Enzymic hydrolysis of sphingolipids: Hydrolysis of ceramide lactoside by an enzyme from rat brain

Ceramide lactoside [1-O-(galactosido-4-β-glucosido)-2-N-acyl-sphingosine] was hydrolysed to ceramide glucoside and galactose by β-galactosidase of rat brain. The reaction was not reversible, required cholate or taurocholate, had optimum pH5·0 and K_m 2·2×10⁻⁵m. It was inhibited by γ-galactonolactone and galactose as well as by ceramide, sphingosine and fatty acid. Ceramide lactoside could be degraded to ceramide, galactose and glucose by mixtures of rat-brain β-galactosidase and ox-brain β-glucosidase.     

Enzymic alanylation of an isoxazolinone glucoside by legume seedling extracts

Enzymic synthesis of the natural product β-(2-β-d-glucopyranosyl-3-isoxazolin-5-on-4-yl) alanine is described, using the natural isoxazolinone glucoside and O-acetyl-l-serine as substrates and extracts from seedlings as enzyme preparations. Lathyrus odoratus extracts show a higher activity than those of Pisum sativum, Citrullus vulgaris and Leucaena leucocephala.     

Signaling events mediating activation of brain ethanolamine plasmalogen hydrolysis by ceramide.

Ceramide is a lipid second messenger that acts on multiple-target enzymes, some of which are involved in other signal-transduction systems. We have previously demonstrated that endogenous ceramide modifies the metabolism of brain ethanolamine plasmalogens. The mechanism involved was studied. On the basis of measurements of breakdown products, specific inhibitor effects, and previous findings, we suggest that a plasmalogen-selective phospholipase A2 is the ceramide target. Arachidonate-rich pools of the diacylphosphatidylethanolamine subclass were also affected by ceramide, but the most affected were plasmalogens. Concomitantly with production of free arachidonate, increased 1-O-arachidonoyl ceramide formation was observed. Quinacrine (phospholipase A2 inhibitor) and 1-O-octadecyl-2-O-methyl-rac-glycerol-3-phosphocholine (CoA-independent transacylase inhibitor) prevented all of these ceramide-elicited effects. Therefore, phospholipase and transacylase activities are tightly coupled. Okadaic acid (phosphatase 2A inhibitor) and PD 98059 (mitogen-activated protein kinase inhibitor) modified basal levels of ceramide and sphingomyelinase-induced accumulation of ceramide, respectively. Therefore, they provided no evidence to determine whether there is a sensitive enzyme downstream of ceramide. The evidence shows that there are serine-dependent and thiol-dependent enzymes downstream of ceramide generation. Furthermore, experiments with Ac-DEVD-CMK (caspase-3 specific inhibitor) have led us to conclude that caspase-3 is downstream of ceramide in activating the brain plasmalogen-selective phospholipase A2.     

Kinetic-controlled hydrolysis of Leu-Val-Val-hemorphin-7 catalyzed by angiotensin-converting enzyme from rat brain

Leu-Val-Val-hemorphin-7 (LVV-H7, LVVYPWTQRY), an opioid peptide, was found to be hydrolyzed sequentially by rat brain angiotensin-converting enzyme (ACE) in three steps through dipeptidyl carboxypeptidase activity. The kinetic constants evaluated were in order of: k(1) (0.19 min(-1))>k(2) (0.0008 min(-1)) approximately k(3) (0.0006 min(-1)) in 10 mM NaCl at pH 7.5 giving rise to LVV-H5 almost quantitatively. The decapeptide was noted to be hydrolyzed 164- and 346-fold more efficiently than angiotensin I (Ang I) in k(cat) and kcat/Km values, respectively, at their optimal conditions. The kinetic-controlled preferential action of the brain enzyme on LVV-H7 is suggestive of its multiple roles in vivo.     

Hydrolysis of chitosan sulfate by an enzyme complex from Streptomyces kurssanovii

The possibility of enzymatic hydrolysis of chitosan was shown. The optimum conditions for the process are: sodium acetate buffer pH 6.0, 37 degrees C, 24 h, and the chitosan sulfate-protein volume ratio of 500:1 in the enzyme preparation. During hydrolysis, the intrinsic viscosity of chitosan sulfate solution decreased by a factor of 2.7.     

