A continuous spectrophotometric assay for the determination of cellulase solubilizing activity

A cellulase assay was developed for the continuous measurement of colored cellulose oligosaccharides (total carbohydrates) released during enzymatic hydrolysis of dyed crystal-line cellulose. Several cellulosic substrates were uniformly dyed by Remalzol brilliant blue R salt without altering their physical properties. Dyed Avicel (6.5%, w/w) was selected as the most representative substrate for the assay procedure. The assay was performed continuously in a simple, thermally controlled apparatus designed for filtration of the reaction mixture via a 5-μm-pore-size nylon filter to retain the crystalline dyed cellulose while spectrophotometrically monitoring the absorbance at 595 nm of the reaction filtrate. Crude supernatant cellulase of Trichoderma viride QM9414 was used to test the assay procedure. The activity of cellulase on dyed Avicel as measured by ΔA_595nm correlated directly with the total carbohydrates formed. The initial reaction rate of cellulase solubilizing activity was readily determined with high sensitivity. The continuous assay has utility for the study of cellulase kinetics and for the comparison of activities from different microorganisms.     

A high-performance liquid chromatography method for the assay of cytidine monophosphate-sialic acid synthetase

An HPLC method has been developed for the assay of cytidine monophosphate-sialic acid synthetase (EC 2.7.7.43) using ion-pair chromatography and gradient elution. This procedure permits the assay of alternative substrates and inhibitors of the enzyme and is not subject to the limitation of the colorimetric method. The newly synthesized N-acetyl-9-deoxy-9-fluoro-D-neuraminic acid was found to be a good substrate of the enzyme with a Km of 6.35 mM as compared to 1.84 mM for N-acetylneuraminic acid.     

A rapid sampling method for measuring efflux from erythrocytes.

A method is described for obtaining kinetic data using a water-jacketed Hamilton gas-tight syringe as a reaction vessel and delivering aliquots of the reaction mixture to a quench solution at intervals as small as 2 sec apart by means of a repeating dispenser attachment. This apparatus has been used to measure the rate of [¹⁴C]uridine equilibrium exchange efflux from human erythrocytes at 15°C and at uridine concentrations near the K_m for the transport process. It should be useful for kinetic studies of any reaction having a half time of the order of 4 sec or more, provided a method of rapidly quenching the reaction is available.     

A centrifugal column assay for thymidylate synthetase using the active site titrant 5-fluoro-2′-deoxyuridylate

Thymidylate synthetase has been titrated in mouse liver extracts with [³H]fluorodeoxy-uridine monophosphate using centrifugally eluted gel filtration columns. The method is quick, efficient, and yields a linear dependence upon added extracted protein. The stability of the complex showed a pH maximum near 7.5. Specific binding of the tritiated nucleotide derivative to thymidylate synthetase was shown by denaturation of the complex in sodium dodecyl sulfate and gel electrophoresis of the denatured complex. Centrifugal elution of the sodium dodecyl sulfate-quenched complex from gel filtration columns was used to estimate the kinetics of complex formation at 0 and 12°C.     

A fluorometric-high-performance liquid chromatographic assay procedure for several enzymatic activities using formycin analogs of adenosine 5'-mono, 5'-tri, and cyclic 3',5'-monophosphate as substrates

