ABSTRACT
A colorimetric reagent, 4-(4'-nitro-2'-methylsulfonylphenylazo)phenyl phosphate (NMPP), has been shown to be an effective substrate of alkaline phosphatase. NMPP and p-nitrophenyl phosphate were applied in comparative studies using enzyme immunoassays for the detection of viral antigens and antiviral antibodies. The new substrate exhibited similar, or even higher, sensitivity than p-nitrophenyl phosphate depending on the substrate concentrations used. Positive and negative reactions were easier to define, even without cumbersome equipment. The enzyme reaction was terminated by the uncompetitive inhibitor, theophylline.
Subject(s)
Azo Compounds , Enzyme-Linked Immunosorbent Assay , Alkaline Phosphatase , Antibodies, Viral/analysis , Antigens, Viral/analysis , Colorimetry , Indicators and Reagents , Nitrophenols , Organophosphorus Compounds , Substrate SpecificityABSTRACT
Sperm acrosin proteolytic activity in single sperm can be detected by a protein-free halo on a gelatin-substrate film. With current techniques, halos have variable sizes and are often absent because of unevenness of the hand-spread gelatin-substrate film. We prepared gelatin-substrate films with a coating machine. Using these films, halos were formed uniformly throughout the gelatin-substrate films in the vicinity of single mammalian sperm. The level of acrosin activity as determined by halo diameters was human greater than dog greater than squirrel monkey greater than mouse greater than rat. This simple and reproducible technique may be used to diagnose infertility due to decreased acrosin activity, as a screening method for identifying compounds with male sterility effects, and for identifying agents with developmental and/or genetic effects.
Subject(s)
Acrosin/analysis , Endopeptidases/analysis , Spermatozoa/enzymology , Animals , Dogs , Gelatin , Humans , Male , Methods , Mice , Rats , SaimiriABSTRACT
We have prepared a dry film for the enzymic determination of total serum cholesterol. It consists of a transparent support bearing a buffered gelatin layer, and a white reflective spreading layer that contains all of the necessary components for the detection of cholesterol. The method is based on (a) hydrolysis of cholesterol esters to cholesterol by cholesterol ester hydrolase (EC 3.1.1.13), (b) oxidation of cholesterol to cholest-4-en-4-one and hydrogen peroxide by cholesterol oxidase (EC 1.1.3.6), and (c) oxidation of a triarylimidazole leuco dye with hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) to produce a dye with maximum absorption at about 650 nm. For use over a wider range of concentration, the dye density is read at 540 nm. With reflection densitometry and appropriate mathematical transformation, readings and cholesterol concentrations are linearly related to 5500 mg/L. Results correlate well with those by the Abell-Kendall comparison method (slope 0.97, intercept 92.5, correlation coefficient 0.974, Sy.x = 250.7), and the method is precise (CV of 1.2-2.3% for a control fluid and patients' samples) and relatively free of interferences.
Subject(s)
Cholesterol/blood , Hyperlipidemias/blood , Chemical Phenomena , Chemistry , Chromogenic Compounds , Humans , Methods , Reference Values , Spectrophotometry , Triglycerides/bloodABSTRACT
When rat liver cytosolic P-enolpyruvate carboxykinase is purified, its activity is no longer enhanced by incubation with 30 muM Fe2+. Ferrous ion stimulation of the purified enzyme is restored by the addition of rat liver cytosol. The agent responsible is a cytosolic protein, named P-enolpyruvate carboxykinase ferroactivator, that was readily separated from the enzyme during purification of the latter. A quantitative assay for P-enolpyruvate carboxykinase ferroactivator is described. Subcellular fractionation of livers from fasted rats shows that 98% of the combined mitochondrial and cytosolic P-enolpyruvate carboxykinase ferroactivator activity resides in the cytosol. Fasting does not produce significant change in this cytosolic activity when compared to that of fed animals. Examination of various tissue homogenates shows that the ferroactivator is found in liver, kidney, erythrocytes, adipose tissue, and brain. No activity was detected in blood serum or skeletal muscle. The ability to enhance the activity of purified rat liver cytosolic P-enolpyruvate carboxykinase in the presence of Fe2+ is not species specific. P-enolpyruvate carboxykinase ferroactivator may have an important function in regulating enzyme activity in vivo.
