A phase I study of pegylated liposomal doxorubicin hydrochloride (Caelyx) in combination with cyclophosphamide and vincristine as second-line treatment of patients with small-cell lung cancer.

The purpose of this study was to determine the recommended phase II dose of liposomal doxorubicin (Caelyx ; Doxil in the United States) in combination with cyclophosphamide and vincristine for previously treated patients with good performance status with relapsed or refractory small-cell lung cancer. Twenty-one eligible patients were enrolled between November 1999 and September 2001 and received liposomal doxorubicin 25-40 mg/m2, cyclophosphamide 750-1000 mg/m2, and vincristine 1.2 mg/m2 intravenously (I.V.) every 21 days. At doses of liposomal doxorubicin 40 mg/m2, cyclophosphamide 750 mg/m2, and vincristine 1.2 mg/m2 I.V., 1 of 6 patients had dose-limiting neutropenia and fever in cycle 2 and 2 of 6 developed grade 3 hand-foot syndrome during cycle 3. Therefore, the recommended phase II doses are liposomal doxorubicin 35 mg/m2, cyclophosphamide 750 mg/m2, and vincristine 1.2 mg/m2 I.V. every 21 days. Antitumor activity was seen at all dose levels. This combination is well tolerated and has evidence of antitumor activity. A phase II evaluation is ongoing.

Aromatic dental monomers affect the activity of cholesterol esterase.

The dental restorative monomer, BISGMA (2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane), and bisphenol A diglycidyl ether (BADGE) increase the velocity of the reaction catalyzed by pancreatic cholesterol esterase (CEase, bovine). The metabolite of these monomers, bisphenol A bis(2,3-dihydroxypropyl) ether, and a common plasticizer, di-2-ethylhexyl phthalate (DEHP), also increase the velocity of CEase-catalyzed ester hydrolysis. BISGMA at concentrations of 1.5-8.0 microM increases the velocity to 126-169% of its value in the absence of BISGMA. Increasing BISGMA above 8 microM caused no further increase in velocity. BADGE at 7-25 microM increases the velocity to 112-205% of its value without BADGE. The metabolite of BISGMA and BADGE at concentrations of 2.0-7.1 microM increases the velocity to 103-113% of its value without metabolite. DEHP at concentrations of 0.52-4.3 microM increases the velocity to 108-187% of its value without DEHP. On the other hand, bisphenol A dimethacrylate is a competitive inhibitor of CEase, with a K(i) of 3.1 microM.

Molecular dynamics simulation of n-dodecyl phosphate aggregate structures.

Aggregates of n-dodecyl phosphate present an attractive model system of simple phospholipid amphiphile supramolecular structures for study by molecular dynamics simulation, since these systems have previously been studied experimentally under various conditions. A detailed molecular dynamics description of the properties of planar bilayer membranes (as a model for unilamellar vesicular membranes) and spherical micelles under various simulated conditions is presented. It is shown that the united-atom model of GROMOS96 applying the force-field parameter set 43A2 for biomolecular systems yields properties in agreement with experimental ones in most cases. Hydrogen bonding plays a role in stabilizing the bilayer aggregates at low pH, but not for the micelles, which are energetically favoured at high pH. NMR -S(CD) order parameters for a lipid bilayer system, the diffusion of amphiphiles within aggregates and of counterions, and lifetimes of hydrogen bonds between amphiphiles and to water are estimated from the MD simulations.

Enzymes inside lipid vesicles: preparation, reactivity and applications.

