2. The pro-apoptotic effects of statin and fibrate were measured by flow cytometry with biotin-annexin V, followed by addition of avidin-fluorescein isothiocyanate and propidium iodide. Apoptosis was confirmed using karyopyknotic staining, as well as detection of DNA fragmentation and caspase 3 activation. 3. Incubation of IM-9 cells with both 0.1 micromol/L cerivastatin and 200 micromol/L clofibrate had a synergistic effect compared with 0.1 micromol/L cerivastatin alone or 200 micromol/L clofibrate alone. The magnitude of apoptosis induced by various combinations of statins and clofibrate were as follows: cerivastatin (0.1 micromol/L) + clofibrate (200 micromol/L) > atorvastatin (0.1 micromol/L) + clofibrate (200 micromol/L) > pravastatin (100 micromol/L) + clofibrate (200 micromol/L). Other fibrates (bezafibrate and clinofibrate) did not show any synergistic effect. Furthermore, karyopyknotic staining, caspase 3 activation and DNA fragmentation demonstrated synergistic pro-apoptotic effects of statin and fibrate. 4. The results of the present study suggest that simultaneous treatment with statins and clofibrate could provide improved therapeutic efficacy in leukaemia patients.

The purpose of this study was to evaluate the apoptosis and necrosis induced by five kinds of statins in IM-9 human lymphoblasts with fluorescence-enhanced flow cytometry using avidin–biotin complex. IM-9 human lymphoblasts (2 × 10 4 cells/cm 2 ) were seeded into tissue culture plates and incubated with five kinds of statins. Statin-treated cells were first incubated with biotin-annexin V, followed by addition of avidin-FITC and propidium iodide, and then subjected to flow cytometry. The fluorescence intensity was enhanced using an avidin–biotin complex system, resulting in successful separate determination of the statin-induced apoptosis and necrosis by flow cytometry, which enabled us to quantitatively evaluate the statin-induced cell damage. Flow cytometric analysis results in the intensity of statin-induced apoptosis in IM-9 cells as follows: atorvastatin ≒ cerivastatin > fluvastatin ≒ simvastatin > pravastatin. The intensity of statin-induced necrosis in IM-9 cells was expressed as follows: atorvastatin ≒ cerivastatin > fluvastatin ≒ simvastatin > pravastatin. The total damage of IM-9 cells induced by five kinds of statins were expressed as the sum of both percentages of apoptosis and necrosis as follows: atorvastatin ≒ cerivastatin > fluvastatin ≒ simvastatin > pravastatin. Our studies show that fluorescence enhancement with avidin–biotin complex is useful for the identification and quantitation of annexin-positive apoptosis cells and thus, the fluorescence-enhanced flow cytometry was shown to be applicable for screening of statins as new anti-leukemia agents.

Publisher Summary This chapter describes the structural chemistry and the biological aspects of carboxypeptidase G3. Carboxypeptidase G3 consists of four subunits of identical molecular mass of 15 kDa. The pI is estimated to be 7.2. The enzyme is suspected to be a zinc-dependent metallopeptidase. The amino acid sequence and gene for carboxypeptidase G3 are not known yet. The enzyme exists on the cell membrane of Pseudomonas sp. bacteria and seems to be a constitutive enzyme. The enzyme shows in vitro antitumor activity. Carboxypeptidase G3 very slightly inhibits the growth of murine leukemia, but shows remarkable antitumor activities against human carcinoma KB and PC-3 cells among ten kinds of human carcinoma cell lines. Its antitumor activity mechanism seems to be based on the degradation of folic acid. Unlike carboxypeptidases G, G1 and G2, the carboxypeptidase G3 acts not only on the L-form of acidic amino acids, but also on the D-form and slightly hydrolyzes Bz-DL-Glu. However, some properties such as C-terminal glutamate-releasing activity and the requirement for Zn2+ are quite similar to those of the other enzymes.

