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 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 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.     

Evidence for a single catalytic site on the "beta-D-glucosidase-beta-D-galactosidase" of almond emulsin.

An isoenzyme of glycosidase obtained from almond emulsin, which is both a β-d-glucosidase and a β-d-galactosidase, has now been shown to possess β-D-fucosidase activity. It has been concluded that all three activities reside in a single catalytic site for the following reasons. (i) d-Glucosylamine, d-galactosylamine, and d-fucosylamine (a newly discovered potent inhibitor of this enzyme) each act competitively against all three of the substrates. (ii) Any given inhibitor exhibits the same K_i value when tested in the presence of any of the three substrates, (iii) When the enzyme is incubated with any two of the p-nitrophenyl glycoside substrates, at or above their respective K_m values, the rate of p-nitrophenol formation is not additive, but rather is equal to the value calculated on the basis of the individual K_m values and relative maximum velocities.     

Separation and identification of subpicomole amounts of methotrexate polyglutamates in animal and human biopsy material

A rapid, quantitative procedure for separating methotrexate (MTX) polyglutamates utilizing molecular-sieve column chromatography has been developed. MTX polyglutamates were separated in 25 min and detected by a radioassay with a sensitivity of 0.1 pmol MTX, allowing analysis on 2 to 5 mg of tissue. This technique has been used to demonstrate and quantitate the levels of MTX polyglutamates in liver, kidney, and brain tissues of rats treated with weekly low doses of MTX, and in liver and red cells of patients with acute leukemia treated with weekly low doses of MTX.     

Enzymatic assays of L-serine in biological material

Affinity chromatography of adenosine deaminase (EC 3.5.4.4.) on agarose-bound inosine with biospecific elution of the enzyme using linear gradients of adenosine or inosine leads via chromatographic parameters to a dissociation constant of the binary complex of K_diss = 3.5 × 10^?3m and to a binding enthalpy of ΔH = ?3.9 kcal mol^?1. These values can be explained by formation of two hydrogen bonds between immobilized inosine and the enzyme. The measurement of height equivalents of theoretical plates of the affinity column with dependence on the flow rate leads to the assumption that the velocity with which the equilibrium is reached is high compared with the flow rate; the high specificity of the affinity resin is not first of all due to a high number of theoretical plates but to the selectivity of the heterogenous enzymic reaction.     

Improvements on the Prescott-Jones method for the colorimetric analysis of ureido compounds

The colorimetric assay procedure of Prescott and Jones ((1969) Anal. Biochem.32, 408–419) has been modified to make the standard curve more consistently linear. Improved results are obtained when the assay tubes are exposed to yellow light at an intensity of 45–50 fc (480–540 lx) during color development. Somewhat longer incubation times are required for color development with yellow light.     

Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

The amino acid sequence of the $\text{Spirulina maxima}$ ferredoxin has been determined. $\text{Spirulina maxima}$ is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The $\text{S. maxima}$ ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.     

Altered interaction between binding and catalytic subunits of a cyclic AMP-stimulated protein kinase from hepatoma cells.

Binding activity obtained from an established line of hepatoma tissue culture (HTC) cells has a lower apparent affinity for cyclic AMP at physiological pH than has the analogous binding activity from rat liver. However, the apparent binding affinity of HTC preparations can be reversibly increased by adding NaCl or guanidine · HCl. In the presence of such activating substances, a macromolecular inhibitory activity has been chromatographically separated from the cyclic AMP-binding activity. Removal of this inhibitory component causes the apparent affinity of the cyclic AMP-binding activity from HTC cells to increase and resemble that observed with liver preparations. Before treatment with salt, the inhibitory activity seems to be physically associated with the binding activity. Adding the isolated inhibitory component back to a suitably activated binding preparation from HTC cells results in a decrease in the apparent affinity for cyclic AMP. The isolated inhibitory component is devoid of cyclic AMP-binding and cyclic AMP phosphodiesterase activities and has an apparent minimal molecular weight of about 30,000 by gel filtration. It possesses protein kinase activity and seems to be identical to the catalytic subunit of a cyclic AMP-stimulated protein kinase on the basis of chromatographic properties and sensitivities to heat and low pH. This catalytic subunit represents only a minor portion of total cellular protein kinase activity and is also present in liver extracts. However, the binding activity from liver is not inhibited significantly under conditions where the binding from HTC cells is affected by the catalytic subunit. The difference in this inhibitory response between liver and HTC preparations appears to reflect differences in the cyclic AMP-binding proteins themselves.     

