Synthesis of leucine enkephalin derivatives: structure-function studies

Nucleoli isolated from livers of rats injected intraperitoneally with one dose of thioacetamide had a five-fold increase in the rate of RNA synthesis $\text{in vitro}$ when compared with livers of rats treated with saline or CCl₄. The stimulation was maximal 24 hours after treatment and decreased to control values 73 hours after treatment. The enhanced level of nucleolar activity was maintained at that level when thioacetamide was injected daily. Along with the increase in the endogenous activity there was a 7-fold increase in the “free” RNA polymerase I activity determined by blocking the bound enzyme with actinomycin D (7). The nucleoli of the thioacetamide-treated rats offer a useful model of modulation of ribosomal gene function.     

Sensitivity of Escherichia coli acrA mutants to psoralen plus near-ultraviolet radiation

The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E. coli strains differing at the acrA locus. Survival was determined for both bacteria and phage lambda. AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA. Free lambda phage exposed to PUVA survived as well when plated on acrA mutants as on wild type. In contrast, prophage lambda CI857 ind carried in lysogenic acrA strains was hypersensitive to PUVA. The enhanced sensitivity of bacterial and lambda DNA, when inside acrA cells, was paralleled by an increased photobinding of radiolabelled psoralens in the mutant. Binding was increased specifically to DNA rather than to nucleic acids in general. The difference in psoralen-binding ability determined by the acrA gene persisted after permeabilizing treatment of the cells. The results suggest that the acrA mutation causes an alteration specifically in the environment of the cellular DNA so as to allow increased intercalation and photobinding of psoralens.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

Dimers of nicotinamide adenine dinucleotide: New evidence for the structure and the involvement in an enzymatic redox process

The composition and the structure of the product from the known electrochemical dimerization of the NAD⁺ have been conclusively demonstrated. A detailed analysis of the ¹H and ¹³C nmr spectra has in fact led to the conclusion that the product contains three diastereoisomeric dimers of the 4,4′-tetrahydrobipyridyl type. Furthermore, the cytoplasmic fraction obtained from a standard mitochondrial preparation of rat liver has been shown to catalyze the oxygen uptake by the dimers. A 1 : 1 molar ratio of the reagents in the redox process is indicated by manometric data on oxygen uptake complemented by spectrophotometric analysis of the oxidized substrates, suggesting that H₂O₂ is the reduction product. NAD⁺ was identified as the oxidation product by an enzymatic method.     

Modification of an essential arginine of carbamate kinase

Carbamate kinase from Streptococcus faecalis is inactivated by butanedione in borate buffer, which implies the presence of an essential arginine at the active site of the enzyme. The inactivation reaction is first order in [butanedione] and a replot of the inactivation rate data infers that one arginine is modified. The enzyme is protected against inactivation by ADP, ATP, the metal-nucleotides and carbamyl phosphate but not by carbamate. Amino acid analyses reveal that one of three arginines is modified by butanedione in the absence of protecting agents, and the binding of ADP to the enzyme prevents modification. Thus, analysis of the data suggest that (i) substrate binding to arginine and (ii) protein conformational changes at the active site are responsible for protection of an essential arginine against modification by butanedione.     

Preparation and spectroscopic characterization of molecular species of brain phosphatidylserines

This study describes the first preparation and spectroscopic characterization of naturally occurring phospholipids separated according to degree of unsaturation. Phosphatidylserines (PS) have been prepared from bovine brain and shown to be pure by extensive thin layer chromatographic analysis as well as by infrared spectroscopy and fatty acid analysis. The PS has been separated according to degree of unsaturation and prepared using AgNO3-impregnated silica gel H thin-layer chromatography. Fatty acid analysis of the two principal PS subfractions indicates that they are enriched in the molecular species 1-octadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphorylserine. The identity of the two PS subfractions was further verified by rechromatographing on several thin layer systems and by infrared spectroscopy. With the use of a 100 MHz Fourier transform nuclear magnetic resonance (NMR) spectrometer, the spectra of bovine whole brain, white matter, gray matter, monoenoic, and hexaenoic PS were obtained. Distinct proton resonances were assigned to double bond protons, protons adjacent to a double bond, and protons between two double bonds, using fatty acid methyl ester standards. The various PS preparations gave different intensities of the various proton resonances which correlated with differences in fatty acid composition. The method provides a convenient, non-destructive spectroscopic method for distinguishing monoenoic and polyunsaturated species of intact phospholipids. Electron spin resonance studies of nitroxide-labelled cholestane in sonicated PS vesicles showed greater probe motion as the unsaturation of the acyl chains was increased. The hexaenoic PS vesicles were more fluid than monoenoic PS vesicles at all temperatures in the range 10-55 degrees C. These results suggest that neuronal membranes are more fluid than myelin membranes as neuronal membranes contain more hexaenoic phospholipids.     