The present study describes the synthesis of formycin 5′-triphosphate (FoTP), formycin 5′-monophosphate (FoMP), and formycin 3′,5′-cyclic monophosphate (cFoMP) from formycin A (FoA). These compounds, analogs of ATP, AMP, cAMP, and adenosine, respectively, are all fluorescent and differ chemically from the adenosine compounds by the reversal of the carbon atom at position 8 and the nitrogen at position 9 of the purine ring. Both FoMP and cFoMP were synthesized by chemical procedures from FoA while FoTP was made from FoMP enzymatically. All the analogs could be separated from each other using a high-performance liquid chromatographic (hplc) reverse-phase isocratic system that includes a μBondapak C-18 column as stationary phase and a solution of 0.01 m KH₂PO₄ adjusted to pH 5.5 with NaOH containing methanol as a mobile phase. At a flow rate of 2 ml/min, FoTP had a retention time of about 1 min followed by FoMP (2 min), cFoMP (3.5 min), and finally FoA (5.5 min). The analogs were detected by fluorometry using an excitation wavelength of 300 nm and an emission wavelength above 320 nm. This detection system proved to be more sensitive than absorbtion spectroscopy and as little as 2 pmol of the compounds could be measured.The analogs, together with the hplc system, were used to develop fluorometric (nonradioactive) assays for several enzymes including 3′5′-cyclic nucleotide phosphodiesterase (PDase), ATP pyrophosphohydrolase, and alkaline phosphatase. With these enzymes, the conversion of cFoMP to FoMP, FoTP to FoMP, and FoMP to FoA, respectively, could be followed. The conversion of FoA to formycin B (FoB), an analog of inosine, was also followed. The intracellular PDase activity isolated from the eukaryotic microorganism Dictyostelium discoideum was studied in some detail, and an apparent K_m of 5 μm and V_max of 0.1 nmol/min/mg protein were obtained for the enzyme at pH 7.5 and 30°. These values are compared to those in the literature.A number of advantages of this fluorometric-hplc assay procedure are discussed, including the facts that it offers an increase in sensitivity over other spectrophotometric assays and is at least equivalent to radiochemical assays currently in use.     

A macrophage receptor for (mannose/glucosamine)-glycoproteins of potential importance in phagocytic activity

Lung macrophages, in the absence of serum factors in vitro, strongly bound and ingested yeast cells ($\text{Candida}\text{krusei}$ and zymosan). Binding was temperature-and calcium-dependent, and was inhibited by the presence of D-mannose, D-glucosamine, horseradish peroxidase and beta-glucuronidase. Pretrypsinization of the macrophages also prevented binding of yeast cells. Binding was not affected by D-mannitol, D-glucose, D-galactose nor L-fucose. I suggest that macrophage binding of yeast cells is mediated by a mannose/glucosamine receptor on the cell membrane. This receptor may be responsible for opsosin-independent phagocytosis of activators of the alternative complement pathway and, as well, the phagocyte-dependent clearance of certain lysosomal enzymes.     

Mechanism of action of thrombin on fibrinogen. The reaction of thrombin with fibrinogen-like peptides containing 11, 14, and 16 residues

The following peptides were synthesized by classical methods in solution: Ac-Gly-Gly- Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (A), Ac-Ala-Glu-Gly-Gly-Gly-Val- Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (B), and Ac-Phe-Leu-Ala-Glu-Gly-Gly- Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (C). The rates of hydrolysis of the Arg-Gly bond of these three peptides by thrombin were measured, and the values of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ were found to be 0.05 × 10^?7 (A), 0.02 × 10^?7 (B), and 1.6 × 10^?7 (C) [(NIH units/ liter)s]^?1. The value of$\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ for peptide C is less than 1% of that for fibrinogen [although the value of k_cat itself, for peptide C (but not for A or B), is comparable to that for fibrinogen]. These results indicate that phenylanine and leucine at positions P₉ and P₈, respectively, play a key role in the reaction of thrombin with fibrinogen. The data also show that factors outside of the 16 residues of peptide C are important in determining the rate of hydrolysis of fibrogen by thrombin.     

A rapid,sensitive assay for starch phosphorylase and ADPglucose pyrophosphorylase

Starch phosphorylase (EC 2.4.1.1) and ADPglucose pyrophosphorylase (EC 2.7.7.27) activities can be measured very accurately and quickly using a recording pH meter. Activities less than 0.28 nmol/min are readily measured.     

A comparison of alternative measures of mutagenic potency in the Salmonella (Ames) test

Both the spontaneous and the induced mutation rates in Salmonella tester strains vary among different laboratories, and also within the same laboratory over time. If there is an association between spontaneous and induced mutagenesis, a measure of mutagenic potency that incorporates the background may be more consistent than the simple measure of the induced slope. We have used the statistical procedures recently described by Bernstein et al. (1982), and a large data-base of Salmonella test results to examine the association between spontaneous and induced mutation and to compare several alternative measures of mutagenic potency. A correlation analysis indicated an association between spontaneous and induced mutation for TA98, TA1537 and TA1535; TA1538 was close to being significant. This was observed over a wide range of chemicals. In addition, for TA98, for which we observed the strongest association, we obtained a rough estimate of the relationship between slope and intercept by using least squares to fit K and p in the power curve beta = k alpha p. We then chose 3 simple potency measures: the slope, the ratio of slope to spontaneous background, and the ratio of slope to the square-root of spontaneous background. These corresponded to the range of p's estimated from the least-squares fit procedure. The reproducibility of these measures was compared and no significant differences were found. Though there were some differences in the relative potency ranking of chemicals using the different measures, they were highly correlated.     

