A radioenzymatic assay for quinolinic acid

A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing [3H]ATP, further to [3H] deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.     

Accurate assay of dopa decarboxylase by preventing nonenzymatic decarboxylation of dopa

The nonenzymatic decarboxylation of dopa was completely blocked by both 2-mercaptoethanol and EDTA together over the wide range of pH. This finding made it possible to measure the activity of dopa decarboxylase precisely even at an alkaline pH value. The pH optimum of dopa decarboxylase was found to be pH 7.0 and the Km value for dopa was determined to be 4 X 10(-5) M.     

Electron Microscopic Observations on the Ribonucleic Acid of Murine Leukemia Virus

The ribonucleic acid (RNA) of murine leukemia virus (MLV) Rauscher strain was observed by the aid of electron microscopy with the use of the protein monolayer technique. RNA was observed directly after release from virus particles or after isolation by sedimentation in sucrose density gradients. Molecules were found in an extended linear form. Many of the RNA filaments released by detergent treatment contained curled regions, suggesting the linear filaments were originally coiled within the virus particle. The relationship of the curled areas to the containment of the RNA within the virus particle is discussed, and a mechanism for the inclusion of RNA in the budding virion is proposed. Treatment of the extended MLV-RNA with dimethyl sulfoxide resulted in the collapse of the molecule forming a tangled complex. Treatment with urea or heating at 50 C in 3 mm NaCl also produced this effect. Also under the conditions in which MLV-RNA was linear, RNA from Rous sarcoma virus also was linear, but Newcastle disease virus RNA and ribosomal RNA of rat liver had collapsed structures. The results indicated that the RNA of MLV, and perhaps other RNA-containing tumor viruses, has a specific unique conformation dependent upon hydrogen bonds.     

Tissue-Specific Isoforms of Catalase Subunits in Castor Bean Seedlings

Catalases purified from endosperm glyoxysomes and non-specializedmicrobodies from hypocotyls of castor bean seedlings differedin their specific activity [90–164 and 0.89–4.9kunits (mg protein)^–1, respectively] and in their constituentsubunits [two subunits of 54 and 56 kDa for the endosperm enzymeand only one of 56 kDa for the hypocotyl enzyme]. Immunoblotanalysis also showed that particulate fractions from the endospermsand from etiolated and green cotyledons contained two catalasesubunits of 54 and 56 kDa, whereas such fractions from the hypocotylsand roots contained only the 56-kDa subunit. Leaf peroxisomesfrom green leaves had two catalase subunits of around 55 kDaeach. Results of translation in vitro indicated that the 54-and 56-kDa subunits were translated from distinct mRNAs andlevels of both mRNAs increased in the endosperms during germination,prior to increases in levels of catalase proteins. In the hypocotyls,the 56-kDa subunit seemed to be synthesized constitutively. ¹Present addresses: YO, Toyota Central Institute, 31-9 Musashizuka,Nagabuchi, Nagakute, Aichi 480-11, Japan     

Abscisic acid from Pinus densiflora pollen

(+)-Abscisic acid was isolated as the methyl ester from Pinus densiflora pollen and identified spectroscopically.     

One molecule of diphtheria toxin fragment a introduced into a cell can kill the cell

Erythrocyte ghosts containing a known number of molecules of purified fragment A of diphtheria toxin with a constant amount of FITC-BSA as a fluorescence marker were prepared by dialyzing a mixture of erythrocytes and these substances against hypotonic solution. These substances were then introduced into diphtheria toxin-resistant mouse L cells by virus-mediated cell fusion of the cells with the ghosts, and mononuclear recipients that had fused with only one erythrocyte ghost were separated in a fluorescence-activated cell sorter (FACS) on the basis of their cell size and fluorescence intensity. After separation, the viability of cells containing known numbers of fragment A was examined by measuring colony-forming ability. The results demonstrated that a single molecule of fragment A was sufficient to kill a cell.This fact was confirmed by introduction into cells of fragment A from an immunologically related mutant toxin, CRM 176 (fragment A-176); this has a completely functional fragment B region, but in cell extracts, the enzymic activity of its fragment A is about 10 fold less than that of wild toxin. The cytotoxicity of CRM 176 is about two hundredths of that of the wild-type (Uchida, Pappenheimer and Greany, 1973). As expected, about 100–200 fold excess of fragment A-176 was needed to kill the cells.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.     

Conversion of Tyrosine Hydroxylase to Stable and Inactive Form by the End Products

We have reported previously that tyrosine hydroxylase in the crude extract from rat striatum exists in the inactive form showing almost no activity at the physiological pH and that the inactive form is produced by the action of the end products of the enzyme, such as dopamine. The incubation of the enzyme with the end products resulted in not only the inactivation but also a remarkable stabilization of the enzyme. Catechols possessing amino groups but no negatively charged groups on the side chains (catecholamine-type catechols) were effective at a concentration as low as 10(-7) M in both the inactivation and stabilization of the enzyme. In contrast, catechols not possessing positively or negatively charged side chains (3,4-dihydroxyphenylethyleneglycol-type catechols) were ineffective at a concentration of 10(-7) M but effective at a concentration of 10(-6) M for both the inactivation and stabilization. Catechols possessing negatively charged groups (3,4-dihydroxyphenylacetic acid-type catechols) were ineffective even at a concentration of 10(-6) M. Thus, the end products of tyrosine hydroxylase appear to serve to keep the enzyme inactive and stable. The reaction mechanism of the conversion of the enzyme from the active/labile form to the inactive/stable form by dopamine was also investigated.