Canadian journal of biochemistry最新文献

Cell-free enzyme preparations from Streptococcus faecium UNH 564P and Phycomyces blakesleeanus strain C5-car10(-) were used to study the incorporation of [14C]isopentenyl pyrophosphate and either all-trans-[4,8,12-3H]farnesyl pyrophosphate (FPP) or all-trans--[4,8,12,16-3H]geranylgeranyl pyrophosphate (GGPP) into squalene and the carotenoids of the organisms. It was found that the triterpenoid (C30) carotenoids of S. faecium are formed by condensation of two molecules of FPP similar to squalene biosynthesis rather than by condensation of two molecules of GGPP with subsequent degradation. Additional studies have shown that carotenoid glucoside biosynthesis in S. faecium extracts is stimulated by the addition of glucose and UDP-glucose. Such glucoside biosynthesis appears maximal in systems exposed to aeration. These results confirm that the triterpenoid carotenoids in S. faecium are symmetrical and are representatives of a unique new class of carotenoids.

Anomalies both kinetic and equilibrium in nature are described for the inhibition of cytochrome c oxidase activity by sulphide in the isolated enzyme and in submitochondrial particles. These anomalies are related to the involvement of more than 1 mol of sulphide in the blockage of one cytochrome aa3 centre. Sulphide reduces resting cytochrome a3, a reaction that results in oxygen uptake and the loss of a sulphide molecule. Sulphide can also reduce cytochromes c and a; in the former case, a part of the one-equivalent oxidation product, presumed to be the SH radical, reacts with oxygen. Such oxygen uptake is also seen under aerobic conditions when ferricyanide reacts with sulphide. Three phases are identified in the inhibitory interaction of sulphide with the cytochrome c oxidase enzyme itself: an initial rapid reaction involving sulphide oxidation, oxygen uptake, and conversion of cytochrome aa3 into the low-spin "oxyferri" form; a subsequent step in which sulphide reduces cytochrome a; and the final inhibitory step in which a third molecule of sulphide binds the a3 iron centre in the cytochrome a2+ a3 3+ (oxy) species to give cytochrome a2+ a3 3+ H2S. the initial events parallel some of the events in the interaction of the cytochrome c-cytochrome aa3 system with monothiols; the final inhibitory event resembles that with cyanide.

We have examined the ability of a topoisomerase purified from chicken erythrocyte nuclei to mediate nucleosome core assembly in vitro at physiological ionic strength (0.15 M NaCl). Although we have detected limited amounts of spontaneously assembled nucleosome cores at this salt concentration, the addition of this topoisomerase does not increase the amount of assembly observed. Nucleosome assembly was assayed by quantitating the amount of core particle length DNA accumulated with time upon the nuclease digestion of histone-DNA complexes. In addition, the amount of negative supercoils introduced into relaxed closed circular DNA upon nucleosome core particle assembly was determined. Correctly assembled complexes do not protect more DNA from nuclease digestion than random histone-DNA complexes but shift the heterogeneous size distribution of protected fragments to a more homogeneous distribution centred around 145 base pairs. Under our conditions of nucleosome assembly, a second histone-DNA complex which is distinct from the nucleosome core can be detected under physiological ionic strength conditions. This particle does not form in high salt assembly experiments. Similarly, the assembly of this particle is unaffected by the presence or absence of topoisomerase.

A rapid and practical method has been developed for the gas-liquid chromatographic determination of the sn-1,2-diacylglycerol moieties of natural glycerophospholipids using polar wall-coated open tubular columns. The method gives complete resolution and quantitative estimates for all species according to molecular weight and degree of unsaturation, including stearoyl docosahexaenoylglycerol and related polyunsaturates. For this purpose the sn-1,2-diacylglycerols are obtained from the glycerophospholipids by hydrolysis with phospholipase C and are converted into the trimethylsilyl or tertiary-butyldimethylsilyl ethers. The silyl ethers are separated by gas-liquid chromatography on the capillary glass columns coated with a polar cyanopropylsiloxane polymer, in the temperature range 175-250 degrees C, using hydrogen as the carrier gas. Practical applications of the method are illustrated by analyses of the sn-1,2-diacylglycerol moieties of the phosphatidylcholines of soybean phosphatides, egg yolk, and rat liver. The method of analysis is applicable to other classes of glycerophospholipids and the total time requirements for the analysis of any one phospholipid class are comparable to those for a fatty acid analysis.