The type,amount, location,and energy transfer properties of the carotenoid in reaction centers from Rhodopseudomonas sphaeroides

Analysis of photosynthetic reaction centers from Rhodopseudomonas sphaeroides strains 2.4.1 and Ga shows that each contains approx. 1 mol of a specific carotenoid per mol of reaction center. In strain 2.4.1. the carotenoid is spheroidene (1-methoxy-3,4-didehydro-1,2,7′,8′-tetrahydro-ψ,ψ-carotene); in strain Ga, it is chloroxanthin (1-hydroxy-1,2,7′,8′-tetrahydro-ψ,ψ-carotene). The carotenoid is bound to the same pair of proteins as are the bacteriochlorophylls and bacteriopheophytins of the reaction center. This binding induces strong circular dichroism in the absorption bands of the carotenoid. The carotenoid is close enough to the other pigments of the reaction center so that light energy transfers efficiently from the carotenoid to the bacteriochlorophyll, sensitizing bacteriochlorophyll fluorescence. The fluorescence polarization spectrum of the reaction centers shows that the transition vectors for the visible absorption bands of the carotenoid lie approximately parallel to the 600 nm ($\text{Q}{}_{\mathrm{<mtext>x</mtext>}}$) transition of the bacteriochlorophyll complex.     

A rapid assay for prolyl 3-hydroxylase activity.

An assay is reported for prolyl 3-hydroxylase activity. The method is based on the release of tritiated water (THO) during 3-hydroxylation of a 2,3-T-l-proline-labeled (T = tritium) polypeptide substrate in which all prolyl residues recognized by prolyl 4-hydroxylase have been converted to 4-hydroxyprolyl residues. The formation of THO was essentially linear with enzyme concentration and time, and the K_m for the polypeptide substrate was about 3.4 × 10^?8m. A linear correlation was found between THO release and the synthesis of 3-hydroxyproline, the latter being analyzed by amino acid analyzer. The assay is simple, rapid, sensitive, and reproducible, and it is specific even in tissue samples containing a large excess of prolyl 4-hydroxylase activity.     

Optimization of Folin-Ciocalteu reagent concentration in an automated Lowry protein assay

The Lowry method (G. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, 1951, J. Biol. Chem.193, 265–275) for protein concentration measurement has been automated to permit assay of samples with concentrations from 1 to 400 μg/ml. Calibration with solutions of bovine serum albumin resulted in a nonlinear (quadratic) curve. The quantity of color developed in the assay was found to be strongly dependent on the concentration of the Folin-Ciocalteu phenol reagent. Color yield peaked sharply at a reagent concentration 40% lower than that used in the Lowry procedure. Optimization of the reagent concentration is necessary to obtain maximum sensitivity from the Lowry assay.     

A simple and rapid radio-receptor assay for the estimation of acetylcholine

The high potency with which acetylcholine (ACh) inhibits the binding of the specific muscarinic agonist, [³H]$\text{cis}$ methyldioxolane ([³H]CD), has provided the basis for the development of a radio-receptor assay for estimation of ACh. A synaptosomal preparation of the rat cerebral cortex was used as a source of muscarinic receptors. When binding assays were run at 0°C, the IC₅₀ value of ACh was approximately 5 × 10^?9 M, which corresponds to 2.5 – 10 pmoles of ACh, depending upon the assay volume. The ACh content of the rat cerebral cortex and corpus striatum was measured following fast microwave irradiation. By measuring the displacement of [³H]CD binding caused by aliquots of the supernatant from tissue homogenates and comparing the displacement values with an ACh standard curve, the ACh content of the cerebral cortex and corpus striatum was calculated to be 19 and 55 nmoles/g wet tissue weight, respectively.     

Colorimetric assay of liver and Escherichia coli asparatate transcarbamylases in the presence of 2-mercaptoethanol

A modification of the method of Prescott and Jones (1) for the colorimetric determination of carbamyl aspartate has been developed to permit the assay of aspartate transcarbamylases in the presence of 2-mercaptoethanol. Interference by this compound is eliminated by means of N-ethylmaleimide. The usefulness of the modified method is illustrated by examination of the contrasting properties of the Escherichia coli and rat liver enzymes.     

