Effect of cadmium and lead on the membrane potential and photoelectric reaction of Nitellopsis obtusa cells

The effects of Cd and Pb on membrane potential (E(m)) and photoelectric reaction of Nitellopsis obtusa cells were investigated. It was found that Cd and Pb at 1.0 mM caused a depolarization of the E(m), whereas both metals at lower concentrations changed the E(m) in a different way. Pb at 0.1 mM and 0.01 mM hyperpolarized the E(m), whereas Cd at the same concentrations depolarized and did not change the E(m), respectively. In the presence of 0.01 mM Pb, the light-induced hyperpolarization of the E(m) was by 18% higher as compared to the control, whereas at 1.0 mM Pb it was by 40% lower. Pb at 0.1 mM and Cd at 0.01 mM or 5 × 0.01 mM did not change the light-induced membrane hyperpolarization. However, in the presence of Cd at 0.1 mM and 1.0 mM this hyperpolarization was 2-fold lower or was completely abolished, respectively. These results suggest that at high Cd and Pb concentrations both depolarization of the E(m) and decrease of light-induced membrane hyperpolarization in Nitellopsis obtusa cells are probably due to inhibition of the plasma membrane H(+)-ATPase activity, whereas both metals at lower concentrations differ in mechanism of membrane potential changes.     

Nickel and Ultraviolet-B Stresses Induce Differential Growth and Photosynthetic Responses in Pisum sativum L. Seedlings

Enhanced level of UV-B radiation and heavy metals in irrigated soils due to anthropogenic activities are deteriorating the environmental conditions necessary for growth and development of plants. The present study was undertaken to study the individual and interactive effects of heavy metal nickel (NiCl(2)·6H(2)O; 0.01, 0.1, 1.0?mM) and UV-B exposure (0.4 W m(-2); 45?min corresponds to 1.08 KJ m(-2)) on growth performance and photosynthetic activity of pea (Pisum sativum L.) seedlings. Ni treatment at high doses (0.1 and 1.0?mM Ni) and UV-B alone reduced chlorophyll content and photosynthetic activity (oxygen yield, carbon fixation, photorespiration, and PSI, PSII, and whole chain electron transport activities), and declining trends continued with combined doses. In contrast to this, Ni at 0.01?mM appeared to be stimulatory for photosynthetic pigments and photosynthetic activity, thereby enhanced biomass was observed at this concentration. However, combined dose (UV-B + 0.01?mM Ni) caused inhibitory effects. Carotenoids showed different responses to each stress. Nickel at high doses strongly inhibited PSII activity and the inhibition was further intensified when chloroplasts were simultaneously exposed to UV-B radiation. PSI activity appeared to be more resistant to each stress. High doses of Ni (0.1and 1.0?mM) and UV-B alone interrupted electron flow at the oxygen evolving complex. Similar damaging effects were caused by 0.01 and 0.1?mM Ni together with UV-B, but the damage extended to PSII reaction center in case of 1.0?mM Ni in combination with UV-B. In conclusion, the results demonstrate that low dose of Ni stimulated the growth performance of pea seedlings in contrast to its inhibitory role at high doses. However, UV-B alone and together with low as well as high doses of Ni proved to be toxic for P. sativum L.     

Effect of selected alcohol dehydrogenase inhibitors on the human heart lactate dehydrogenase activity--an in vitro study

Metabolic acidosis complicates methanol, ethylene glycol and other alcohol intoxications. It is caused firstly by acid metabolites and secondly by the lactate elevation. The aim of the study was to evaluate the effect of alcohol dehydrogenase (ADH; EC 1.1.1.1) inhibitors and substrates: 4-methylpyrazole (4-MP), cimetidine, EDTA, ethanol and methanol on lactate dehydrogenase (LDH; EC 1.1.1.27) activity. The activity of LDH was determined spectrophotometrically in in vitro human heart homogenates with the mentioned compounds at 0.01, 0.1, 1.0 mM concentrations of 4-MP, cimetidine, EDTA, and 12.5, 25.0, 50.0 mM of ethanol and methanol. The LDH activity was significantly inhibited by 0.1 mM (p<0.05) and 1.0 mM (p<0.01) 4-MP and 1.00 mM EDTA (p<0.05). Higher LDH activity vs. control was observed in the samples incubated with all studied ethanol and methanol concentrations but these differences were not statistically significant. Thus, 4-MP was found to be the most effective inhibitor of LDH of all compounds tested. Therefore, such effect of 4-MP seems to be an additional advantage in methanol and ethylene glycol intoxications.     