The determination of dissociation constants by affinity electrophoresis on Cibacron Blue F3G A-agarose-polyacrylamide gels

The application of copolymerized agarose-polyacrylamide gels as the support for immobilized Cibacron Blue F3G A is demonstrated for the analytical electrophoresis of proteins possessing an affinity for this dye. Bovine serum albumin was used as a model protein to develop this technique. The optimal conditions for preparing matrices are described. These conditions produce gels with suitable mechanical strength and which allow rapid electrophoresis of proteins. The dye-agarose-polyacrylamide gels permit the determination of dissociation constants. The ease of preparation of these matrices recommends them for a variety of quantitative analytical investigations.     

A secondary attachment site for bacteriophage lambda in trpC of E. coli.

We have determined the nucleotide sequence of a secondary lambda attachment site in trpC. Direct sequence analysis of lambdatrp transducing phage DNA fragments carrying the two prophage attachment sites reveals a 6 nucleotide homology in the crossover region which is a subset of the 15 nucleotide core sequence in the primary lambda attachment site: GCTTTTTTATACTAA. This 6 nucleotide sequence is also present in the intact trpC genome at the attachment site, as shown by analysis of trpC mRNA spanning this region.     

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.     

A simple, automated apparatus for the rapid multiple flushing of reaction (assay) vessels with gases

Folin and Ciocalteu's phenol reagent may be reduced by a variety of compounds, which therefore interfere with the Lowry method of protein determination (1,2). Peters and Fouts (3) reported that two of the zwitterionic biological buffers described by Good et al. (4) reacted strongly with the Folin reagent and thus seriously interfered with protein determinations. The zwitterionic buffers have many properties which lead to their use in biological work, where protein estimations will be required. Since the publication of Good et al. (4), the range of zwitterionic buffers has been increased. The possibility that these other buffers may also produce artefacts has therefore been investigated.     

Increased concentration of CTP synthetase in hepatoma 3924A: immunological evidence

CTP synthetase (UTP:L-glutamine ligase, EC 6.3.4.2) was purified 370-fold from rapidly growing rat hepatoma 3924A. A major band was demonstrated by acrylamide gel electrophoresis which corresponded to this enzymic activity. It was estimated that the enzyme was 90% pure. Antibodies were produced in rabbit using this purified hepatoma enzyme. The specificity of the anti-serum was proved by the absence of the reaction between control serum and CTP synthetase. The amount of anti-serum required to inactivate completely the cytosolic CTP synthetase of hepatoma 3924A was 11-fold of that required for normal liver which is in good agreement with the 11-fold increase in CTP synthetase activity in this hepatoma. These results demonstrate that the liver and hepatoma 3924A CTP synthetases were immunologically similar or identical and that the markedly increased enzymic activity in hepatoma 3924A reflected an increase in the enzyme protein amount. These studies provide further evidence that in the neoplastic transformation a reprogramming of gene expression takes place which is manifested in the emergence of increased concentrations of CTP synthetase which should provide selective advantages to cancer cells by increasing the capacity for this rate-limiting step in $\text{de novo}$ CTP biosynthesis.     

A novel arrangement of the 18S and 28S sequences in a repeating unit of Drosophila melanogaster rDNA.

