Cultures enzyme assays

Enzyme Assay The crude enzyme was extracted from the solid state culture with 100 ml of 0.33% toluene at 4°C. The enzyme activity was assayed by the determination of substrate viscosity diminishing using Ostwald viscometer (5). The enzyme reaction was done at 37°C in 0.05 N acetate buffer pH 5.25. One unit of enzyme was defined as the amount of enzyme that could reduce the viscosity of 2% pectin by 50% in 10 min. 

The mycelia, of six days old culture, were harvested by filtration through Whatman paper n 1 and kept at -80 C for RNA assays. The culture filtrates were used for enzyme assays. 

Extracellular PG activity was not detected in cultures on glucose, a similar situation to that of Fusarium moniliforme (18). In contrast, all our data confirm the presence of the protein and the mRNA of PG in non-inducing conditions our assays did reveal no differences between FORL PG growing on both conditions, regarding migration or other detectable characteristics that could justify the presence of the enzyme and its lack activity. It is posible that a very low concentration of the enzyme results in undetectable activity in enzyme assays. 

Inhibitors of several enzymes in the glycolytic pathway, upon which survival of T. brucei is dependent, have been described. Lonidamine (29) has been shown to inhibit T. brucei hexokinase (IC50 = 850 pM) and be toxic to the parasite (T.b. LD50 = 50 pM) in culture [31]. A series of mannitol derivatives have been discovered, which inhibit T. brucei phos-phofructokinase (TbPFK) [32]. The most potent compound (30) within this series exhibits an IC50 = 23 pM in a recombinant enzyme assay and inhibits parasite growth in vitro (IC50 = 30 pM). 

Prenatal and postnatal diagnoses can be made by enzyme assay of cultured amniocytes, fibroblasts or white blood cells. Treatment remains symptomatic. Sodium bicarbonate is necessary to correct the acidosis. Aspartic acid supplementation will improve the systemic condition but has no effect on the neurological disturbances. Biotin supplementation is of no value. 

Enzyme assays—both kinetic and end-point radiocoordination of proteins, lipid assays, receptor binding assays and tissue-culture techniques... 

Figure 6. HPLC kinetics of polygalacturonic acid depolymerization by extracellular pectate lyases from crude supernatants of Erwinia chrysanthemi and Lachnospira multiparus cultures. A panels are full scale representations of products found over the reaction time sequence. B panels have expanded ordinates to better demonstrate the kinetics of minor products. Area unit refers to integration from HPLC tracings of product absorbance at 235 nm. Numbers in the panels refer to the degree of polymerization for individual products. Conditions for enzyme assay and product detection were the same as described for Figure 5.

Among these latter inhibitors, a series of monocyclic (3-lactams I (compound la being the prototype, Fig. 49), has resulted in highly potent derivatives in the isolated enzyme assay, but their efficacy in cell culture was quite limited, as described for all inhibitors of this enzyme [367, 414-416]. 

Cell Culture. KB cells were maintained in a humidified atmosphere of 5% carbon dioxide - 95% air at 37°C in the presence of modified Eagle s medium containing calf serum (10%), penicillin (100 jug/ml) and streptomycin (100 units/ml). Cells were routinely subcultured with 0.25% trypsin and stocks were discarded after twenty passages. All drugs were administered with fresh media 24 hours after subculture in the following concentrations TPA, 1.6 uM RA, 1.6 juM butyric acid, 2mM. Drug treatments were for 20-24 hours. Cells were harvested for enzyme assays with phosphate-buffered saline containing 0.05% EDTA and stored at -20°C in 0.32 M sucrose. 

Both H- and L-NHase genes are expressed under the control of a lac promoter in E. coli only when the transformants are cultured in the presence of CoCl2 [63], However, the level of NHase activity in their cell-free extracts is much lower than those of H- and L-NHases in R. rhodochrous Jl. Most of H-NHase is produced as an insoluble form in the E. coli transformant, and there is only a little L-NHase in either the supernatants or precipitates of the extracts. Establishment of an effective host-vector system in Rhodococcus [71] facilitates the development of strains with improved NHase activities. In this connection, the transformation system in Rhodococcus has been investigated. Enzyme assays of recombinant Rhodococcus cells showed that a downstream region of the Rhodococcus sp. N-774 NHase gene is indispensable for the production of active... 

Twenty percent (v/v) mycelium suspension was used to inoculate 500-mL conical flasks containing 15 g of corncob as carbon source and 22.5 mL of PPMKC medium (pH6.0) as optimized by Damaso et al. (6). After inoculation, the flasks were incubated in a stationary manner at 45°C for 6 d in a laboratory electric incubator. At each sampling time, the culture medium was vacuum filtered using filter paper (Whatman, no. 4, fast-flow rate), and the filtrate was used for further enzyme assays. During the cultivation, two or more flasks were sampled daily. 

The importance of zinc for a normal functioning of the Cu-Zn-SOD was shown in Lemna gibba. In zinc-deficient culture media the activity of Cu-Zn-SOD was strongly inhibited whereas in copper-deficient media little change was found in the enzyme activity (Vaughan et al., 1982). In extracts of zinc-deficient plants, restoration of enzyme activity was possible by supplying zinc to the enzyme assay medium. 

CAT assay. An enzyme assay. CAT stands for chloramphenicol acetyl transferase, a bacterial enzyme which inactivates chloramphenicol by acety-lating it. CAT assays are often performed to test the function of a promoter. The gene coding for CAT is linked onto a promoter (transcription control region) from another gene, and the construct is transfected into cultured cells. Largely supplanted by the reporter gene luciferase. 

Antenatal diagnosis for fetuses at risk for the urea cycle enzyme disorders can be made by appropriate enzyme assays and DNA analysis in the cultured amniocytes. 

The answer is a. (Murray, pp 259-267. Scriver, pp 3827-3876. Sack, pp 97—158. Wilson, pp 287-320.) Most enzymes are expressed in chorionic villi or amniocytes and allow prenatal diagnosis of metabolic disorders through cell culture and enzyme assay. Percutaneous umbilical blood sampling (PUBS), or cordocentesis, offers another strategy if the enzyme is normally present in leukocytes. However, transabdominal aspiration of the umbilical cord is difficult and must be performed later in pregnancy (18-1-weeks) than CVS (8 to 10 weeks). OC-fetoprotein (AFP) is not known to be involved in any metabolic disorders, but it is used as an index of fetal tissue differentiation and integrity. Amniotic or maternal serum OC-fetoprotein (MSAFP) is most often used to detect, respectively, neural tube defects or... 

Activator proteins can also be quantified with immunochemical techniques if suitable antisera are available. Enzyme-linked immunosorbent assays (ELISA) bave been developed for the sulfatide/Gvii activator (Gardas et al., 1984) and for the Gm2 activator (Banerjee et al., 1984). The ELISA for the Gm2 activator was more than 10 times as sensitive as enzymic assays and permitted the precise determination of Gm2 activator in very dilute samples such as subcellular fractions of cultured skin Fibroblasts (Banerjee et al., 1984). 

New azaphilones named isochromophilones III-VI (18-21) were isolated from the culture broth of Penicillium multicolor FO-3216 as inhibitors of ACAT. Their structures were elucidated by NMR and other spectroscopic analyses. The IC50 values of isochromophilones (17-20) (ACAT) activity in an enzyme assay using rat liver microsomes were calculated to be 110, 50, 50 and 120 pM, respectively [79]. 

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