Effect of cinnamate on nitrate reductase activity in isolated cucumber cotyledons

The effect of cinnamic acid on in vivo nitrate reductase activity and protein content in cucumber cotyledons was studied. Cinnamate increased in vivo nitrate reductase activity and also the total protein content at lower concentrations (0.01–0.1 mM). Higher concentration, however, proved inhibitory. The effect of cinnamate on nitrate reductase activity has been discussed.     

Comparison of reactive oxygen scavenging systems between a cetacean (DKN1) and a porcine renal epithelial cell line (LLC-PK1)

A transformed renal epithelial cell line, (DKN(1)), from an Atlantic Bottlenose Dolphin, Tursiops truncatus was established in this laboratory and has been used for in vitro genomic analysis and initial toxicological evaluations of dolphin cells. Studies were initiated to compare maintenance of normal antioxidant mechanisms in DKN(1) with similar mechanisms in cells of a pig kidney line, LLC-PK(1). Levels of catalase, glutathione peroxidase, and of reduced glutathione in these dolphin cells were significantly lower than in the porcine cells. Both cell lines were then challenged with hydrogen peroxide at 0.01, 0.1, and 1.0 mM concentrations. The dolphin cells exhibited increased cytotoxicity with a concurrent increase in apoptosis at lower concentrations (0.1 mM) than those required to initiate cytotoxicity in the porcine cells (1.0 mM). Taken together, these results would indicate that the dolphin cells are more susceptible to the damaging effects of certain reactive oxygen species than their terrestrial counterparts.     

Histamine Acting on H2 Receptors Stimulates Phospholipid Methylation in Synaptic Membranes of Rat Brain

Histamine stimulated [3H]methyl group incorporation into phospholipids in crude synaptic membranes of rat whole brain (without cerebellum) in modified Krebs-Ringer solution containing the methyl donor S-adenosyl-[methyl-3H]methionine. The transient increase of [3H]methyl incorporation into lipids peaked within 45 s after addition of histamine (5 or 10 microM) and decreased the basal level in 60 s. Histamine-stimulated [3H]methyl incorporation was increased linearly in a protein concentration-dependent manner. The stimulation was temperature and histamine concentration dependent. TLC analysis of a chloroform/methanol extract indicated that radioactive phospholipids (phosphatidylcholine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidyl-N-monomethylethanolamine) accounted for 60-65% of the total radioactivity recovered. The synaptosomal fraction had the highest specific activity of all the subfractions of crude synaptic membranes (P2). Histamine-induced [3H]methyl incorporation was inhibited by addition of cimetidine (0.01-10 microM) or famotidine (0.01-1.0 microM) in a concentration-dependent manner but not by mepyramine (0.1-10 microM) or diphenhydramine (0.1-10 microM). The stimulation of [3H]methyl incorporation was also observed by addition of impromidine (0.01-10 microM) or dimaprit (1.0 microM-1.0 mM) in a concentration-dependent manner but not by 2-pyridylethylamine (1.0 microM-1.0 mM). These results indicate that phospholipid methylation is induced by histamine acting on H2 receptors in rat brain synaptosomes.     

Changes in phytohormone contents in chickpea seeds germinating under lead or zinc stress

The present work describes the changes that take place in phytohormone contents in germinating chickpea (Cicer arietinum cv. Aziziye-94) seeds in response to heavy metal stress. For this aim, endogenous abscisic acid (ABA), gibberellic acid (GA₃), zeatin (Z) and zeatin riboside (ZR) contents were followed for 24, 48 and 72 h in chickpea seeds germinating at the concentrations of 0.1, 1.0 and 5.0 mM Pb or 0.1, 1.0 and 10 mM Zn. The results showed that Pb and Zn significantly delayed and impeded the germination of chickpea seeds. The negative effect of Pb on germination was higher than that of Zn. Further, Pb increased ABA and Z contents while decreased GA₃ content in the germinating seeds. The high concentrations of Zn (1.0 and 10 mM) decreased contents of Z, ZR and GA₃ while 0.1 mM Zn increased the content of the same hormones. The ABA content was enhanced by Zn in all concentrations used.     