There are a number of methods that can be used for the preparation of enzyme-containing lipid vesicles (liposomes) which are lipid dispersions that contain water-soluble enzymes in the trapped aqueous space. This has been shown by many investigations carried out with a variety of enzymes. A review of these studies is given and some of the main results are summarized. With respect to the vesicle-forming amphiphiles used, most preparations are based on phosphatidylcholine, either the natural mixtures obtained from soybean or egg yolk, or chemically defined compounds, such as DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) or POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine). Charged enzyme-containing lipid vesicles are often prepared by adding a certain amount of a negatively charged amphiphile (typically dicetylphosphate) or a positively charged lipid (usually stearylamine). The presence of charges in the vesicle membrane may lead to an adsorption of the enzyme onto the interior or exterior site of the vesicle bilayers. If (i) the high enzyme encapsulation efficiencies; (ii) avoidance of the use of organic solvents during the entrapment procedure; (iii) relatively monodisperse spherical vesicles of about 100 nm diameter; and (iv) a high degree of unilamellarity are required, then the use of the so-called 'dehydration-rehydration method', followed by the 'extrusion technique' has shown to be superior over other procedures. In addition to many investigations in the field of cheese production--there are several studies on the (potential) medical and biomedical applications of enzyme-containing lipid vesicles (e.g. in the enzyme-replacement therapy or for immunoassays)--including a few in vivo studies. In many cases, the enzyme molecules are expected to be released from the vesicles at the target site, and the vesicles in these cases serve as the carrier system. For (potential) medical applications as enzyme carriers in the blood circulation, the preparation of sterically stabilized lipid vesicles has proven to be advantageous. Regarding the use of enzyme-containing vesicles as submicrometer-sized nanoreactors, substrates are added to the bulk phase. Upon permeation across the vesicle bilayer(s), the trapped enzymes inside the vesicles catalyze the conversion of the substrate molecules into products. Using physical (e.g. microwave irradiation) or chemical methods (e.g. addition of micelle-forming amphiphiles at sublytic concentration), the bilayer permeability can be controlled to a certain extent. A detailed molecular understanding of these (usually) submicrometer-sized bioreactor systems is still not there. There are only a few approaches towards a deeper understanding and modeling of the catalytic activity of the entrapped enzyme molecules upon externally added substrates. Using micrometer-sized vesicles (so-called 'giant vesicles') as simple models for the lipidic matrix of biological cells, enzyme molecules can be microinjected inside individual target vesicles, and the corresponding enzymatic reaction can be monitored by fluorescence microscopy using appropriate fluorogenic substrate molecules.

Conformationally changed cytochrome c-mediated fusion of enzyme- and substrate-containing liposomes.

The fusion between enzyme-containing liposomes and substrate-containing liposomes was studied, utilizing conformationally altered cytochrome c as fusion mediator under stress conditions. The liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and liposome aggregation and subsequent liposome fusion were induced by the addition of cytochrome c, which was partially denatured by 0.5 M guanidinium hydrochloride (GuHCl). In the presence of 0.5 M GuHCl, cytochrome c was found to have a significantly large local hydrophobicity which was determined with the aqueous two-phase partitioning method. Under these conditions, cytochrome c could efficiently bind to POPC bilayer membranes as quantitatively evaluated by immobilized liposome chromatography (ILC). The retardation of cytochrome c treated with 0, 0.5, and 1 M GuHCl on ILC could be correlated with the corresponding local hydrophobicity of cytochrome c. The enzymatic reaction triggered by liposome fusion involved the proteolytic enzyme alpha-chymotrypsin and its substrate succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-pNA), which were separately trapped in POPC liposomes. Addition of partially denatured cytochrome c (most likely in the molten globule state) to the mixture of enzyme- and substrate-containing liposomes resulted in the release of one of the hydrolysis products, p-nitroaniline, to the outer phase of the fused liposomes, indicating that the enzymatic reaction occurred during the liposome fusion process. Such a coupled fusion-reaction system may have specific advantages over the conventional fusion analysis and may find application as drug delivery system.

Modeling of enzymatic reactions in vesicles: the case of alpha-chymotrypsin.

The kinetic behavior of the alpha-chymotrypsin-catalyzed hydrolysis of the two p-nitroanilide substrates succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-Ala-Ala-Pro-Phe-pNA) and benzoyl-L-Tyr-p-nitroanilide (Bz-Tyr-pNA) was modeled and simulated for two different systems, namely for an aqueous solution and for a vesicle system, which was composed of phospholipid vesicles containing entrapped alpha-chymotrypsin. In the case of the vesicles, the substrate was added to the bulk, exovesicular aqueous phase. The experimentally determined time-dependence of product (p-nitroaniline) formation was modeled by considering the kinetic behavior of the enzyme and-in the case of vesicles-the substrate permeability across the bilayer membrane. In aqueous solution-without vesicles-the kinetic constants kcat and KS (respectively KM) were determined from fitting the model to experimental data of batch product concentration-time curves. The results were in good agreement with the corresponding values obtained from initial velocity measurements. For the vesicle system, using the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), simulation showed that the substrate permeation across the bilayer was rate limiting. Using experimental data, we could obtain the substrate permeability coefficient for Bz-Tyr-pNA by parametric fitting as 2. 45 x 10(-7) cm/s.