A rapid assay for epoxide hydratase activity with benzo(a)pyrene 4,5-(K-region-)oxide as substrate

A rapid radiometric assay for epoxide hydratase activity has been developed using the highly mutagenic [³H]benzo(a)pyrene 4,5-(K-region-)oxide as substrate. By addition of dimethylsulfoxide after the incubation, conditions were found where the unreacted substrate could be separated from the product benzo(a)pyrene-4,5-dihydrodiol(trans) simply by extraction into petroleum ether. The product is then extracted into ethyl acetate and, radioactivity is measured by scintillation spectrometry. This assay allows a rapid measurement of epoxide hydratase activity with an epoxide derived from a carcinogenic polycyclic hydrocarbon as substrate and is at the same time sensitive enough for accurate determination of epoxide hydratase activity in preparations with extremely low enzyme levels such as rat skin homogenate (8–14 pmol of product/mg of protein/min).     

Antiestrogenic and antifertility actions of anordrin (2α,17ga-diethynyl-A-nor-5α-androstane-2β,17β-diol 2,17-dipropionate)

Anordrin, administered in a single s.c. dose of 62.5 μg in sesame oil, stimulated sustained uterine growth (wet weight) when measured at 24 and 72 hr, but total soluble protein and total DNA per uterus was not increased. By comparison, 3 μg of estradiol-17β under the same conditions significantly increased all three parameters of uterine growth. Both of the above steroid treatments significantly increased nuclear estrogen receptor content of the uterus, but only the estradi-ol-17β treatment resulted in significantly elevated cytosol receptor content per uterus. Anordrin binds to the 8S estrogen receptor with an affinity of about 2 × 10⁵ M^-1 as determined by competition with [³H]estradiol-17β. The abortifacient activity of Anordrin when given orally (8 mg/kg b.w.) to mice on the 7th day of pregnancy was almost completely blocked by simultaneous oral administration of estradiol-17β (0.8 mg/kg b.w.). It is concluded that the actions of Anordrin on the uterus can be attributed to its antiestrogenic activities.     

Terminal deoxynucleotidyl transferase activity in a cell line (molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia

A terminal deoxynucleotidyl transferase having a sedimentation coefficient of 3–4S has been found associated with the chromatin from a cell line (Molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia.     

Formation of 3-(2'-deoxyribofuranosyl) and 9-(2'-deoxyribofuranosyl) nucleosides of 8-substituted purines by nucleoside deoxyribosyltransferase

Earlier results suggested that although the N-deoxyribosyltransferase from lactobacilli is a convenient tool for the preparation of analogs of 2'-deoxyadenosine, 8-substituted purines do not act as substrates. However, eight of nine 8-substituted purines that were examined proved to be substrates for the transferase from Lactobacillus leichmannii, and deoxyribonucleosides of four of these bases have been prepared. The substituents at C-8 of the purine greatly affect the rate of deoxyribosyl transfer to the base, and in all cases the rate is slower than transfer to purines lacking an 8-substituent. The 8-substituent also affects the nature of the nucleoside formed. With the electron-donating methyl group at position 8 of adenine, the transferase forms the expected 8-methyl-9-(2'-deoxyribofuranosyl)adenine. However, when purines bearing an electron-withdrawing substituent at the 8-position are used as substrates, the deoxyribosyl moiety is preferentially transferred to N-3 of the base. In the case of 8-trifluoromethyladenine the 3-deoxyribonucleoside is the only product detectable. With 8-bromo or 8-chloroadenine as substrate the 3- and 9-deoxyribonucleosides can both be isolated from the enzymatic reaction mixture. Time course studies indicated that with thymidine and 8-bromoadenine as substrates the 3-deoxyribonucleoside is initially the major product, but that the 9-deoxyribonucleoside becomes the major product after long incubation periods. Negligible interconversion of these nucleosides occurs in the absence of transferase, but conversion in either direction occurs readily in the presence of the enzyme. Significant hydrolysis of pyrimidine and purine deoxyribonucleosides occurs in the presence of the transferase. This was more obvious during the course of reactions involving 8-substituted purines because the slowness of deoxyribosyl transfer required longer incubation periods and larger amounts of enzyme. The hydrolysis is proportional to enzyme concentration, little affected by the nature of the base and is attributed to hydrolysis of a deoxyribosyl derivative of the transferase which is an obligatory intermediate of deoxyribosyl transfer. 8-Trifluoromethyl-3-(2'-deoxyribofuranosyl)adenine, 8-methyl-9-(2'-deoxyribofuranosyl)adenine, and 8-bromo-9-(2'-deoxyribofuranosyl)adenine were tested for their ability to inhibit the growth of CCRF-CEM cells in culture. Unlike the potent 2-halogeno-2'-deoxyadenosine derivatives, these three nucleosides cause less than 50% inhibition at concentrations up to 100 microM.     