Thermostable succinyl-Coenzyme A synthetase from Nitrosomonas europaea ATCC 25978: Purification and properties

The ammonia-oxidizing chemoautotrophic bacterium Nitrosomonas europaea possesses prominant succinate-reducing activity of succinyl-Coenzyme A synthetase (SCS, EC 6.2.1.5). SCS was purified as an electrophoretically homogeneous protein from Nitrosomonas europaea strain ATCC 25978 about 275-fold, with a 3.9% activity yield. The molecular mass of the native enzyme was estimated to be about 130 kDa by gel filtration, whereas SDS-PAGE gave two protein bands with M_r values of 29 (α) and 36 kDa (β). The isoelectric point of the enzyme was 5.3. The apparent K_m values of the enzyme for ATP, succinate and CoA were 0.4 mM, 5 mM and 0.1 mM, respectively. The pH and temperature optima of the SCS were about 5.0 and 55°C, respectively. The SCS was stable in the pH range of 8.0–10.0 and up to 70°C. The enzyme was thermostable; 50% of the enzyme activity was retained at 90–100°C for 10 min. The SCS was activated by Mg²⁺ at 1.0–100 mM, but inhibited by Cu²⁺ (0.1 mM) and SDS (1.0 mM). The enzyme utilized ATP as the preferred substrate.     

The Respiratory Activity of Rhodococcus rhodochrousM8 Cells Producing Nitrile-Hydrolyzing Enzymes

The respiratory activity of Rhodococcus rhodochrousM8 cells containing nitrile hydratase and amidase was studied in the presence of nitriles and amides of carbonic acids. The culturing of cells with acrylonitrile and acrylamide yielding maximum respiratory activity was studied. The optimum conditions for measurements and maintenance of respiratory activity were found. Curves for the linear concentration dependence of cell respiratory activity on 0.01–0.5 mM acrylonitrile, 0.025–1.0 mM acetonitrile, and 0.01–0.1 mM acrylamide were plotted. The selectivity of cell respiratory activity for some substrates was analyzed.     

The respiratory activity of Rhodococcus rhodochrous M8 cells producing nitrile-hydrolyzing enzymes]

The respiratory activity of Rhodococcus rhodochrous M8 cells containing nitrile hydratase and amidase was studied in the presence of nitriles and amides of carbonic acids. Culturing of cells with acrylonitrile and acrylamide yielding their maximum respiratory activity was studied. The optimum conditions for measurements and maintenance of respiratory activity were found. Curves for the linear concentration dependence of cell respiratory activity on 0.01-0.5 mM acrylonitrile, 0.025-1.0 mM acetonitrile, and 0.01-0.1 mM acrylamide were plotted. The selectivity of cell respiratory activity for some substrates was analyzed.     

Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32

X-prolyl-dipeptidyl aminopeptidase, which hydrolyzed Gly-Pro-p-nitroanilide (relative activity [RA] = 100%) and Arg-Pro-p-nitroanilide (RA, 130%), was purified to homogeneity from the cell extract of Lactobacillus helveticus CNRZ 32. The enzyme also hydrolyzed Ala-Pro-Gly (RA, 11%) and Ala-Ala-p-nitroanilide (RA, 2%) but was not active on Ala-Leu-Ala, dipeptides, and endopeptidase and carboxypeptidase substrates. The enzyme was purified 145-fold by streptomycin sulfate precipitation, ammonium sulfate fractionation, and a series of column chromatographies on DEAE-cellulose, arginine-Sepharose 4B, and glycyl-prolyl-AH-Sepharose 4B. The purified enzyme appeared as a single band on native polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses and had a molecular weight of 72,000. Optima for activity by the purified enzyme were pH 7.0 and 40°C. The enzyme was incubated at 40°C for 15 min with various metal ions. It was activated by Mg²⁺ (2.5 mM), Ca²⁺ (0.1 to 2.5 mM), Na⁺ (10 to 50 mM), and K⁺ (10 to 50 mM) and was inhibited by Hg²⁺ (0.1 to 2.5 mM), Cu²⁺ (0.1 to 2.5 mM), and Zn²⁺ (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40°C), 1,10-phenanthroline (1.0 mM, 15 min at 40°C), phenylmethylsulfonyl fluoride (1.0 mM), N-ethylmaleimide (1.0 mM), and iodoacetate (1.0 mM). It was completely inhibited by diisopropyl fluorophosphate (1.0 mM, 2 h at 40°C) and p-chloromercuribenzoate (1.0 mM, 15 min at 40°C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM).     

