Enzymic oxidation of unconjugated bilirubin by rat liver.

The presence of the enzyme bilirubin oxidase, which degrades bilirubin in vitro, was demonstrated in the liver. Subcellular-fractionation experiments indicate that bilirubin oxidase is located in both the inner and outer membranes of the mitochondria. The mean rate of the reaction is 1.57 +/- 0.38 (S.D.) nmol of bilirubin degraded/min per mg of mitochondrial protein (munits/mg of protein). With respect to the overall breakdown of bilirubin, the enzyme has a Km' of 136 microM-bilirubin and a Vmax.' of 9.13 munits/mg of protein. Its activity is influenced by the ionic strength of the media and is inhibited by KCN, thiol reagents, NADH and albumin. The enzyme is aerobic, and between 1 and 1.5 mol of O2 are consumed per mol of bilirubin degraded. The products of the reaction include propentdyopents. The hepatic bilirubin oxidase activity of the jaundiced Gunn-rat liver is not significantly different from that of the Sprague-Dawley rat, and it is not induced by beta-naphthoflavone.     

Enzymic hydrolysis of acetylcarnitine in liver from rats, sheep and cows

1. The enzymic utilization of O-acetyl-l-carnitine other than via carnitine acetyltransferase (EC 2.3.1.7) was investigated in liver homogenates from rats, sheep and dry cows. 2. An enzymic utilization of O-acetyl-l-carnitine via hydrolysis of the ester bond to yield stoicheiometric quantities of acetate and l-carnitine was demonstrated; 0.55, 0.53 and 0.30mumol of acetyl-l-carnitine were utilized/min per g fresh wt. of liver homogenates from rats, sheep and dry cows respectively. 3. The acetylcarnitine hydrolysis activity was not due to a non-specific esterase or non-specific cholinesterase. O-Acetyl-d-carnitine was not utilized. 4. The activity was associated with the enriched outer mitochondrial membrane fraction from rat liver. Isolation of this fraction resulted in an eightfold purification of acetylcarnitine hydrolase activity. 4. The K(m) for this acetylcarnitine utilization was 2mm and 1.5mm for rat and sheep liver homogenates respectively. 6. There was a significant increase in acetylcarnitine hydrolase in rats on starvation and cows on lactation and a significant decrease in sheep that were severely alloxan-diabetic. 7. The physiological role of an acetylcarnitine hydrolase is discussed in relation to coupling with carnitine acetyltransferase for the relief of ;acetyl pressure'.     

Effect of Triton X-100 on the hydrolysis of sphingomyelin by sphingomyelinase of rat brain.

Mixed dispersions of the nonionic detergent Triton X-100 and sphingomyelin were used as substrate for sphingomyelinase of rat brain. The dependence of the rate of hydrolysis on the concentration of sphingomyelin was measured in two ways: at a fixed concentration of Triton X-100 or at varying concentrations of this detergent, while maintaining a fixed molar ratio of Triton X-100 to sphingomyelin. In either case, the upsilon vs. S curves deviated from the hyperbolic shape predicted by the Michaelis-Menten kinetic theory. These deviations are discussed and interpreted on the basis of the physicochemical properties of the mixed dispersions of detergent and lipid studied in previous papers.     

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.     

Biosynthesis of sphingomyelin in rat liver endoplasmic reticulum.

Rat liver microsomal sphingomyelin synthetase (CDPcholine: N-acylspingosine choline phosphotransferase (EC 2.7.8.3)) has been shown to be markedly stimulated by ATP and pantothenic acid derivatives such as CoA, pantethine, pantetheine and 4'-phosphopantetheine.     

Enzymic hydrolysis of the bacterial polysaccharide xanthan by cellulase

In this paper the mechanism of enzymic hydrolysis by a cellulase on xanthan is investigated. It is demonstrated that in salt free solution there is a random breakdown of the main chain when the polysaccharide is in the unordered conformation. The apparent rate of hydrolysis followed by the decrease of the solution viscosity depends on the quality of the solution. In addition, the rate of hydrolysis may be directly correlated with the degree of local order expressed by its specific rotary power. It is shown that there is no hydrolysis on the ordered helical conformation.     

Correlation between the thermotropic behavior of sphingomyelin liposomes and sphingomyelin hydrolysis by sphingomyelinase of Staphylococcus aureus

The hydrolysis of d-erythro beef brain sphingomyelin and $\text{d,l}\text{-erythro}\text{-N-}\text{palmitoylsphingomyelin}$ dispersed as multilamellar liposomes by sphingomyelinase of Staphylococcus aureus is correlated with the thermotropic behavior of the sphingomyelins. In both cases maximal enzymatic hydrolysis was achieved at the beginning of the gel to liquid crystalline phase transition (30°C for beef brain sphingomyelin and 41°C for N-palmitoylsphingosinephosphorylcholine) with much lower activity both below and above these temperatures. The enzymatic activity was depressed in the presence of cholesterol in the bilayer which also depressed the phase transition. The profile of the enzymatic activity is explained by the uniqueness of the lipid molecules arrangement at the phase transition.     

Enzymatic hydrolysis of immobilized sphingomyelin by three bacterial phospholipases C

Through hydrophobic interaction, sphingomyelin was adsorbed to agarose beads containing octyl groups by a stepwise dilution procedure. This immobilized lipid was used as a substrate for three bacterial phospholipases C (E.C. 3.1.4.3.). The degradation with time of this substrate showed two different fractions of the substrate according to hydrolysing velocity in the early part of the time-curve when phospholipases C from Bacillus cereus and Clostridium perfringens were used. The early fractions could be predigested by the enzymes, a procedure which resulted in linear time-curves. The corresponding early part of the time-curve for phospholipase C from Staphylococcus aureus was linear, indicating a comparatively large early fraction of the substrate for this enzyme. The stock gel of the immobilized lipid substrate could be stored for months. It was easily and reproducibly handled as a water suspension. After enzymatic hydrolysis the substrate was rapidly separated from enzyme and product by filtration. The enzyme assay presented thus represents a convenient way to avoid the difficulties connected with the use of temporary sonicated suspensions as substrate for bacterial phospholipases C.