The function and activity of certain membrane enzymes when localized on – and off – the membrane

Abstract

A group of enzymes known to be involved in group translocation-type transport mechanisms for the uptake of a variety of nucleotide precursors are enzymatically active both in their natural membrane milieu and in aqueous solution. The activity in aqueous solution markedly differ, however, from the enzymatic activity when the enzyme is membrane localized. The adenine phosphoribosyltransferase (PRT) of E. coli (Hochstadt-Ozer and Stadtman, 71a) is capable of carrying out an exchange reaction between the base moieties of adenine and AMP without requiring P-ribose-PP as an intermediate; the enzyme in aqueous solution requires P-ribose-PP, indicating a different reaction mechanism in the two environments. Like the adenine PRT of E. coli, the hypoxanthine PRT of Salmonella typhimurium (Jackman and Hochstadt, '76) also carried out an exchange reaction on the membrane only and also is more sensitive to a number of inhibitors in aqueous solution relative to the sensitivity when embedded in the membrane. In addition, however, the hypoxanthine PRT, while restricted to hypoxanthine as a substrate in the membrane, also accepts guanine as substrate in its soluble form. The membrane capacities reas determined in a guanine PRT deletion strain (Jackman and Hochstadt, '76). Finally, in mammalian cell lines purine nucleoside phosphorylase, which translocates the ribose moiety of inosine across the plasma membrane of mouse fibroblasts undergoes a 30-fold increase in substrate turnover number upon liberation from the membrane. These data raise two important caveats with respect to study of membrane enzymes and transport. Firstly, an enzyme once solubilized and found to differ kinetically from substrate transport in situ cannot be excluded from participating in translocations in the membrane on the basis of its activity in aqueous solution. Secondly, an enzyme which "appears" largely soluble upon cell rupture cannot be assumed to be a cycloplasmic enzyme because of majority of the solubilized activity may represent only a small fraction of the enzyme molecules highly activated concomitant to their solubilization. In this latter case the ability to activate enzyme still residing on the membrane (e.g., with detergents) would be necessary in order to estimate total membrane associated activity after cell rupture.

The activity of the intrinsically water-soluble enzyme ADAMTS13 correlates with the membrane state when bound to a phospholipid bilayer

“…This holds especially for enyzmes. When they are bound to a lipid membrane, many enzymes change their properties and temperature dependencies 2 . In the 1970s, many studies have found deviations from a pure Arrhenius-behavior.…”

Specificity and control of uptake of purines and other compounds in Bacillus subtilis

“…The phosphoribosylation process, with the high-energy metabolite phosphoribosylpyrophosphate, may simultaneously provide the driving force for the active transport of Hyp and Gua (by group translocation) (13). We have not examined this apparently controversial proposal (15,20), but we have found biphasic Michaelis-Menten plots that suggest the existence of an additional low-affinity transport mechanism for Hyp and Gua (but not for the nucleosides).…”

“…The uptake, which was linear for at least 5 min in all cases, is expressed as nanomoles x (OD6oo x minutes)-'; 1 OD6w unit is the amount of cells that gives an OD6w of 1 if the cells are suspended in 1 ml (this corresponds to about 0.5 mg [dry weight] of cells). Except where otherwise stated, the labeled compounds were used at the following concentrations: [2-`4C]Ura, 10 ,uM, 0.1 ,uCi/ml; [8-14CJAde, 10 ,uM, 0.1 ,uCi/ml; [8-14C]Ado, 40 FLM, 0.5 ,Ci/mn; [8-14C]Gua, 20 ,uM, 0.1 ,Ci/ml; [8,5'-3H]Guo, 15 ,uM, 0.5 uCi/ml; [5,6_3H]Uri, 25 ,uM, 0.5 ,Ci/ml; [5-3Hlcytidine, 15 ,uM, 0.5 ,uCi/ml; 1-0-methyl-(a-D-[U-14C]gluco)pyranoside, 50 ,uM, 0.15 ,Ci/ml; or [3-4C]a-aminoisobutyric acid, 100 ,M, 0.1 ,uCi/ml.…”

Mechanism of purine nucleoside handling and transport in isolated membrane vesicles from polyoma transformed BHK/21 cells

“…The resultant inosine is metabolized by a purine nucleoside phosphorylase activity, which may or may not be the same as that used in the first mechanism which resulted in extravesicular hypoxanthine, leading to hypoxanthine and additional ribose-l-P. The initial I^evious studies from this laboratory have shown that, when inosine interacts with isolated plasma membrane vesicles from cultured mouse fibroblast cells grown to high cell density, the predominant intravesicular transport product is ribose-l-P1 (Li and Hochstadt, 1976a,b;Hochstadt and Quinlan, 1976). The mechanism involves membrane-localized purine nucleoside phosphorylase acting in a group translocation reaction during which hypoxanthine is released on the exterior membrane surface while the ribose moiety is phosphorylated in the process of being transported across the membrane.In contrast, in polyoma transformed baby hamster kidney (BHK) cells there exist two mechanisms of inosine handling: one, similar to that we have described for the mouse fibroblast cells, in which group translocation occurs and depends on a transmembranal purine nucleoside phosphorylase and for which internal inosine is not a substrate (Hochstadt and Quinlan, 1976;Li and Hochstadt, 1976b) and a second mechanism which involves the uptake of intact inosine.…”

“…The resultant inosine is metabolized by a purine nucleoside phosphorylase activity, which may or may not be the same as that used in the first mechanism which resulted in extravesicular hypoxanthine, leading to hypoxanthine and additional ribose-l-P. The initial I^evious studies from this laboratory have shown that, when inosine interacts with isolated plasma membrane vesicles from cultured mouse fibroblast cells grown to high cell density, the predominant intravesicular transport product is ribose-l-P1 (Li and Hochstadt, 1976a,b;Hochstadt and Quinlan, 1976). The mechanism involves membrane-localized purine nucleoside phosphorylase acting in a group translocation reaction during which hypoxanthine is released on the exterior membrane surface while the ribose moiety is phosphorylated in the process of being transported across the membrane.…”