Lipid intermediates, labeling

In contrast, the transglycosylase activity of the class A PBPs is poorly characterized, mainly because of the unavailability and complexity of its substrates, lipid intermediates of stem peptides [1-6,8,10,15], The transglycosylase activity is routinely assayed by a complicated procedure involving the use of bacterial membrane preparations that contain radioisotope-labeled native substrates for the enzyme [3,6,8,10], Unlike the transpeptidase domain of PBPs, a crystal structure has not yet been described for a transglycosylase domain of any PBPs. 

Suppose that glucose 1-phosphate labeled with is added to a cellular system designed to study the synthesis and processing of N-glycosylated proteins. When bacitracin is added to the system, a lipid-soluble intermediate labeled with accumulates. In the absence of bacitracin, the label appears in inorganic phosphate. Explain these results, and identify the lipid-soluble intermediate that accumulates. (Refer to Section 11.3.3 in the text.)... 

Successive experiments have been carried out therefore with purified rat brain microsomes incubated with radioactive SAM at different pH values, with the aim of examining the rate of labeling of lipid intermediates, such as PME and PDE, and of Ptd-choline. As known,microsomes represent in liver and other tissues the main subcellular fraction responsible for the metabolic pathway. 

For the synthesis of a small library of palmitoylated and isoprenylated N-Ras peptides in solution, a modular strategy was adopted, with the tetrapeptide MGLP 38 as a key intermediate. This tetrapeptide allowed further elongation at its C-terminus with lipidated or nonlipidated cysteine methyl esters, as well as the addition of various N-terminally MIC-labeled dipeptides, consisting of different GC lipidated units 39—41... 

Fig. 11. Evidence that a membrane-associated immunochemical reaction (complement fixation) depends on the mobility of the target hapten (IX) in the plane of a model membrane. The extent of the immunochemical reaction, complement fixation, is measured by A Absorbance at 413 nm. Temperature is always 32°C, which is above the chainmelting temperature (23°C) of dimyristoylphosphatidylcholine used for the data given in A and below the chain-melting transition temperature (42°C) of dipalmitoylphosphatidyl-choline used for the data in B. Thus A refers to a fluid membrane and B refers to a solid membrane. The numbers by each curve are equal to c, the mole % of spin-label hapten IX in the plane of the lipid membrane. It will be seen that complement fixation, as measured by A Absorbance at 413 nm is far more effective in the fluid membrane than in the solid membrane at low hapten concentrations (i.e., c 0.3 mo e%). In C the lipid membrane host is a 50 50 mole ratio mixture of cholesterol and dipalmitoylphosphatidylcholine. The immunochemical data suggest that this membrane is in a state of intermediate fluidity. Specific affinity-purified IgG molecules were used in these experiments. (For further details, see Ref. 5.)... 

Fig. 4.5. Sphingomyelin synthesis in Hymenolepis diminuta distribution of label in various intermediates, separated by thin-layer chromatography, after incubation with cytidine-5 -diphospho [methyl-13C]choline. The position of the lipid standards is indicated by the arrows, (a) sphingomyelin (b) dihydrosphingosine (c) sphingosine (d) ketosphingosine (e) ceramide. The origin is at band O. d.p.m., disintegrations/min. (After Bankov Barrett, 1985.)...

Figure 1 The retrobiosynthetic principle. Labeling patterns of central metabolic intermediates (shown in yellow boxes) are reconstructed from the labeling patterns of sink metabolites, such as protein-derived amino acids, storage metabolites (starch and lipids), cellulose, isoprenoids, or RNA-derived nucleosides. The reconstruction is symbolized by retro arrows following the principles of retrosynthesis in synthetic organic chemistry. The figure is based on known biosynthetic pathways of amino acids, starch, cellulose, nucleosides, and isoprenoids in plants. The profiles of the central metabolites can then be used for predictions of the labeling patterns of secondary metabolites. In comparison with the observed labeling patterns of the target compounds, hypothetical pathways can be falsified on this basis.

Products were identified as 2,2,2-trichloroethanol and 2,2,2-trichloro-acetaldehyde by GC-MS and GC, respectively. Trichloroethanol (6.7 mu M) was completely degraded in 72 hours. When toluene was added to reaction mixtures, there was a 50% increase in the mineralization of (sup 14 C) TCE. M. vaccae s ability to cometabolize 2,4,6-trinitrotoluene (TNT) was also investigated. Two novel metabolites, as well as known reduction products, have been identified during catabolism of TNT with propane as cosubstrate. When M. vaccae was incubated with propane and (sup 14 C) TNT, 50% of the labelled carbon was found in the lipid fraction of cells. Analysis of this fraction demonstrated metabolism of sup 14 C into known phosphatides and other polar lipids indicating that ring cleavage had occurred. TNT or reduced intermediates were not found in any portion of the lipid fractions. 

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