To study the effect of instillation of latanoprost on the diurnal variation in the intraocular pressure(IOP), blood pressure, and pulse rate in patients with normal-tension glaucoma(NTG).The diurnal variation in the IOP was determined in 23 eyes in 23 NTG patients after a washout period of 4 weeks or longer. The diurnal variation in IOP was then remeasured after latanoprost monotherapy of 8 weeks or longer. The IOP was measured by the Goldmann applanation tonometer at 10:00, 13:00, 16:00, 19:00, 22:00, 01:00, 03:00, 07:00, and 10:00 before and after treatment, and the IOP at each time point, mean diurnal IOP, maximum IOP, minimum IOP, and range of variation in IOP were compared before and after treatment. The blood pressure and pulse rate before and after treatment were also measured and compared.The IOP decreased significantly at all time points. The treatment caused significant decreases in the mean diurnal IOP, maximum IOP, minimum IOP, and range of variation in IOP. There were no differences in the blood pressure and pulse rate before and after treatment.Latanoprost significantly decreases the IOP throughout the day in NTG patients, and has no effects on the blood pressure and pulse rate. It is therefore useful in the treatment of NTG.

Antimicrobial activities of Rokitamycin (RKM) in the urine were decreased extremely after the samples were stored at -20°C for a long time. Therefore, a reaction of macrolides with urine was studied under the condition of freezing at -20°C. 1. Each of RKM, Midecamycin acetate (MOM), Josamycin (JM) or other macrolides was added to human urine and stored at -20°C. Their antimicrobial activities in human urine were decreased to 12%-45% for 1 week and to 3%-22% for 8 weeks. 2. RKM in human urine was analyzed by thin-layer chromatography after storage at -20°C. It had been found that the amount of RKM was decreased and a new compound, which was more hydrophilic than RKM, was generated according to the decrease of its activity. 3. Each of RKM, MOM, JM or other macrolides was added to a 1.4 % (w/v) aqueous urea solution and stored at -20°C. Their antimicrobial activities in the urea solution were decreased in the same way as in human urine. 4. By the thin-layer chromatography analysis, it had been found that the amount of each macrolides was decreased and new compounds were generated. 5. From the Mass spectra and NMR spectra data, these new compounds were determined as macrolide-18-bis-urea adducts. 6. The bis-urea adducts were substantially hydrolyzed and converted into parent compounds after the heat treatment at 121°C for 15 minutes in the pH 4.5 solution. 7. Antimicrobial activities in the urine samples, which were collected from humans after oral administration of RKM and stored at -20°C for 8 weeks, were also recovered approximately 100% of the initial activities by the heat treatment.

A new internal-surface reversed-phase (ISRP) silica support has been designed for direct injection analysis of drugs in biological fluids by liquid chromatography. The support, prepared by using a new enzyme, polymyxin acylase, has N-octanoylaminopropyl phases, bound only to the internal surfaces of the porous silica, and N-(2,3-dihydroxypropyl)-aminopropyl phases on the external surfaces in order to be nonadsorptive to proteins. The average pore diameter of the prepared ISRP silica support was 50 A, which is small enough to exclude macromolecules such as serum proteins from the pores. The new ISRP support can be used for the direct injection analysis of hydrophilic and hydrophobic drugs in serum or plasma without destructive accumulation of proteins over the eluent pH range of 3 to 7. The recovery of drugs from serum was almost 100%, regardless of the difference in their protein bindings.

An enzyme useful for preparing semisynthetic polymyxins was found to be produced by a Pseudomonas sp. strain, M-6-3 (closely related to Ps. acidovorans). This enzyme, tentatively named polymyxin acylase, is the first enzyme known which can remove the acyl moiety from an acyl peptide without affecting the peptide moiety. It can be produced in a cell-bound form in excellent yield in a medium containing citric acid as the sole source of carbon. The activity of the acetone-dried cell powder of the bacterium was optimum at pH 7.5 and remained stable up to about 50°C for 30 min. The apparent Vmax and Km values for colistin (polymyxin E) were 8.7 nmol/min/mg and 2.8 mm, respectively. The suspension of the cell-bound enzyme in colistin solution gave a good yield of deacyl colistin and fatty acid(s), and no chemical change occurred in the peptide moiety during the enzyme reaction. This enzyme deacylated not only polymyxins B, M, and P, but also several other fatty acyl peptides. It should be valuable as a tool for...

