Stereochemistry of the hydrolysis of α,α-trehalose by trehalase,determined by using a labelled substrate

(1,1′-¹³C)α,α-Trehalose was obtained in 37% yield from the Pavia condensation of 2,3,4,6-tetra-O-benzyl-d-(1-¹³C)glucopyranose, in dichloromethane in the presence of trifluoromethanesulfonic anhydride, followed by the usual deprotection techniques. The hydrolysis of this substrate by cockchafer trehalase was monitored at 37° by using ¹³C-n.m.r. spectroscopy with short recording times. Equimolecular amounts of α- and β-d-glucopyranose are released simultaneously by the action of the enzyme. This result is consistent with a bimolecular substitution mechanism, taking into account previous results involving C-2 asymmetric participation in the catalytic step of hydrolysis of α,α-trehalose. For comparative evaluation of its accuracy, the usual polarimetric technique was also used for the determination of the anomeric configuration of the d-glucose released by the action of the enzyme on α,α-trehalose.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

Evoked Release of Proteins from Central Neurons In Vivo

Push-pull cannulae were implanted in both substantiae nigrae and caudate nuclei of the halothane-anesthetized cat. The release of total protein, acetylcho-linesterase, and nonspecific cholinesterases was examined. Following direct application of potassium to one substantia nigra, changes occurred in the local release of total protein and acetylcholinesterase, but not nonspecific cholinesterases; changes also were observed in both caudate nuclei and the contralatera/ substantia nigra. The local evoked release of acetylcholinesterase and of total protein differed in the extent to which they were calcium-dependent. Control studies suggest that release of these compounds, both spontaneous and evoked, is related, at least in part, to neuronal activity. The significance of the neuronal release of proteins is discussed.     

Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA₂ ^Ile and tRNA^Ala map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs^Leu and the three for tRNAs^Ser, as well as the two isoaccepting species for tRNA^Asn, tRNA^Gly, tRNAs^Ile, tRNAs^Met, tRNAs^Thr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were³²P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.     

Enrichment and characterization of the mRNAs of four aminoacyl-tRNA synthetases from yeast.

We have partially purified the messenger RNAs for yeast arginyl-, aspartyl-, valyl-, alpha and beta subunits of phenylalanyl-tRNA synthetases in order to study their biosynthesis and ultimately, to isolate their genes. Sucrose gradient fractionation of poly U-Sepharose selected mRNAs resulted in a ten fold enrichment of the in vitro translation activity of these mRNAs. The translation products of messenger RNAs for arginyl- and valyl-tRNA synthetases have the same molecular weight as the purified enzymes; translation of aspartyl-tRNA synthetase messenger RNA yielded a 68 kD molecular weight polypeptide (while the purified cristallisable enzyme appears as a 64-66 kD doublet, which, as we showed is a proteolysis product). The translation of the mRNAs for alpha and beta phenylalanyl-tRNA synthetase gave polypeptides having the same molecular weight as those obtained from the purified enzyme, but the major translation products are slightly heavier, indicating that they may be translated as precursors. As estimated from centrifugation experiments mRNAs of arginyl-, aspartyl-, alpha and beta subunits of phenylalanyl-tRNA synthetase were 1700-2000 nucleotides long, indicating that alpha and beta are translated from two different mRNAs.     

Incompatibility in Podospora anserina: Comparative Properties of the Antagonistic Cytoplasmic Factors of a Nonallelic System

In the fungus Podospora anserina, the interaction between the nonallelic incompatible R and V genes has two consequences: a lytic reaction due to the synthesis of specific proteolytic enzymes, and a quenching in protein and ribonucleic acid synthesis. The incompatibility reaction when vegetative or sexual R and V cells fuse is asymmetric: it is induced only in the R protoplasm. The cessation in ribonucleic acid and protein synthesis was investigated in heterokaryotic strains carrying the antagonistic R and V genes and their "neutral" r and v alleles. Asymmetry between R and V genes lies in the fact that the strains homozygous for the R genes are the only strains that cannot grow. From these results it is postulated that the V-gene product is a diffusible cytoplasmic factor and that the R-gene product, which is nonautonomous, is a ribosomal component.