The nonenzymatic hydrolysis of oligoribonucleotides. VII. Structural elements affecting hydrolysis

Several elements of oligoribonucleotide structure are important for efficient hydrolysis. We have found that the following factors influence oligoribonucleotide hydrolysis: (i) single-stranded structure of RNA flanking the scissile phosphodiester bond, (ii) the substituent on atom C-5 of the uridine adjacent to the cleaved internucleotide bond, (iii) the position of the scissile UA phosphodiester bond within a hairpin loop, (iv) the concentration of formamide, urea, ethanol and sodium chloride.     

Acetylcholinesterase-catalyzed hydrolysis of an amide.

In this paper we report that acetylcholinesterase catalyzes hydrolysis of amides, an observation which had not been made previously. The amide used is an analog of acetylcholine, 2-acetoaminoethyltrimethylammonium iodide. The experiments were performed with an enzyme preparation obtained from electroplax of Electrophorus electricus. Inhibition of the enzyme by a specific organic phosphate inhibitor abolished both the esterase and the amidase activity of the enzyme. The effect of hydrogen ions between pH 5 and pH 10 on the steady-state kinetic parameters, Km and kcat, has been investigated. These parameters show essentially the same dependence on pH as is observed in catalytic hydrolysis of acetylcholine. k-cat is controlled by an ionizing group of the enzyme with an apparent pK of approximately 6.3, and reaches a pH-independent maximum value of 3.6 sec- minus 1 above pH 8. The value for Km of 1 mM at pH 7 and 25 degrees is about five times greater than that for catalytic hydrolysis of the ester at the same pH and temperature. Preliminary electrophysiological experiments indicate that the amide analog binds to the receptor less well, by several orders of magnitude, than acetylcholine does.     

Enzyme-accelerated hydrolysis of polyglycolic acid.

In a preliminary study of the enzyme-polymer interactions, the role of 15 enzymes in the in vitro hydrolysis of polyglycolic acid has been investigated. Carboxypeptidase A, alpha-chymotrypsin, clostridiopeptidase A and ficin increase the rate of hydrolysis of this synthetic polymer, illustrating the ability of enzymes to influence polymer degradation.     

Intralysosomal hydrolysis of glycyl-L-phenylalanine 2-naphthylamide.

Glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap), a cathepsin C substrate, induces an increase of the free and unsedimentable activities of this enzyme when incubated with a total mitochondrial fraction of rat liver. 1 mM-ZnSO4 considerably inhibits the cathepsin C total activity, measured with Gly-L-Phe-2-NNap as the substrate, in the presence of Triton X-100. The inhibition is markedly less pronounced when the free activity is determined; a high activity remains that depends on the integrity of the lysosomes; it decreases as the free activity of N-acetylglucosaminidase increases when lysosomes are subjected to treatments able to disrupt their membrane. Cathepsin C activity is reduced when thioethylamine hydrochloride is omitted from the incubation medium. Under these conditions at 37 degrees C, the free activity equals the total activity, although the lysosomes are intact, as indicated by the low free activity of N-acetylglucosaminidase. 1 mM-ZnSO4 strikingly inhibits the total activity, whereas more than 80% of the free activity remains. These observations are presented as evidence that Gly-L-Phe-2-NNap can possibly cause a disruption of the lysosomes as a result of its hydrolysis inside these organelles. In the presence of ZnSO4, intralysosomal hydrolysis becomes apparent, owing to a preferential inhibition by Zn2+ of extralysosomal hydrolysis; in the absence of thioethylamine hydrochloride, it is measurable because the disruption of lysosomes by Gly-L-Phe-2-NNap is delayed as a result of a slow-down of the reaction. The usefulness of Gly-L-Phe-2-NNap and related dipeptidyl naphthylamides in lysosomal-membrane-permeability studies is emphasized.     

Kinetics of chitinase production. I. Chitin hydrolysis

As part of the development of a comprehensive mathematical model for chitinase production by Serratia marcescens QMB 1466 growing on chitin, the different mass transport and kinetic steps involved during chitin hydrolysis were studied. The experimental results for the hydrolysis of chitin by a crude preparation of chitinase show a system kinetically limited by the overall rate of chitin hydrolysis. This rate is linearly related to the concentration of enzyme adsorbed on the chitin particle. Adsorbed and bulk enzyme concentration were found to be related through a Langmuir type of isotherm.     

Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of [3H]choline and [3H]phosphorylcholine ([3H]Pchol) from cells containing [3H]choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of [3H]phosphatidic acid ([3H]PA) in cells containing [3H]myristate-labeled PC. [3H]Diacylglycerol ([3H]DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with [3H]myristate and [14C]arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol. By analyzing the increase in 3H versus 14C in DAG, we estimate that the DAG that is formed in response to PMA arises largely from PC. Muscarinic receptor activation also causes formation of DAG from PC, but approximately 20% of carbachol-stimulated DAG appears to arise from hydrolysis of the phosphoinositides.     

Brefeldin A promotes hydrolysis of sphingomyelin.