Bilayer permeability-based substrate selectivity of an enzyme in liposomes.

Liposomes were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which contained the water soluble proteinase alpha-chymotrypsin. This liposome entrapped enzyme showed selectivity for externally added substrates in that only small substrates (benzoyl-l-Tyr-p-nitroanilide or acetyl-l-Phe-p-nitro-anilide)-for which the liposome bilayer was permeable-were transformed into products. Large substrates (succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide or casein) could not penetrate from the external aqueous phase into the liposomes, and were not hydrolyzed. This substrate selectivity is entirely based on the compartimentation and permeability properties of the liposome microreactor.

A phase I study of gemcitabine/cisplatin/etoposide in the treatment of small-cell lung cancer.

PURPOSE: To define the maximum tolerated dose and toxicity of combined cisplatin, etoposide, and gemcitabine in patients with small-cell lung cancer. METHODS: We undertook a phase I study in patients with either extensive small-cell lung cancer with or without prior chemotherapy, or limited disease who had progressed or recurred despite prior treatment. Patients received cisplatin 75 mg/m2 i.v. day 1, etoposide 50-100 mg/m2 i.v. day 1 followed by oral administration of 50-100 mg/m2 days 2 5, and gemcitabine at either 800 or 1000 mg/m2 i.v. days 1 and 8, on a 3 weekly cycle. RESULTS: We treated 20 patients, 14 at the 800 mg/m2 gemcitabine dose level, and six at the 1000 mg/m2 dose level. The protocol initially used an etoposide dose of 100 mg/m2 etoposide daily (i.v. day 1 and orally days 2-5), but the first two patients died of septic complications. With reduction of the etoposide dose to 50 mg/m2 daily x 5, the regimen was well tolerated. At this etoposide dose, neutropenia, mucositis, and gastrointestinal toxicity occurred in one patient at each of the two gemcitabine dose levels. In addition, one patient receiving gemcitabine at the 1000 mg/m2 level experienced a possible allergic reaction. The overall response rate was 54%. Patients on gemcitabine at the 800 mg/m2 level who had not received prior chemotherapy had the highest response rate, at 75%. CONCLUSION: The recommended phase II doses for this regimen are cisplatin 75 mg/m2 i.v. day 1, etoposide 50 mg/m2 i.v. day 1 and orally days 2-5, and gemcitabine 800 mg/m2 i.v. days 1 and 8. Future trials should further examine the optimal relative doses and schedule of gemcitabine and etoposide.

Randomized trial of cyclophosphamide, methotrexate, and fluorouracil chemotherapy added to tamoxifen as adjuvant therapy in postmenopausal women with node-positive estrogen and/or progesterone receptor-positive breast cancer: a report of the National Cancer Institute of Canada Clinical Trials Group. Breast Cancer Site Group.

PURPOSE AND METHODS: By the mid 1980s, tamoxifen alone was considered standard adjuvant therapy for postmenopausal women with node-positive, estrogen receptor (ER)- or progesterone receptor (PgR)-positive breast cancer. From 1984 through 1990, 705 eligible postmenopausal women with node-positive, ER- or PgR-positive breast cancer were randomized to a National Cancer Institute of Canada Clinical Trials Group (NCIC CTG) study that compared tamoxifen 30 mg by mouth daily for 2 years (TAM) versus TAM plus chemotherapy with all-intravenous cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600 mg/m2 given every 21 days for eight cycles (CMF). RESULTS: There were no significant differences in overall survival, recurrence-free survival, locoregional recurrence-free survival, or distant recurrence-free survival between the two treatment arms. However, there was significantly greater severe toxicity, which included leukopenia (P < .0001), nausea and vomiting (P < .0001), and thromboembolic events (P < .0001), as well as significantly more mild or greater toxicity, which included thrombocytopenia (P = .04), anemia (P = .02), infection (P = .0004), mucositis (P = .0001), diarrhea (P = .0001), and neurologic toxicity (P = .006), in women who received TAM plus CMF. CONCLUSION: The addition of CMF to TAM adds no benefit and considerable toxicity in this group of women.