Characterization of cytosolic sialidase from Chinese hamster ovary cells: part I: cloning and expression of soluble sialidase in Escherichia coli

The cDNA of Chinese hamster ovary (CHO) cell cytosolic sialidase was amplified by RT-PCR and cloned into the pGEX-2T plasmid vector encoding for glutathione S-transferase (GST). Screening revealed transformed Escherichia coli clones with the constructed plasmid encoding the CHO cell sialidase sequence. After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, SDS-PAGE of the total protein extracts revealed a new protein of about 70 kDa, correlating with the molecular weight of a fusion protein composed of the GST (26 kDa) and the cloned cytosolic CHO cell sialidase (43 kDa). A soluble fusion protein was purified from sonified E. coli homogenates by one-step affinity chromatography on Glutathione Sepharose 4B, which showed sialidase activity towards 4-methyl-umbelliferyl-alpha-D-N-acetylneuraminic acid (MUF-Neu5Ac) substrate. Induction of cells with 0.1, 0.5, and 1.0 mM IPTG revealed highest total protein amounts after induction with 1.0 mM IPTG, but highest specific activity for affinity chromatography purified eluates from cultures induced with 0.1 mM IPTG. Therefore, large scale production was performed by inducing cells during exponential growth in a 25 L bioreactor for 3 h with 0.1 mM IPTG after chilling the cell suspension to 25 degrees C. The amount of 26.46 mg of 40-fold purified GST-sialidase with a specific activity of 0.999 U/mg protein was obtained from crude protein extracts by one-step affinity chromatography. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) and Neu5Ac were competitive inhibitors for the sialidase, the former being the more effective one using MUF-Neu5Ac as the substrate. The cytosolic sialidase is capable of desialylating a wide spectrum of different types of gangliosides using a thin-layer chromatography overlay kinetic assay without detergents. This is the subject of the accompanying paper (Müthing, J.; Burg, M. Carbohydr. Res. 2001, 330, 347-356).     

Effects of salicylic acid and salinity on apoplastic antioxidant enzymes in two wheat cultivars differing in salt tolerance

The effects of salicylic acid (SA) and salinity on the activity of apoplastic antioxidant enzymes were studied in the leaves of two wheat (Triticum aestivam L.) cultivars: salt-tolerant (Gerek-79) and salt-sensitive (Bezostaya). The leaves of 10-d-old seedlings grown at nutrient solution with 0 (control), 250 or 500 mM NaCl were sprayed with 0.01 or 0.1 mM SA. Then, the activities of catalase (CAT), peroxidase (POX) and superoxide dismutase (SOD) were determined in the fresh leaves obtained from 15-d-old seedlings. The NaCl applications increased CAT and SOD activities in both cultivars, compared to those of untreated control plants. In addition, the NaCl increased POX activity in the salt-tolerant while decreased in the salt-sensitive cultivar. In control plants of the both cultivars, 0.1 mM SA increased CAT activity, while 0.01 mM SA slightly decreased it. SA treatments also stimulated SOD and POX activity in the salt-tolerant cultivar but significantly decreased POX activity and had no effect on SOD activity in the saltsensitive cultivar. Under salinity, the SA treatments significantly inhibited CAT activity, whereas increased POX activity. The increases in POX activity caused by SA were more pronounced in the salt-tolerant than in the salt-sensitive cultivar. SOD activity was increased by 0.01 mM SA in the salt-tolerant while increased by 0.1 mM SA treatment in the salt-sensitive cultivar.     

The effect of diuretics and vasopressin on the osmotic permeability of the frog urinary bladder.

1. Large concentrations (in mM) of ethacrynic acid (0.1), furosemide (1.0), theophylline (5.0) and osmotic diuretics (100.0) sharply increased the flux of water along an osmotic gradient through the frog urinary bladder wall. Spironolactone (0.1), and hydrochlorothiazide (5.0) showed only a weak action on osmotic permeability. MercusalR, clopamide and triamterene did not affect water transport. 2. The presence of 0.2--1.0 mU/ml vasopressin (ADH) after pretreatment with a diuretic did not result in summation of the effects of both drugs used. 0.01--0.1 mM ethacrynic acid and 0.01 mM MercusalR significantly decreased the reaction to ADH. 1.0 mM furosemide, 0.1 mM spironolactone, 0.01 mM clopamide and 0.8 mM acetazolamide did not change the reaction to ADH. A reduction in the cellular response to ADH and a decrease in the osmotic permeability of the tubular wall may be responsible in part for the diuretic action of ethacrynic acid and MercusalR.     