Kinetically determined competitive inhibitor constants, which were estimates of the binding capacity of inhibitors interacting with the free enzyme form of beta-galactosidase (Escherichia coli), showed that amino sugars and alcohols inhibited the enzyme much more than did their respective parent sugars and alcohols. In most cases the inhibition was 10-30 times greater, but the inhibition by 1-aminogalactopyranose was about 300 times greater than that of galactopyranose. When the amino groups were acetylated the inhibitory advantage was completely eliminated and partial neutralization of the amino group of an inhibitor by the presence of a group with a negative charge on the same inhibitor decrease the inhibitory advantage. Studies of binding to the galactosyl form of the enzyme (rather than to the free form) indicated that the binding at the "glucose" site was increased only slightly by the presence of the amino group. Overall, the findings suggested that beta-galactosidase has a negative charge near the anomeric carbon binding position of the "galactose" site. Since negative charges are unlikely to be of any importance in binding the normally neutral beta-galactosidase substrates, this charge may be important in stabilizing a positively charged reaction analogous to an oxonium-carbonium ion intermediate.

A cell-free enzyme extract from Streptococcus faecium UNH 564P has been prepared. The extract incorporates either [2-14C]mevalonic acid (MVA) or [1-14C]isopentenyl pyrophosphate (IPP) into squalene and the carotenoids of the bacterium. ATP and manganese ion were found to be absolute requirements for MVA incorporation by the extract. Only manganese ion was found to be an absolute requirement for IPP incorporation by the extract. Other cofactors including magnesium ion, glutathione, potassium fluoride, NADP, and FAD significantly increase the incorporation of both substrates into the S. faecium terpenoids. Isolation and purification of the radioactive terpenoids from the cell-free system confirmed that the carotenoids of S. faecium are triterpenoids.

In earlier studies it was found that the association of basic polypeptides with lipid vesicles drastically altered ultraviolet absorption by the olefinic bonds of the lipid. Such an effect suggested that the polypeptide was increasing the polarity of the chromophore environment by either direct interaction with the acyl chains or by inducing their hydration. It is reported here that freeze-thaw cycling, which was expected to allow hydration of the olefinic-bond region of the membranes, caused the same spectral alteration as vesicle interaction with basic polypeptides. When these vesicles were subsequently placed under conditions that would be expected to accelerate the escape of water entrapped within the membranes (i.e., by placing them under vacuum or adding sucrose to establish a high osmotic gradient to their exterior), the absorption spectrum was rapidly restored to that for olefinic bonds in a nonpolar environment. Since placing the polylysine- dioleoylphosphatidylcholine (DOPC) vesicle interaction product under the same conditions restored the spectral intensity, at 190 nm, to between 80 and 85% of that for the lipid in a nonpolar environment, it seems that a major effect of polylysine on DOPC membranes may be though induction of hydration of their interior.

In broadening our research on the inhibition of cathepsin B (EC 3.4.22.1) by rat haptoglobin, we have used the haptoglobin-hemoglobin complex and asialohaptoglobin. The inhibition of cathepsin L (EC 3.4.22.15), another lysosomal thiol proteinase, by haptoglobin and its related molecules has also been investigated. With azocasein as substrate, both enzymes were inhibited by both haptoglobin and its related molecules. When azocasein was used as a substrate, the apparent Michaelis constant (Km, app.) for cathepsin L was 1 X 10(-5) +/- 0.4 X 10(-5) M. When haptoglobin was added, the apparent inhibition constant (Ki, app) was 3 X 10(-8) +/- 2.5 X 10(-8) M. The results suggest that rat haptoglobin specifically inhibits lysosomal thiol proteinases and that it has a regulatory role in tissue proteolysis associated with the inflammatory reaction. On the other hand, these properties would seem to be peculiar to the systems rat haptoglobin-rat liver cathepsin B or L.