Automated fluorometric determination of free and total tryptophan in plasma

We have developed a sensitive automated fluorometric method based upon the manual procedure of Denckla and Dewey [(1967) J. Lab. Clin. Med.69, 160–169] for determining both free and total tryptophan in plasma. Free tryptophan is measured in a series of increasingly diluted aliquots of a sample of plasma after tryptophan bound to albumin is removed by continuous-flow dialysis. Free and total tryptophan are then derived from Scatchard plots of the data. The method can be used for nutritional assessments, clinical investigation of behavioral disorders in which serotonin is implicated in the pathogenesis, and studies on tryptophan transport and metabolism.     

The synthesis of 4-methylumbelliferyl alpha-ketoside of N-acetylneuraminic acid and its use in a fluorometric assay for neuraminidase

4-Methylumbelliferyl α-ketoside of N-acetylneuraminic acid was synthesized by reacting the sodium salt of 4-methylumbelliferone with the 2-chloro-2-deoxy derivative of peracetylated methyl N-acetylneuraminate, followed by preparative silica gel chromatography, deblocking, and purification by gel filtration on Sephadex G-25. The final product was isolated as either the sodium or ammonium salt, and its suitability as a substrate for neuraminidase was evaluated. The optimal pH values for various neuraminidases were 5.6 in acetate buffer (Arthrobacter ureafaciens), 5.0–5.1 in acetate buffer (Clostridium perfringens), and 4.4 in phosphate-citrate buffer (human fibroblasts). K_m values for these enzymes at the optimal pH were 6 × 10^?4m (Arthrobacter), 1 × 10^?4m (Clostridium), and 3 × 10^?4m (human fibroblasts).     

Synthetic and biosynthetic studies of porphyrins: III. Structures of the intermediates between uroporphyrinogen-III and coproporphyrinogen-III: Synthesis of fourteen heptacarboxylic,hexacarboxylic, and pentacarboxylic porphyrins related to uroporphyrin-III

Four heptacarboxylic, six hexacarboxylic, and four pentacarboxylic porphyrins related to uroporphyrin-III by decarboxylation of one, two, or three of the acetic acid side chains have been synthesised as their methyl esters by application of the MacDonald or b-oxobilane methods, as appropriate. Comparison (mixed mp, “mixed” nmr spectra, and hplc) of the synthetic materials with the methyl esters of hepta-, hexa-, and pentacarboxylic porphyrins isolated from natural sources showed that the structures of the latter corresponded to the D-ring methyl, the DA-dimethyl, and the DAB-trimethyl analogs of uroporphyrin-III. Because the naturally occurring porphyrins arise by oxidation of intermediate porphyrinogens, we conclude that the enzymic decarboxylation of uroporphyrinogen-III to coproporphyrinogen-III takes place in a preferred sequential clockwise fashion (both in normal and abnormal metabolism) starting with the acetic acid moiety on the D-ring and followed by those on the A, B, and C rings.     

Relation between single-strand DNA mass and sedimentation distance in alkaline sucrose gradients

Single-strand breaks are introduced into bacteriophage T2 and Bacillus subtilis DNA in dilute solution with gamma rays and the DNA sedimented on alkaline sucrose gradients. Assuming (1) the number of single-strand breaks is linear with dose, and (2) the distance sedimented in alkaline sucrose gradients D is proportional to M^α (M is the single-strand DNA mass), the value of a is determined to be 0.40.     

Staining and destaining polyacrylamide gels: A comparison of Coomassie blue and fast green protein dyes

A method using fast green dye for quantitation of immune serum globulin and its products of fragmentation in polyacrylamide gels has been developed. Although fast green is shown to be somewhat less sensitive than the usually used Coomassie blue stain, the former dye does not suffer from selective loss of dye due to temperature or alcohol content of the destaining solution. Destaining of fast green-stained gels is accomplished rapidly and efficiently by the described destaining procedure without an accompanying loss in quantitation.     

Dihydroorotase from Clostridium oroticum is an allosteric enzyme.