Heparan sulphate sulphotransferase. Properties of an enzyme from ox lung

A heparan sulphate sulphotransferase was partially purified from an ox lung homogenate by (NH(4))(2)SO(4) precipitation. Various glycosaminoglycans were assayed as sulphate acceptors with this enzyme. The highest acceptor activity was obtained with desulphated heparin and heparan sulphate, which indicates that sulphate transfer may be to free amino groups of the substrate. Some heparan sulphate was (35)S-labelled by incubation with the enzyme and re-isolated. On treatment of this heparan [(35)S]sulphate with nitrous acid and separation of the degradation products on Sephadex G-15, a major peak of radioactivity was obtained, and identified as [(35)S]sulphate by high-voltage electrophoresis at pH5.3. The [(35)S]sulphate is believed to be derived from N-[(35)S]sulphated groups of heparan [(35)S]-sulphate. That the ox lung preparation contained an N-sulphotransferase was confirmed by the isolation of 2-deoxy-2-[(35)S]sulphoamino-d-glucose as the major product from the flavobacterial degradation of heparan [(35)S]sulphate.     

Enzymic hydrolysis of sphingomyelin by rat liver

1. An enzyme that hydrolyses sphingomyelin to ceramide (N-acylsphingosine) and phosphorylcholine was isolated from rat liver. 2. The enzyme is particle-bound (mitochondria or lysosomes) and can be solubilized by ultrasonic treatment and freezing and thawing. 3. It has been partially purified by precipitation at pH5.2, neutralization and ammonium sulphate fractionation. 4. The enzyme is activated by Triton X-100 (0.2%) or low concentrations of cetyltrimethylammonium bromide (0.02%), higher concentration being inhibitory. 5. The optimum pH is 5-5.5. 6. Of synthetic substrates tested, the erythro isomers of dl-trans-2-N-palmitoyl-1-O-phosphorylcholinesphingosine or dihydrosphingosine were hydrolysed at a rate similar to the natural compound. The threo isomer was hydrolysed much more slowly. The enzyme had little activity on lecithin. 7. The split products of the hydrolysis have little inhibitory effect.     

Purification of soluble arylsulphatases from ox brain

1. Two soluble arylsulphatases (A and B) have been extracted from ox brain by a modified Albers autolysis method and purified by acetone and ammonium sulphate precipitation and dialysis. 2. A 1600-fold purification was achieved with arylsulphatase A and 320-fold purification with arylsulphatase B. 3. The specific activity of arylsulphatase A was 266000 4-nitrocatechol units/mg. of protein N, and that of arylsulphatase B was 64600units/mg. of protein N. 4. Arylsulphatase A seems to be electrophoretically homogeneous. 5. With 3mm-dipotassium 2-hydroxy-5-nitrophenyl sulphate as substrate the optimum pH for the activity of arylsulphatase A was 4.7, and for arylsulphatase B it was 6.1 with a 60mm solution of the same substrate.     

Hydrolysis of a phosphonate ester catalyzed by an enzyme from Dictyostelium discoideum.

An activity capable of hydrolyzing the phosphonate ester, 4-nitrophenyl phenylphosphonate has been detected in the eucaryotic microorganism Dictyostelium discoideum. This activity (4-nitrophenyl phenylphosphonate-esterase) was found in both membrane-enriched and soluble fractions. Two other activities present in this fraction, namely cyclic AMP-diesterase and an ATP-hydrolase, did not copurify with the 4-nitrophenyl phenylphosphonate-esterase. Also while ATP and cAMP inhibited the hydrolysis of 4-nitrophenyl phenlyphosphonate, they did so in a noncompetitive manner and, furthermore, saturating concentrations of 4-nitrophenyl phenylphosphonate had no effect on the hydrolysis of either cyclic AMP or ATP. The partially purified preparation was more active at pH 7 than at either pH 5 or 9, more active with acetate than chloride, and more active at 22 °C than at either 12 or 30 °C. The activity is inhibited by AMP in a competitive manner, but is not affected by levamisole, an inhibitor specific for alkaline phosphatase activity.