Direct measurement of enzyme activity with infrared spectroscopy

A direct approach to enzyme activity measurements is presented. Vibrational spectroscopy can monitor the progress of enzymatic reactions because the vibrational spectrum of substrates and products usually differs. This is demonstrated by the example of ATP hydrolysis by Ca(2+)-ATPase: The substrate concentration can be followed using the infrared absorption of the alpha- and beta-PO(2)(-) phosphate groups of ATP, and the product concentration can be followed using the PO(3)(2-) absorption of P(i) and of the beta-phosphate of ADP. The results of the infrared spectroscopic measurement of ATPase activity and of an independent activity assay agree very well. The main advantage of the infrared method is that it observes the reaction of interest directly--that is, no activity assay that converts the progress of the reaction into an observable quantity is required.     

Direct fluorometric measurement of hepatitis C virus helicase activity

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNA-stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3' labeled with a Cy3 dye and 5' labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.     

Direct measurement of cardiac sympathetic efferent nerve activity during dynamic exercise

The assumption that tachycardia during light to moderate exercise was predominantly controlled by withdrawal of cardiac parasympathetic nerve activity but not by augmentation of cardiac sympathetic nerve activity (CSNA) was challenged by measuring CSNA during treadmill exercise (speed, 10-60 m/min) for 1 min in five conscious cats. As soon as exercise started, CSNA and heart rate (HR) increased and mean arterial pressure (MAP) decreased; their time courses at the initial 12-s period of exercise were irrespective of the running speed. CSNA increased 168-297% at 7.1 +/- 0.4 s from the exercise onset, and MAP decreased 8-13 mmHg at 6.0 +/- 0.3 s, preceding the increase of 40-53 beats/min in HR at 10.5 +/- 0.4 s. CSNA remained elevated during the later period of exercise, whereas HR and MAP gradually increased until the end of exercise. After the cessation of exercise, CSNA returned quickly to the control, whereas HR was slowly restored. In conclusion, cardiac sympathetic outflow augments at the onset of and during dynamic exercise even though the exercise intensity is low to moderate, which may contribute to acceleration of cardiac pacemaker rhythm.     

Direct measurement of the energy expenditure of physical activity in preterm infants

The energy cost of physical activity (EEA) has been estimated toaccount for 5-17% of total energy expenditure (TEE) in neonates. To directly measure EEA, a force plate was developed and validated tomeasure work outputs ranging from 0.3 to 40 kcal · kg¹ · day¹.By use of this force plate plus indirect calorimetry, TEE and EEA weremeasured and correlated with five activity states in 24 infants withgestational age of 31.6 ± 0.5 (SE) wk and postnatal age of 24.8 ± 3.7 days. TEE and EEA were 69.2 ± 1.5 and 2.4 ± 0.2 kcal · kg¹ · day¹,respectively. EEA per state was 0.5 ± 0.0 (quiet sleep), 2.4 ± 0.2 (active sleep), 2.8 ± 0.4 (quiet awake), 7.5 ± 0.8 (active awake), and 15.1 ± 2.3 (crying)kcal · kg¹ · day¹.This provides the first direct measurement of the contribution ofphysical activity to TEE in preterm infants and will enable measurementof caloric expenditure from muscle activity in various diseaseconditions and development of nursing strategies to minimize unnecessary energy losses.

     

Nitro esterase activity of Pseudomonas fluorescens

The authors have demonstrated the biological nature of a process in which nitroester bonds are hydrolysed in the molecule of nitrocellulose. Pseudomonas fluorescens, strain 1/2, was isolated from a natural cenosis of microorganisms inhabiting active ooze, which was selected in a device for continuous cultivation by immobilisation on nitrocellulose fibers. The strain cleaves some nitro groups off the surface of nitrocellulose and the resultant nitrate ions are used in the process of assimilative reduction.     

Direct measurement of apoprotein B specific activity in 125I-labeled lipoproteins.