A 1H nuclear magnetic resonance method for investigating the phospholipase D-catalyzed hydrolysis of phosphatidylcholine in liposomes.

Liposomes with mean diameters between 45 and 73 nm have been prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at pH 8.0; and a new methodology is described which allows one to quantitatively follow the phospholipase D-catalyzed transformation of POPC to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid and free choline. The method does not require a special sample preparation; it takes advantage of the fact that the chemical shift of the protons of the three methyl groups in free choline differs from the chemical shift of the choline methyl protons in POPC. Measurements have been carried out under different experimental configurations and they have been paralleled by electron and light microscopy studies, partially using a fluorescently labeled phospholipid. It has been found that for a fixed concentration of the Ca2+-independent phospholipase D from Streptomyces sp. AA 586 the initial velocity and the reaction yields depend on the size of the vesicles. The smaller the vesicles, the higher the yields and the lower the initial rates. Furthermore, the size of the liposomes does not change during hydrolysis of the external POPC layer.

Microinjection into giant vesicles and light microscopy investigation of enzyme-mediated vesicle transformations.

BACKGROUND: 'Giant vesicles' have diameters of several micrometers and can be observed by light microscopy. Their size may allow manipulation of individual vesicles and direct observation of the progress of a chemical reaction in real time. We set out to test this possibility using enzymatic hydrolysis of vesicle components as a model system. RESULTS: We describe a novel micromanipulation technique that allows us to microinject femtoliter amounts of a reagent solution adjacent to or into giant vesicles with diameters ranging from 10 to 60 microm. The vesicle transformations can be monitored directly in real time by light microscopy and recorded by video analysis. Snake venom phospholipase A2 was added to vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, and the enzymatic hydrolysis of components of the lipid bilayer was observed over time. A specific effect on the targeted giant vesicle was seen and video recorded, while the neighbouring vesicles remained unaffected. Addition of the enzyme to the outside of a vesicle caused it to burst, whereas injection of the enzyme inside a vesicle resulted in a slow and constant decrease in its size, until it eventually disappeared from the resolution power of the light microscope. CONCLUSIONS: These results show that it is possible to micromanipulate an individual vesicle, and to follow visually the progress of an enzymatic reaction occurring on the vesicle bilayer over time.

Human skin irritation studies of a lecithin microemulsion gel and of lecithin liposomes.

Soybean lecithin microemulsion gels offer promising features for the possible use as matrices in transdermal therapeutic systems. In order to assess the skin irritancy potential of the gel, acute and cumulative irriation tests were performed in human subjects in vivo using as comparison an unilamellar soybean lecithin liposome preparation and the solvent isopropyl palmitate (IPP). Acute irritation was tested in 151 volunteers in a 48-hour patch test, whereas cumulative irritation was assessed in a 21-day human repeated insult patch test in 20 volunteers. In the acute irritation test, discrete irritation (erythema only) developed with the gel in 2 subjects (1.3%), with the liposomes in 3 subjects (2.0%), and with IPP in 2 subjects (1.3%). For the assessment of cumulative irritation, the IT50 (irritation time of 50% of the test population) was calculated. IT50 was 13 days for the gel, 14 days for the liposomes and 17 days for IPP. This study shows a very low acute and a low cumulative irritancy potential for the soybean lecithin microemulsion gel making it a candidate matrix for transdermal therapeutic systems also under toxicological aspects.

Catalytic activity of elastase in reverse micelles.

The activity of porcine pancreatic elastase has been studied in reverse micelles formed by AOT (sodium bis(2-ethylhexyl) sulfosuccinate) in isooctane. For the two substrates succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide and succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, the catalytic constant, kcat, in reverse micelles increases with increasing wo until, at high wo, the value of kcat measured in bulk buffer solution is approached (wo = [H2O/]AOT]). In analogy to alpha-chymotrypsin--and in apparent contrast to many other enzymes--elastase does not show a maximum in the kcat-wo profile. Within the wo range of 8 to 35, for both substrates, the Michaelis constant Km (as expressed relative to the total volume of the solution, Km,overall) increases with increasing wo.