Putrescine Effect on Nitrate Reductase Activity, Organic Nitrogen, Protein, and Growth in Heavy Metal and Salinity Stressed Mustard Seedlings

Putrescine effect on nitrate reductase activity, organic nitrogen and protein contents, and plant growth under Cd or Pb (0.1 – 2 mM) and salinity (5 and 100 mM NaCl) stresses was examined in Indian mustard (Brassica juncea L. cv. RH-30) seedlings. Cd or Pb and salinity inhibited nitrate reductase activity and decreased organic nitrogen and protein contents in leaf tissue. The increased nitrate reductase activity induced by putrescine was correlated with increased organic nitrogen and protein contents and growth of plants.     

Parameters affecting optimum activity and stability of urokinase-bound fibrocollagenous tubes

Urokinase was immobilized by entrapment to fibrocollagenous tubes in order to develop a small-caliber fibrinolytic vascular prosthesis. Several parameters associated with the immobilization process were studied in order to optimize bound urokinase activity and stability. A total of 37% of the absorbing enzyme was attached to the collagen tube and 38% of the attached enzyme retained esterolytic activity, under optimal conditions. In the crosslink step of the entrapment process, the glutaraldehyde concentration was varied from 0.01 to 5.00% (i.e., 0.01, 0.1, 1.0, 3.0, and 5.0%). Urokinase activity was optimized at a 1.0% glutaraldehyde crosslink concentration. Urokinase-bound fibrocollagenous tubes (UK-FCT) prepared at the above glutaraldehyde concentrations were tested for their activity with time. The UK-FCT's with 0.1, 1.0, and 3.0% glutaraldehyde retained constant activity for at least 75 h operation time. The UK-FCT's with 0.1, 1.0, and 3.0% glutaraldehyde retained constant activity for at least 75 h operation time. The UK-FCT's with 5.0 and 0.01% glutaraldehyde remained stable for the first 50 h operation time, but begandeactivating beyond 50 h. UK-FCT'S Crosslinked with 0.01, 0.1, 1.0, and 5.0% glutaraldehyde were recrosslinked with 0.02% glutaraldehyde for 24 h, after they have been operating for 50 h, and the effect of reexposing the crosslink agent on the stability of the UK-FCT's was studied. The results showed that 0.02% glutaraldehyde reexposure had no effect on 0.1, 1.0, and 5.0% glutaraldehyde crosslinked UK-FCT's but exerted an inhibitory effect on a 0.01% crosslink density UK-FCT. Several fibrocollagenous tubes were exposed to various glutaraldehyde concentrations prior to immobilizing urokinase. The subsequent immobilization process occurred under optimal conditions. The effect of the precrosslink step on the activity of the UK-FCT was studied. Results indicated that UK-FCT activity decreases as the precrosslink density increases. The UK-FCT's made under optimal conditions remained stable for at least 75 h operation time, corresponding to ca.1 year of storage time. Ex vivo exposure of UK-FCT's to whole canine blood did not affect catalytic activity. Implantation of a UK-FCT by carotid arterial interposition via an end-to-end anastomosis and subsequent excision after 60 days resulted in an enhanced esterolytic activity which decreased with time to a level close to preoperative levels.     

The action of dibucaine in vitro on fat cell lipolysis and protein kinase activity.

Dibucaine at 0.1 and 0.25 mM markedly inhibited epinephrine-stimulated lipolysis in rat epididymal fat cells $\text{in}$$\text{vitro}$ but did not inhibit protein kinase activity. At 1.0 mM, dibucaine half-maximally stimulated protein kinase of fat cells under basal conditions but did not stimulate lipolysis. It is concluded that dibucaine inhibits lipolysis by a mechanism not involving inhibition of protein kinase.     