A method for determining apoprotein B specific activity in radioiodinated lipoproteins is described and validated. It utilizes organic solvents and tetramethylurea in the isolation of apoprotein B from other radiolabeled contaminants, both lipid and protein, in exogenously labeled VLDL. The contaminants are also removed from those lipoprotein classes subsequently derived from VLDL, namely IDL and LDL. The procedure requires approximately 50 microgram of apoB per analysis, allowing specific activity determinations in triplicate on 3-ml plasma samples with a standard error of less than 6%. Finally, data from a study of apoprotein B turnover in VLDL, IDL, and LDL in a human subject is presented to demonstrate the potential of this method in further elucidating the kinetic interrelationships between these lipoprotein classes.     

Direct measurement of renal sympathetic nervous activity in high-fat diet-related hypertensive rats

The elevation of renal sympathetic nervous activity (SNA) is a possible cause of blood pressure (BP) elevation. Although a high-fat diet (FAT) often induces BP elevation in animals, the effect of FAT on renal SNA in animals is not consistent between studies. Thus, we compared the basal levels of efferent renal SNA and BP in FAT- or high-carbohydrate diet (CHO)-fed rats. Twenty-four male Sprague-Dawley rats were fed FAT (P/F/C=20/45/35% cal) or CHO (20/5/75) from 5 weeks of age. After 20-21 weeks of feeding, a 24-h urine sample was collected to measure sodium excretion. The next day, blood (0.2 ml) was withdrawn from a femoral artery, and basal efferent renal nerve discharges and mean arterial pressure (MAP) were recorded under anesthesia. Immediately after the experiment, abdominal (epididymal, perirenal and mesenteric) adipose tissues were dissected. Total abdominal fat weight was significantly greater in the FAT group than in the CHO group. The plasma level of leptin was significantly higher in the FAT group, but blood glucose and plasma insulin levels did not differ between the two groups. MAP and renal SNA were significantly higher in the FAT group. In addition, the ratio of urinary sodium excretion to dietary sodium intake was significantly lower in the FAT group than in the CHO group. The data suggest that the increased renal SNA may contribute to BP elevation in FAT-fed rats. The present study firstly demonstrated that renal SNA was elevated with FAT-related BP elevation.     

Determination of methanol and its application to measurement of pectin ester content and pectin methyl esterase activity

A simple and precise spectrophotometric determination of methanol in the range 2–40 μg is described. The method has been applied to the determination of the methyl ester content of 50–200 μg of pectin, and is an improvement on previously described methods. The value of the method in studies of pectin and pectin esterase metabolism is indicated.     

Extracellular esterase activity from Bacillus stearothermophilus

Bacillus stearothermophilus has been reported to produce an extracellular esterase with molecular weight of 42–47 kDa. Extracellular esterase activity in Bacillus stearothermophilus (NCIB 13335) was found to reside in protein with a molecular weight less than 10 kDa. This small esterase was responsible for all the esterase activity observed in this strain under the conditions studied.     

Non-specific esterase activity in bovine acrosomes

Synopsis Fixed and unfixed smears of bovine spermatozoa were stained for esterase activity at pH 6.1–6.2 using 1-naphthyl acetate as substrate and hexazonium pararosaniline as coupler. Best results were obtained with unfixed smears prepared from suspensions of spermatozoa washed free of seminal plasma. The most intense staining was obtained when smears were incubated at 4°C for 40 hr with replacement of the incubating medium every 12 hr. No staining was detected in spermatozoa when smears were incubated in medium lacking substrate or when heat-inactivated smears were incubated in complete medium. Enzyme activity was restricted to the acrosomal cap; no final reaction product was detected in or on the post-nuclear cap, the mid-piece or the remainder of the flagellum. Enzyme activity was distributed quite uniformly throughout the acrosome with no indication of the existence of localized regions with high activity. The use of fixatives, especially those containing formalin, is not recommended for esterase studies of bovine spermatozoa.