The sequences corresponding to the 18S and 28S rRNAs have been mapped within a cloned 17 kilobase (kb) fragment formed by Eco R1 cleavage of Drosophila melanogaster rDNA. This fragment, Dm103, represents the longer of two major types of repeating units that are present in the rDNA of this fly, and was cloned as a hybrid plasmid, pDm103, consisting of Dm103 inserted at the Eco R1 site of the pSC101 vector (Glover et al., 1975). Mapping of the 18S and 28S rDNA in Dm103 was accomplished by quantitative determination of the amount of these rDNAs in each member of an ordered set of restriction fragments obtained by Hind III and Eco R1 ccleavage of pDm103. The amounts of 18S and 28S rDNAs were determined by hybridization of the rRNAs to fragments that were purified by cloning, and an unambiguous order of the fragments within pDm103 was established by heteroduplex mapping and from the stoichiometry of the fragment lengths. The resulting map revealed that the 4 kb of 28S rDNA within the long repeating unit represented by Dm103 is divided into two blocks that are separated by 5.4 kb of DNA of unknown function. It is this unusual arrangement of the 28S rDNA that distinguishes the long repeating units (17 kb) from the short units (11.5) kb), whose 4 kb of 28S rDna is confined to a single block, as is shown in the accompanying paper (White and Hogness, 1977). The remainder of the DNA in this long unit appears to be typically arranged, with the 2 kb of 18S rDNA confined to a single block that is separated by about 1 kb from the closest block of 28S rDNA.     

A measurement of the sequence complexity of polysomal messenger RNA in sea urchin embryos

The first measurement has been made of the number of diverse mRNA sequences (mRNA sequence complexity) in the total polysomes of a eucaryotic system, the sea urchin gastrula. mRNA was purified of nuclear RNA and any other heterogeneous RNA contaminants by release from polysomes with puromycin. Trace quantities of labeled nonrepetitive DNA fragments were hybridized with an excess of mRNA. The hybridization reaction followed ideal first order kinetics in mRNA concentration. At completion of the hybridization reaction, 1.35% of the nonrepetitive DNA was present as mRNA-DNA hybrid. The hybridized DNA was extracted and was at least 70% hybridizable with mRNA, demonstrating a 50-fold purification of the expressed sequences. This purified DNA fraction reassociated with excess unfractionated sea urchin DNA at a rate identical to that of the total nonrepetitive DNA tracer. The mRNA had therefore been hybridized to nonrepetitive DNA sequence, and the amount of hybrid could be used as a direct measure of the mRNA sequence complexity.The complexity of the gastrula mRNA can be calculated as about 17 million nucleotides, sufficient to comprise some 14,000 distinct structural genes. This result also provides an estimate of the number of diverse proteins being translated in the gastrula. From the rate of mRNA-DNA hybrid formation, we estimate that about 8% of the mRNA belongs to this complex class, and that less than 500 copies of each species of message in this class exist per embryo. Most of the mRNA population consists of a relatively small number of diverse species represented a much larger number of times.     

A clonal analysis of tergite development in Drosophila of ultraabdominal and paradoxical genotypes.

The developmental parameters of homeotic second abdominal anlage cells in flies with Ultraabdominal and paradoxical genotypes are compared with those of normal second abdominal anlage cells through the use of induced mitotic recombination to mark the clonal descendants of single anlage cells. Homeotic and normal second abdominal anlage cells show the same pattern of mitotic activity during development. The homeotic second abdominal anlage cells with Ultraabdominal genotype proliferate to the same extent as normal anlage cells during hemitergite formation. However, the proliferation of homeotic second abdominal anlage cells with paradoxical genotype is decreased due to the failure of some daughter cells either to divide or to differentiate normally. The number of anlage cells in a homeotic second abdominal histoblast with Ultraabdominal genotype is slightly smaller and more variable than that in a normal second abdominal histoblast. The number of anlage cells in a homeotic second abdominal histoblast with paradoxical genotype is much smaller and much more variable than that in a normal second abdominal histoblast. These results are discussed in relation to mechanisms governing cell determination. In addition, some aspects of pattern formation in incomplete homeotic second abdominal hemitergites are presented and discussed.     

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.