Dihydroorotase from $\text{Clostridium}$$\text{oroticum}$ exhibits allosteric behavior with respect to both of its substrates. L-dihydroorotate dependence reflects a positive homotropic interaction for which the Hill coefficient is 1.3–1.6, depending upon the preparation. Conversely, a negative homotropic response is observed when L-ureidosuccinate serves as substrate, as characterized by a Hill coefficient of 0.65–0.75. Interaction between L-dihydroorotate binding sites is a labile characteristic lost during enzyme purification. Negative cooperativity of ureidosuccinate binding appears to be more stable. The effects of purification and medium are also discussed.     

Peroxidase activity in human red cell: a biological model for excited state molecules generation.

The presence of enzymically generated triplet acetone in red cells and energy transfer to eosin, rose bengal and 9,10-dibromoanthracene-2-sulfonate was indicate by: (1) product distribution; (2) K_ET τ_o, similar to the 2-methylpropanal/peroxidase/O₂ system; (3) correlation between hemolysis, oxygen uptake and photon emission; (4) membrane protection by energy acceptors, and (5) by comparison of the 2-methylpropanal/peroxidase/O₂ system with 2-methylpropanal/red cells/membranes/O₂ and 2-methylpropanal/acid extractable protein from red cells membrane/O₂ systems, which have a high peroxidase activity.This is the first report of a biological system producing a photohemolysis effect in the dark.     

Outer membrane of Salmonella typhimurium: Identification of proteins exposed on cell surface

Proteins exposed on the outer surface of the outer membrane of Salmonella typhimurium were identified by reacting intact cells with a covalent labeling reagent. Since the outer membrane permitted the free diffusion of small hydrophilic molecules, we used a macromolecular reagent, CNBr-activated dextran, as the non-penetrating labeling agent. We also used a mutant producing a lipopolysaccharide with a very short (i.e. hexasaccharide) carbohydrate chain, in order to avoid steric hindrance by the carbohydrates on membrane surface. Results showed that out of the four “major” proteins of molecular weight around 35 000, three were exposed, and that at least six other proteins were also exposed on cell surface. Only two or three outer membrane proteins consistently did not react with the reagent in intact cells.     

Fluorescence and photoelectron studies of the intercalative binding of benz(a)anthracene metabolite models to DNA

An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.     

Facile assay of enzymes unique to the Calvin cycle in intact cells, with special reference to ribulose 1,5-bisphosphate carboxylase.

A procedure for the facile measurement, in intact cells, of two enzymes unique to the Calvin cycle, ribulose 1,5-bisphosphate carboxylase and phosphoribulokinase, is described. The procedure involved a simple toluene treatment to render phototrophic cells permeable to the necessary substrates, effectors, and cofactors. Whole-cell ribulose 1,5-bisphosphate carboxylase activity quantitatively approximates the activity obtained in cell-free extracts. In addition, the activity measured with toluene-treated whole cells results in a stoichiometric carboxylation of ribulose 1,5-bisphosphate to phosphoglyceric acid. The assay procedures described are most convenient for determining enzyme levels as a function of growth. Moreover, such an assay should open the way to further studies on the regulation of CO₂ assimilation by direct measurement of the enzymes concerned within the cell.     

An improved method for the quantitative determination of deoxyribonucleoside triphosphates in cell extracts

The analysis of deoxyribonucleoside 5'-triphosphates (dNTPs) in cell extracts by high-pressure liquid chromatography [C. Garrett , and D.V. Santi (1979) Anal. Biochem. 99, 268-273] requires the prior, selective degradation of ribonucleoside 5'-triphosphates ( rNTPs ) that are present in the extracts in large quantities. When this method was used for quantifying the dNTPs in mammalian cell extracts, the presence of an interfering peak in the HPLC between the peaks for dTTP and dATP was observed. This unwanted peak sometimes overlapped with that of dATP, depending on the pH of the eluant. It was found that the material which gave this peak was formed during the periodate oxidation of rNTPs in the presence of methylamine, and that it could be removed by changing the order of addition of the reagents in the procedure, i.e., the methylamine was added only after the excess periodate was decomposed, instead of adding it together with periodate, as given in the original procedure. Furthermore, an addition of deoxyguanosine to the reaction mixture was found to be effective in preventing the partial loss of dGTP in the oxidation procedure. By using this improved method, the dNTP contents of the extracts of Ehrlich ascites tumor cells have been measured in an accurate and reproducible manner. The analysis requires about 10(6) cells, and all four dNTPs can be quantified in 2.5 h, starting from the harvest of the cells.     

Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation

A method is presented for preparing very large DNA from Bacillus subtilis protoplasts. When the DNA is characterized by sedimentation in neutral sucrose gradients, a fast-sedimenting component is found whose sedimentation coefficient varies with centrifuge speed. By use of [³H]thymine label for the DNA and a ¹⁴C-labeled amino acid, it is shown that less than 5% cellular material other than DNA is associated with this component. Irradiation of this DNA in solution with gamma rays forms a slower component, called the “main peak”, whose sedimentation coefficient also depends on centrifuge speed. More irradiation breaks down this main peak into even slower-sedimenting DNA; it is shown that for low doses, double-strand breaks are formed in both the B. subtilis DNA and in bacteriophage T2 DNA at the same rate linear in dose, 0.018 double-strand breaks per kilorad per mass equal to that of T2 DNA.The speed dependence of the DNA sedimenting at the main peak is compared with an approximate theory of the speed dependence of the sedimentation coefficient of linear DNA by B. H. Zimm (unpublished calculations). The comparison suggests that for sufficiently high centripedal acceleration, DNA molecules larger than a critical mass will sediment at much the same velocity. The theory, and data on the break-up of the DNA with gamma rays, are used to estimate that the DNA extracted is at least 13 times the mass of T2 DNA, and possibly larger.In the Appendix, data from the literature are put together with data taken during this work to make plausible the assumption that the usual theory for the sedimentation of DNA molecules, experimentally tested in salt solutions, may also be applied to sucrose solutions. If, in neutral sucrose gradients, the distance sedimented is proportional to a power α of the mass, the best value of α = 0.38.     

A generalized,circular dichroism rule for nitrogen heterocycles attached to alditol residues

For alditol-1-yl derivatives of nitrogen heterocycles having C-1 of the alditol-1-yl group in the R configuration and having this group lying “below” the base, the sign of the Cotton effect will be negative if, for the most stable conformer, the bond to C-1 and the dipole-moment vector of the base point in the same direction, and positive if they they point in opposite directions. If the alditol-1-yl bond is essentially aligned with the dipole-moment vector of the nitrogen heterocycle, the sign of the Cotton effect is independent of the conformation. Consequently, if the dipole-moment vector of the nitrogen heterocycle and the alditol-1-yl group point in the same direction, the sign of the Cotton effect will be negative; it will be positive if the two oppose each other.     

myo-Inositol metabolism in rat testis in response to streptozotocin-induced diabetes

The effects of streptozotocin-induced hyperglycemia on de novo myo-inositol biosynthesis in rat testis was examined. Testicular glucose and glucose 6-phosphate levels increased significantly 10 and 12 h after stretozotocin injection, respectively. However, testis myo-inositol content did not increase appreciably until 24 h following injection of the drug. Seventy-two hours after streptozotocin administration, testis myo-inositol levels were 2.7-fold higher in diabetic rats than in controls injected with citrate buffer. No changes were observed in the Specific activities of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and 1-l-myo-inositol-1-phosphatase (EC 3.1.3.25). However, hyperglycemic rats displayed testicular glucose and glucose 6-phosphate levels approximately 4- and 2-fold in excess of control values, respectively. Insulin treatment of diabetic rats resulted in the lowering of plasma glucose, and testis glucose 6-phosphate to normal or below normal levels within hours. Inositol levels remained significantly elevated compared with control animals, although slightly lower than that observed for untreated diabetic rats. Streptozotocin diabetic rats had a significantly decreased testis cytosolic $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio compared with control animals 72 h after injection. The potential role of testis hexokinase distribution in the regulation of glucose 6-phosphate and myo-inositol biosynthesis in normal and diabetic rats was investigated. No significant differences in testis hexokinase distribution or in the kinetic characteristics of the soluble and particulate hexokinase activities were observed. Testicular sperm counts in streptozotocin diabetic rats were not significantly different from control values.