The hydrolysis of sphingomyelin (SM) is a key reaction in the "sphingomyelin cycle," which plays a role in the regulation of cell proliferation and differentiation (Okazaki, T., Bell, R. M., and Hannun, Y. A. (1989) J. Biol. Chem. 264, 19076-19080). SM is produced from endoplasmic reticulum-derived ceramide and is delivered to organelle membranes in a regulated manner, presumably through the same endomembrane trafficking system used for sorting and delivery of proteins. Since brefeldin A (BFA) interferes with this endomembrane trafficking system and thus alters normal membrane and organelle distribution, we investigated the effect of BFA on SM levels in HL-60 leukemia cells. BFA caused a dose-dependent decrease of 20-25% in cellular SM levels, with effects observed at concentrations of BFA as low as 0.10 microgram/ml. BFA effects on SM levels were noted as early as 5 min and were maximal by 20 min, with no further SM hydrolysis observed up to 60 min following treatment with BFA, suggesting the presence of a fixed SM-sensitive pool. BFA did not cause SM hydrolysis at 16 degrees C, a temperature that inhibits the effects of BFA on endomembrane mixing. The very early effects and temperature dependence of BFA-induced SM hydrolysis suggest that the mechanism of hydrolysis may be closely related to endomembrane mixing. These studies are beginning to define important interrelationships between membrane trafficking and topology, SM metabolism, and cell regulation.     

Limited hydrolysis of tRNA by phosphodiesterase.

Digestion of tRNA by electrophoretically pure phosphodiesterase is limited to a short sequence of nucleotides at the 3'-terminus. On the average, four percent of all nucleotides can be released from tRNA. The optimum Mg2 concentration is 10mM and the optimum pH 9.2. The mode of action is a random attack by the enzyme on the substrate. The terminal AMP is completely removed at 15 degrees C after short incubation; about 400 mol of AMP were removed per min by 1 mol of enzyme. The following CMP residues are released much more slowly; at 15 degrees C incompletely, and at 37 degrees C more or less completely in 1 h. In about 50% of the tRNA molecules, the fourth nucleotide could be removed in very long incubations or with very high enzyme concentrations.     

Nucleotide hydrolysis in cytoskeletal assembly.

Two major polymers of the cytoskeleton, actin filaments and microtubules, are assembled with expenditure of energy: the ATP/GTP tightly bound to actin/tubulin is irreversibly hydrolyzed to ADP/GTP during the assembly process, and liberation of Pi in the medium occurs subsequent to the incorporation of subunits in the polymer. Pi release acts as a switch, causing the destabilization of protein-protein interactions in the polymer, therefore regulating the dynamics of these fibres. An understanding of this regulation in vivo requires that progress be made in four areas: the chemistry of the NTPase reaction; the structure of the intermediates in nucleotide hydrolysis and the nature of the conformational switch; the regulation of parameters involved in dynamic instability of microtubules; and the possible involvement of nucleotide hydrolysis in the macroscopic organization of these polymers in highly concentrated solutions, compared with the simple case of a equilibrium polymers. Progress made along these lines will define trends for future investigation.     

Computer analysis of Feulgen hydrolysis kinetics.

A least squares fit of Feulgen hydrolysis time curves to the Bateman function is performed using an especially adapted parameter transformation together with a standard conjugate gradients iteration procedure. The method has been applied to a large number of measured data, and the use and limits of the computer evaluation are discussed.     

Chitosanase-catalyzed hydrolysis of 4-methylumbelliferyl beta-chitotrioside.

4-Methylumbelliferyl beta-chitotrioside [(GlcN)(3)-UMB] was prepared from 4-methylumbelliferyl tri-N-acetyl-beta-chitotrioside [(GlcNAc)(3)-UMB] using chitin deacetylase from Colletotrichum lindemuthianum, and hydrolyzed by chitosanase from Streptomyces sp. N174. The enzymatic deacetylation of (GlcNAc)(3)-UMB was confirmed by (1)H-NMR spectroscopy and mass spectrometry. When the (GlcN)(3)-UMB obtained was incubated with chitosanase, the fluorescence intensity at 450 nm obtained by excitation at 360 nm was found to increase with proportion to the reaction time. The rate of increase in the fluorescence intensity was proportional to the enzyme concentration. This indicates that chitosanase hydrolyzes the glycosidic linkage between a GlcN residue and UMB moiety releasing the fluorescent UMB molecule. Since (GlcN)(3) itself cannot be hydrolyzed by the chitosanase, (GlcN)(3)-UMB is considered to be a useful low molecular weight substrate for the assay of chitosanase. The k(cat) and K(m) values obtained for the substrate (GlcN)(3)-UMB were determined to be 8.1 x 10(-5) s(-1) and 201 microM, respectively. From TLC analysis of the reaction products, the chitosanase was found to hydrolyze not only the linkages between a GlcN residue and UMB moiety, but also the linkages between GlcN residues. Nevertheless, the high sensitivity of the fluorescence detection of the UMB molecule would enable a more accurate determination of kinetic constants for chitosanases.