Activity and spectroscopic properties of bovine liver catalase in sodium bis(2-ethylhexyl)sulfosuccinate/isooctane reverse micelles.

Spectroscopic properties such as ultraviolet-visible spectroscopy, circular dichroism, steady-state fluorescence and the catalytic activity of bovine liver catalase (BLC) have been studied in reverse-micelles microemulsions formed by sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. Activity measurements were carried out with the highly water soluble hydrogen peroxide as substrate as a function of temperature and pH, at various concentrations of substrate, enzyme, AOT and at different w0 values (w0 = [H2O]/[AOT]). The folding of catalase in reverse micelles is not drastically altered, although there are changes in the local surrounding of the prosthetic group. Kinetic measurements have been carried out under first-order conditions at low substrate concentration. They indicate that the measured substrate decomposition rate is limited by the exchange of substrate molecules between the substrate-filled and the enzyme-containing micelles. The specificity constant, ks = kcat/KM, as related to water pool concentrations, k(s,wp)rm, was, at all w0 values, considerably lower than ks measured in buffer (ksb). In contrast, overall values of ks in reverse micelles, k(s,ov)rm, were always 2-4-times higher than ksb. This apparent superactivity can, however, be ascribed to concentration effects. No 'bell-curve' to describe the w0 dependence of catalase activity in reverse micelles was found, k or ks increasing monotonously up to w0 = 50.

Spectroscopic and kinetic studies of lipases solubilized in reverse micelles.

The conformation and activity of three different lipases have been studied in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. In the case of human pancreatic lipase, the conformation of the polypeptide chain--as judged from far-UV circular dichroism measurements--is only slightly altered after the enzyme is transferred from a bulk aqueous solution into the microenvironment of reverse micelles. Significant spectral changes in the near-UV circular dichroism and fluorescence spectrum indicate, however, that the solvation of aromatic amino acid side chains is considerably different in reverse micelles. Conversely, the circular dichroism spectra of the lipases from Candida rugosa and Pseudomonas sp. are considerably different in reverse micelles, compared with the spectra in aqueous solution, indicating that both enzymes loose the native structure at the water/AOT/oil interface. Bound substrate and/or product can prevent this denaturation. While Pseudomonas sp. and human pancreatic lipase are inhibited by tetrahydrolipstatin (THL), the lipase from Candida rugosa is not. These data, together with additional activity and inhibition data, indicate that the micellar microenvironment accentuates the difference between the different enzymes in terms of the relation structure/activity.

Lecithin organogel as matrix for transdermal transport of drugs.

Organogels obtained by adding small amounts of water to a solution of lecithin in organic solvents were studied as matrices for the transdermal transport of drugs. Gels obtained from isopropyl palmitate and cyclooctane were used (molar ratios of water to lecithin of 3 and 12, respectively). Preliminarily histological studies showed that the gels have no harmful effect when applied to the skin for prolonged periods. Data relative to the stability of the organogels with time are also presented. Scopolamine and broxaterol were used as model drugs, and the transdermal experiments were done with a Franz diffusion cell and human skin obtained from plastic surgery. The transport rate of scopolamine obtained with the lecithin gels was about one order of magnitude higher than that obtained with an aqueous solution of the drug at the same concentration. In contrast, the transport rates of scopolamine obtained with the microemulsion solution prior to gelation (molar ratio of water to lecithin, 0) were not different from those obtained with the gel. The same variations in transport rates were observed for broxaterol, in which case the flux through the skin was directly proportional to the concentration of drug in the gel. At a concentration of broxaterol of 75 mg/mL in the donor gel, the flux was 47 micrograms.h-1.cm-2. Because preliminary results showed that transdermal transport is successful with amino acids and peptides also, it is concluded that lecithin gels may be efficient vehicles for the transdermal transport of various drugs.

