Microsomal phosphate/pyrophosphate

Microsomal phosphate/pyrophosphate transport protein M.W. 37,000), which transports phosphate, pyrophosphate, and carbamoyl phosphate, which are substrates for glucose-6-phosphatase and... 

Figure 2. Microsomal -p-nitroanisole 0-demethylation. (C), Complete system containing 1.2mM pNA, O.lSmM NADP, 44mM pyrophosphate, 2.4mM nicotinamide, 6mM MgClt, 6mM G6P, 4 units/mL G6PDH, and 0.73 mg/mL microsomal protein in a final volume of 2.5 mL of O.IM sodium phosphate buffer, pH 7.4 (A), Pyrophosphate and nicotinamide deleted (B), Pyrophosphate deleted ...

The existence of mannosylated saccharides linked to dolichol via a pyrophosphate was already well established. Studies by Behrens etal. (1973) had shown that rat liver microsomes could catalyse the synthesis of various lipid-linked oUgosaccharides from GDP mannose and dolichol phosphate, among which were some that were soluble in chloroform-methanol-water (1 1 0.3, v/v). These were more polar than dolichol monophosphate derivatives and could be bound to DEAE-cellulose in the presence of the organic solvent. In an ammonium formate gradient (also in the organic solvent) these polyprenyl saccharides eluted at the position of dolichol pyrophosphate derivatives. Waechter et al (1973) independently showed that a similar class of compounds was produced from GDP-[ C]maimose in the presence of microsomes from hen oviduct and that the labelled products... 

The transfer of mannose to phenyl phosphate was probably mediated by the same enzyme that synthesised dolichyl mannosyl phosphate. It showed similar ionic requirements, subcellular distribution and thermosensitivity. Phenyl phosphate and dolichyl phosphate showed apparent competition, as if for one enzyme. However, the reverse reaction, to synthesise GDPMan from phenyl mannosyl phosphate, could not be demonstrated, unlike the case with dolichyl mannosyl phosphate. Phenyl mannosyl phosphate was water soluble and, unlike dolichyl phosphate, was not retained in the microsomal membranes. Thus phenyl phosphate might trap mannose both by directing it, irreversibly, into the synthesis of a non-metabolite and by draining it away from microsomal membranes into the incubation medium. In the microsomal system it was shown to inhibit the transfer of mannose from dolichyl mannosyl phosphate to p-nitrophenyl a-D-mannoside, in a manner which depended on the concentration of phenyl phosphate. A study of mannosyl transfer to larger oligosaccharides on dolichyl pyrophosphate is needed and detailed kinetic analysis will be required to determine whether the inhibitor exerts its main... 

Fig. 2. (left) Influence of various compounds on phospho adenylylation of p40. Triton X114-extracted rat liver microsomes were incubated under standard conditions (8) pH]P-ADP-ribose at 1(X) iM concentration in the presence of the compounds listed (ADP-ribose 1 mM P-AMP 3 mM CoA 1 mM Na-phosphate 10 mM Na-pyrophosphate 10 mM). Data from (8). 

However, no further evidence for such amino acid phosphates was found, and there was no progress at all in the identification of activated precursors of protein until a fewyears ago when Hoagland et al. 94,97) found amino acid-dependent exchanges of radioactive pyrophosphate with the two terminal phosphates of ATP in the soluble liver fraction required for amino acid incorporation into microsomes. At the same time the existence of such a system in bacterial extracts was demonstrated by DeMoss and Novell 98, 99), and by Berg 100). The pyrophosphate exchange as well... 

---