Acetoacetate concentration in plasma was determined by head-space gas chromatography after decarboxylation by the use of acetoacetate decarboxylase, which was recently isolated from Bacillus polymyxa A-57 strain. Partial purification of acetoacetate decarboxylase solution was carried out by DEAE-cellulose column chromatography of the cell-free extract, which was prepared by ultrasonication. The properties of the enzyme were found to be particularly suitable for acetoacetate assay (optimum pH, 5.8 ; Vmax, 230μmol/min/mg ; Km, 5.9×10-4M). Plasma was deproteinized by Somogyi's method. Acetone in the mildly basic supernatant was assayed by head-space gas chromatography. Acetoacetate was calculated by subtracting blank acetone from total acetone after enzymatic decarboxylation of acetoacetate. The minimal detectable concentration was 1μM. This method gave better reproducibility (CV=2.0-8.0%) and recovery (96.0-101.8%) than chemical decarboxylation with perchloric acid. Normal subjects (n=31) showed plasma acetone levels of 7.2±3.4μM and acetoacetate levels of 22.5±9.7μM. Diabetic patients (n=44), treated by diet control alone without drug therapy, gave plasma acetone levels of 8.1±3.5μM and acetoacetate levels of 25.0±8.0μM. There was no significant difference between the two groups.

Acetoacetate decarboxylase is a valuable tool for clinical analysis of ketone bodies in human plasma. After screening many microorganisms, we found Bacillus polymyxa A-57 to be a new enzyme source with a good yield of acetoacetate decarboxylase. After purifying the intracellular enzyme by three-stage column chromatography, we identified it as a single protein by SDS-polyacrylamide gel electrophoresis. The enzyme had a molecular weight of approximately 280, 000 and consisted of 10 (±2) identical subunits. It had an optimum pH of 5.9 and was stable up to about 60°C for 30min. The apparent Km and Vmax values for lithium acetoacetate were 0.94mM and 296μmol/min/mg, respectively. In addition, the decarboxylase activity was found in the broth. After purification, we found that it was due to an active peptide which we named A-57-9, which we identified as the antibiotic polymyxin M1. However, the Vmax value (0.25μmol/min/mg) of the peptide was very much lower and the Km value (400 mM) was higher than those of real acetoacetate decarboxylase.

Polymyxin acylase from Pseudomonas sp. M-6-3 can deacylate not only polymyxin antibiotics, but also A-fatty acyl-peptides and N-fatty acyl-amino acids. We found that this enzyme causes intramolecular N2⇄N6 acyl transfer in monooctanoyl-l-lysine; when N2-octanoyl-l-lysine is the substrate, N6-octanoyl- l-lysine is produced at pH 10.5, but when N6-octanoyl- l-lysine is the substrate, N2-octanoyl- l-lysine is produced at pH 8.0. In these reactions, the deacylation proceeded gradually at the final stage and eventually, both N2-octanoyl- l-lysine and N6-octanoyl- l-lysine were hydrolyzed to l-lysine and octanoic acid. Furthermore, this enzyme showed intermolecular acyltrans- ferase activity, transferring several N-octanoyl- dl-amino acids to N-octanoyl-hydroxylamine. This acyltransfer ability of polymyxin acylase offers a new method of enzymic N-acylation of compounds containing amino components.

Polymyxin acylase, which produces deacylated polymyxins by hydrolyzing only the fatty acyl groups of polymyxin antibiotics without affecting the peptide moiety, was purified from acetone-dried cell powder of Pseudomonas sp. M-6–3. The cell-free enzyme, solubilized by Triton X-100, was further purified on successive DEAE-cellulose, hydroxyapatite, and Sephacryl S-300 columns to a homogeneous state. This purified enzyme (Type I) had a single band with an MW of 62,000 in SDS- polyacrylamide gel electrophoresis. Gel filtration on a calibrated Sephacryl S-300 column also gave an estimated MW of 62,000. The isoelectric point of the enzyme was 5.7. Enzyme activity was optimal at pH 9.0 for colistin A (polymyxin E^ and was stable up to 50°C for 24 hr. The observed Vmax and Km values were 1750nmol/min/mg and 3.85 mm, respectively. This enzyme was almost not affected by metal chelators and various thiol-enzyme inhibitors (other than /7-chloromercuribenzoate), and showed high tolerance for organic modifiers; for exa...