Kinetic behaviour of alpha-chymotrypsin in reverse micelles. A stopped-flow study.

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of alpha-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stopped-flow spectrophotometry for the hydrolysis of two substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester and p-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.

Phase I-II study of high-dose epirubicin in advanced non-small-cell lung cancer.

PURPOSE: A phase I multicenter trial was performed to determine the maximum-tolerated dose (MTD) of epirubicin, given on 3 consecutive days every 3 weeks to previously untreated patients with advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: After appropriate staging and a baseline multiple-gated angiogram (MUGA) scan, at least four patients were entered at each dose level, starting at 35 mg/m2 of epirubicin given intravenously (IV) daily for 3 days (105 mg/m2) and escalating by 5 mg/m2 per injection in each dose level (15 mg/m2 per course). Epirubicin was administered up to a maximum dose of 60 mg/m2/d for 3 days (180 mg/m2). The MTD was determined to be 55 mg/m2/d for 3 days (165 mg/m2) after treating a total of 35 (33 assessable) patients. Nadir granulocyte counts and associated febrile episodes comprised the dose-limiting toxicity, but there were no treatment-related deaths. A phase II trial was performed using a dose of 50 mg/m2/d for 3 days (150 mg/m2) every 3 weeks with no dose escalation, but with dose reduction for toxicity as required. A total of 30 patients were entered onto this phase of the study. RESULTS: The major toxicity, as in the phase I trial, was neutropenia with five febrile episodes, again with no treatment-related deaths. An overall response rate of 12 of 63 (19%) was noted in the combined patient population of the phase I-II trial, with 95% confidence intervals of 10% to 31%. When the response rate was analyzed by histology, only one of 17 (6%) patients with squamous histology, as compared with 11 of 46 (24%) with non-squamous histology, responded, but this did not reach statistical significance (P = .15). CONCLUSIONS: High-dose epirubicin is tolerable and is an active single agent in NSCLC. It should be combined with relatively nonmyelosuppressive agents such as cisplatin to try to obtain higher response rates and extend the survival in this disease.

Substrate effects on the enzymatic activity of alpha-chymotrypsin in reverse micelles.

Six different substrates have been used for measuring the activity of alpha-chymotrypsin in reverse micelles formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in isooctane. The substrates were glutaryl-Phe p-nitroanilide, succinyl-Phe p-nitroanilide, acetyl-Phe p-nitroanilide, succinyl-Ala-Ala-Phe p-nitroanilide, succinyl-Ala-Ala-Pro-Phe p-nitroanilide and acetyl-Trp methyl ester. It has been shown that the dependence of the kinetic constants (kcat and Km) on the water content of the system, on wo (= [H2O]/[AOT]), is different for the different substrates. This indicates that activity-wo profiles for alpha-chymotrypsin in reverse micelles not only reflect an intrinsic feature of the enzyme alone. For the p-nitroanilides it was found that the lower kcat (and the higher Km) in aqueous solution, the higher kcat as well as Km in reverse micelles. "Superactivity" of alpha-chymotrypsin could only be found with the ester substrate and with relatively "poor" p-nitroanilides. The presence of a negative charge in the substrate molecule is not a prerequisite for alpha-chymotrypsin to show "superactivity".

Product inhibition of alpha-chymotrypsin in reverse micelles.

The alpha-chymotrypsin-catalyzed hydrolysis of succinyl-L-alanyl-L-alanyl- L-prolyl-L-phenylalanyl p-nitroanilide has been studied in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in isooctane. It has been found that alpha-chymotrypsin is strongly inhibited competitively by the acidic peptide product which is formed during the course of the reaction. It has also been shown that the application of the integrated form of the Michaelis-Menten equation can be useful to detect possible inhibition effects and abnormal kinetic behavior of enzymes in reverse micelles. Furthermore, it has been shown that the turnover number (kcat) at low water content is lower than in water and increases as the water content in the system is lower than in water and increases as the water content in the system (wo = [H2O]/[AOT]) increases, kcat reaching the value in water at high wo. If however, initial velocity data, as obtained under conditions where the enzyme is not saturated with substrate, are plotted against wo, the curves are bell-shaped, with a maximum around wo = 15.