Decreased Histamine-Stimulated Phosphoinositide Hydrolysis in the Cerebral Cortex of a Rat Line Selectively Bred for High Alcohol Preference

This study sheds light on the comparative analysis of agonist-stimulated phosphoinositide (PI) hydrolysis in the cerebral cortex of alcohol-naive rats from established lines selectively bred for low alcohol preference (LAP) and high alcohol preference (HAP). The effect of histamine (1.0 mM), but neither norepinephrine (0.1 mM) nor carbachol (0.5 mM), on PI hydrolysis was significantly reduced in HAP rats (0.4 +/- 5.0 fmol/mg protein [3H]inositol phosphates formed over basal) compared with LAP rats (25.5 +/- 10.0 fmol/mg protein). The contents of monoamines (dopamine, norepinephrine, and serotonin) and histamine in the cerebral cortex did not significantly differ between LAP and HAP rats, nor did the contents of their metabolites, except 3-methoxy-4-hydroxyphenylglycol (one of the metabolites of norepinephrine) and N(tau)-methylhistamine, which was not detected in our system. The histamine stimulatory effect was unchanged in the cerebral cortex of an intact Wistar rat that was treated with intraperitoneal injection of alcohol (1.0 g/kg once per day for 14 days). The results of the current study indicate that the decrease in the histamine effect on PI hydrolysis in HAP rats might be attributed to that particular rat line.     

Relative efficacies of indole antioxidants in reducing autoxidation and iron-induced lipid peroxidation in hamster testes

Increased iron stores are associated with free radical generation and carcinogenesis. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of tumor initiation. Melatonin and structurally related indoles are effective in protecting against oxidative stress. The aim of the study was to compare the relative efficacies of melatonin, N-acetylserotonin (NAS), indole-3-propionic acid (IPA), and 5-hydroxy-indole-3-acetic acid (5HIAA) in altering basal and iron-induced lipid peroxidation in homogenates of hamster testes. To determine the effect of the indoles on the autoxidation of lipids, homogenates were incubated in the presence of each agent in concentrations of 0.0, 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1.0, 2.0, 2.5, or 5.0 mM. To study their effects on induced lipid peroxidation, homogenates were incubated with FeSO(4) (30 microM + H(2)O(2) (0.1 mM) + each of the indoles in the same concentrations as above. The degree of lipid peroxidation was expressed as concentrations of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. The indoles decreased both basal and iron-related lipid peroxidation in a concentration-dependent manner. Melatonin reduced basal MDA + 4-HDA levels when used at the concentrations of 0.25 mM or higher, and prevented iron-induced lipid peroxidation at concentrations of 1.0, 2.0, 2.5, or 5.0 mM. The lowest effective concentrations of NAS required to lower basal and iron-related lipid peroxidation were 0.05 mM and 0.25 mM, respectively. IPA, only when used in the highest concentrations of 2.5 mM or 5 mM inhibited basal lipid peroxidation levels and it was ineffective on the levels of MDA + 4-HDA due to iron damage. 5HIAA reduced basal lipid peroxidation when used at concentrations of 0.25 mM or higher, and it prevented iron-induced lipid peroxidation only at the highest applied concentration (5 mM). In conclusion, melatonin and related indoles at pharmacological concentrations protect against both the autoxidation of lipids as well as induced peroxidation of lipids in testes. In doing so, these agents would be expected to reduce testicular cancer that is initiated by products of lipid peroxidation.     

Interaction of alpha-chymotrypsin with dimyristoyl phosphatidylcholine vesicles

The amidolytic activity of chymotrypsin for Suc-Ala2-Pro-Phe-MCA was somewhat enhanced by dimyristoyl PC at low ionic strength, but not at high ionic strength. The activity was strongly inhibited by pure egg yolk PA. The inhibition by 200 ng PA was neutralized by addition of 1 microgram dimyristoyl PC or pure egg yolk PC, which formed vesicles with the PA. The Km and kcat (s-1) values of chymotrypsin for hydrolysis of Suc-Ala2-Pro-Phe-MCA changed from 15 microM to 42 microM, 0.1 mM and 0.5 mM, and from 1.5 to 2.7, 3.7, and 1.0 in the presence of 1 microgram dimyristoyl PC, 0.5 micrograms pure egg yolk PE and 0.2 microgram egg yolk PA, respectively. Gel-filtration chromatography showed that dimyristoyl PC formed a complex with chymotrypsin, but did not interact with the substrate, indicating that the basic globular protein, chymotrypsin, interacted with net-neutral PL.