Regulation of rat liver phosphofructokinase by glucagon-induced phosphorylation

In perfused rat liver, the effects of various hormones on the stimulation of phosphorylation and allosteric properties of purified phosphorfructokinase were investigated. Rat livers were perfused with [³²P]phosphate followed with various hormones or cyclicAMP, and ³²P-labeled phosphofructokinase was isolated. ³²P incorporation into the enzyme and enzyme inhibition by ATP or citrate were determined. Only glucagon increased the ³²P incorporation into phosphofructokinase and this increase was approximately threefold. The cyclicAMP level was increased simultaneously approximately four- to fivefold compared to the control perfused liver. Similar results were obtained by perfusing the liver with cyclicAMP (0.1 mm). The phosphorylated phosphofructokinase showed a decrease in the K_i values for ATP (from 0.4 to 0.2 mm) and citrate (from 2 to 0.6 mm). Neither epinephrine nor insulin affected the extent of phosphorylation or the allosteric properties of the enzyme. The half-maximal concentration of glucagon required for phosphorylation of phosphofructokinase and modification of its allosteric properties was approximately 6 × 10^?11m. It is concluded that glucagon increases the inhibition of liver phosphofructokinase by ATP and citrate through phosphorylation of the enzyme involving a β-receptor-mediated cyclicAMP-dependent mechanism.     

Comparison of phospho- and dephospho-ATP citrate lyase

The dephospho- form of rat liver citrate lyase has been prepared by treating purified [³²P]-ATP citrate lyase with a partially purified phosphatase. A comparison of the properties of the phospho- and dephosphoenzyme has been performed. The pH optima were the same for both forms of the enzyme in four different buffer systems although the optimum values varied identically for both enzyme forms with the buffer. Both the phospho- and dephosphoenzymes show the same kinetic properties except for the K_m observed for ATP in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer system where it was 54 μm for the phosphoenzyme and 292 μm for the dephosphoenzyme. The present study also indicates that both enzymes are cleaved by trypsin and lysosomal proteases in a similar manner. Both forms of the enzyme tend to associate with mitochondria to the same extent and both enzymes have identical temperature stability curves.     

Crystalline aggregation of a proteolytic fragment of the major head protein of bacteriophage T4.

An analysis has been made of the composition and structure of the two types of sheets assembled from material from dissociated bacteriophage T2 (Poglazov &; Mesyhanzhinov, 1967) and T4 capsids. Serological techniques have been used to show that both types of sheet are assembled from proteolytic fragment of P23¹, the major capsid constituent. The two types of sheets have been found to interconvert depending on the concentration of Mg²⁺ ions in the buffer. Computer modelling experiments show that the “hexagonal” and “rectangular” morphologies observed in the negative stain are due to in-register and staggered associations, respectively, of a single basic hexagonal lattice. Analysis by polyacrylamide gel electrophoresis of samples of sheets and dissociated capsids, together with previous results from immune electron microscopy (Kistler et al., 1978), suggest that hexamers of the proteolytic fragment are derived conservatively from capsomers of the phage head.The value of this proteolytic P23 fragment has been twofold: (1) it has proved to be a useful peptide in the ongoing primary sequence determination of P23 and (2) antibodies raised against it have been employed to follow the fate of P23 antigenic sites during various steps of phage capsid maturation (Kistler et al., 1978).