Antibody to a sperm surface glycoprotein inhibits the egg jelly-induced acrosome reaction of sea urchin sperm

When the surface of sea urchin (Strongylocentrotus purpuratus) sperm is radioiodinated, 75% of the protein-incorporated radioactivity is associated with two glycoproteins of M_r 84,000 (84K) 64,000 (64K) (Lopo and Vacquier 1980). Antibodies were prepared against these two components by separating a Triton X-100 extract of sperm on SDS-polyacrylamide gels, cutting out the band containing the glycoprotein and injecting the homogenized gel into rabbits. Both anti-84K and anti-64K sera agglutinate sperm. Light and EM immunoperoxidase localization show both antigens are distributed over the entire sperm surface. By the immunoperoxidase technique there is some degree of cross-reactivity of both antisera with sperm of other Strongylocentrotus species, but not with those of other genera. Living sperm incubated with anti-84K Fab fragments are completely inhibited from undergoing the egg jelly-induced acrosome reaction and fertilizing eggs. Anti-64K Fab fragments have no effect on the ability of the sperm to undergo the acrosome reaction or fertilize eggs. Sperm incubated in anti-84K or anti-64K Fab fragments undergo the acrosome reaction in response to the Ca²⁺ ionophore A23187, or when the extracellular pH is increased to 9.2 with NH₄OH, indicating that the inhibition of the egg jelly-induced acrosome reaction results from the binding of the anti-84K Fab to an external molecule involved in the initiation or propagation of the acrosome reaction. The 84K glycoprotein is the first sperm surface component identified that might have a role in the induction of the acrosome reaction.     

Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia

The tissue distribution and the effects of starvation and streptozotocin-induced diabetes on insulin B chain-degrading neutral peptidase activity in the rat have been studied. The neutral peptidase activity in tissue extracts was determined by measuring the formation of trichloroacetic acid-soluble radioactivity from ¹²⁵I-labeled B chain of insulin in 0.1 m Tris buffer (pH 7.2). Inhibition by several different compounds (EDTA, dithiothreitol, and potassium phosphate) which are known to inhibit the purified enzyme and the effects of pH suggest that the B chain-degrading activity measured in each of 12 tissue extracts may be similar to the neutral peptidase recently purified from rat kidney (P. T. Varandani and L. A. Shroyer, 1977, Arch. Biochem. Biophys., 181, 82–93). Neutral peptidase activity was observed in all tissues examined and varied in the order kidney ? intestine > pancreas, testis > liver > thymus > heart, skeletal muscle, diaphragm > lung, spleen > fat. Neutral peptidase activity in kidney, liver, fat, and skeletal muscle from diabetic animals was significantly depressed when compared with the levels in these tissues from normal animals. Insulin treatment of diabetic animals raised the neutral peptidase activity in kidney, liver, and fat to levels equivalent to or even exceeding normal levels; however, activity in skeletal muscle persisted at depressed levels. Heart muscle neutral peptidase activity was not significantly affected in either diabetes or starvation. In the liver, starvation reduced the level of neutral peptidase activity while subsequent refeeding raised the activity to a level exceeding the control. Opposite effects were observed in kidney: starvation increased neutral peptidase activity while refeeding brought the activity back to normal levels. Only small decreases in neutral peptidase activity were observed in fat and skeletal muscle after 24 h starvation, but were not evident after 64 h starvation. The changes in neutral peptidase activity correlated well with the changes in glutathione-insulin transhydrogenase activity previously reported in liver and kidney.