Interaction of secretory protein precursors with phospholipids in liposomes

The precursors of secretory proteins were synthesized in a reticulocyte lysate system programmed with rat serum albumin or human placental lactogen mRNA and their interaction with phospholipids in liposomes was studied. The precursor proteins could bind to acidic phospholipids that have an exposed phosphate such as dicetyl phosphate and phosphatidic acid or a phosphate that is covered by a small moiety such as phosphatidylglycerol. The binding of precursor proteins was dependent on the mol% of acidic phospholipids in lecithin-liposomes, increased with elevation of temperature in the range of 0 to 45 degrees C, and was not inhibited by the addition of a large excess of mature proteins. Mature proteins or proalbumin showed no significant binding to the liposomes containing acidic phospholipids. About 15% of the acid-precipitable radioactivity bound to the liposomes was resistant to protease digestion. This radioactivity was shown to correspond to methionine-containing peptides with molecular weights of 2,500 to 3,500. These results indicate that the post-translational insertion of a small part of the precursor proteins into the membrane did occur with the present model system, but the post-translational transfer of precursor proteins across the membrane did not.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli. In vitro and in vivo incorporation into phospholipids

sn-Glycerol 3-phosphorothioate, a bacteriocidal analog of sn-glycerol 3-phosphate in strains of Escherichia coli with a functioning glycerol phosphate transport system, was investigated for its ability to be incorporated into phospholipid under in vitro and in vivo conditions. A cell-free particulate fraction from E. coli strain 8 catalyzes the transfer of sn-[3H]glycerol 3-phosphoro[35S]thioate to chloroform-soluble material in the presence of either CDP-diglyceride or palmitoyl coenzyme A. With CDP-diglyceride as the co-substrate, the product of the reaction was tentatively identified as phosphatidylglycerol phosphorothioate. No formation of phosphatidylglycerol was observed, suggesting that the specific phosphatase required for the synthesis of phosphatidylglycerol does not catalyze, or else at a greatly reduced rate, the hydrolysis of the phosphorothioate monoester linkage. The kinetics of incorporation of sn-[3H]glycerol 3-phosphate and phosphorothioate into chloroform-soluble material in the presence of CDP-diglyceride are almost identical. In the presence of palmitoyl coenzyme A, sn-[3H]glycerol 3-phosphoro[35S]thioate was converted to the phosphorothioate analog of phosphatidic acid. Kinetic analysis showed that the apparent Km values for the incorporation of the phosphate and the phosphorothioate derivatives into phospholipid were 0.4 and 0.8 mM, respectively. The Vmax for the phosphorothioate analog was approximately half that for the phosphate derivative. Chemically synthesized thiophosphatidic acid was not a substrate for CTP:phosphatidic acid cytidylyltransferase. sn-[3H]Glycerol 3-phosphoro[35S]thioate was incorporated into phospholipid by cultures of E. coli strain 8. The major phosphorothioate-containing phospholipid synthesized in vivo was identified as 1,2-diacyl-sn-[3H]glycerol 3-phosphoro[35S]thioate. The phosphorothioate analog of phosphatidylglycerol phosphate was not observed despite our observations that this analog can be synthesized in vitro. Our results indicate that the phosphorothioate analog is an effective sn-glycerol 3-phosphate surrogate and suggest that a major reason for its toxicity toward E. coli strain 8 may be due to a total blockade of endogenous phospholipid biosynthesis.     

Study of interaction of export initiation domain of Escherichia coli mature alkaline phosphatase with membrane phospholipids during secretion

The efficiency of secretion of alkaline phosphatase from Escherichia coli depending on the primary structure of its N-terminal region and the content of zwitterionic phospholipid phosphatidylethanolamine and anionic phospholipids in membranes has been studied in this work to establish the peculiarities of interaction of mature protein during its secretion with membrane phospholipids. It has been shown that the effect of phosphatidylethanolamine but not anionic phospholipids on the efficiency of alkaline phosphatase secretion is determined by the primary structure of its N-terminal region. The absence of phosphatidylethanolamine appreciably reduces the efficiency of secretion of wild type alkaline phosphatase and its mutant forms with amino acid substitutions in positions +5+6 and +13+14. In contrast, secretion of the protein with amino acid substitutions in positions +2+3, significantly decreased as a result of such mutation, in the presence of phosphatidylethanolamine, reaches the level of wild type protein secretion in the absence of phosphatidylethanolamine. The results suggest an interaction of the N-terminal region of the mature protein under its translocation across the membrane with phosphatidylethanolamine.     

Plasma membrane localization of alkaline phosphatase in HeLa cells.

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.     

Interaction of human red cell membrane acetylcholinesterase with phospholipids

Liposomes of phospholipids fully sustain the enzyme activity of the amphiphile-dependent dimers of human erythrocyte membrane acetylcholinesterase; no head group specificity exists. Diacylglycerides, glycerophosphorylcholine, or free fatty acids do not sustain the catalytic activity. It could be shown that the dimeric acetylcholinesterase with an exposed hydrophobic region can penetrate the lipid bilayer of liposomes and thus becomes stabilized by the surrounding phospholipid molecules.     

Chick embryo anchored alkaline phosphatase and mineralization process in vitro.

Bone alkaline phosphatase with glycolipid anchor (GPI-bALP) from chick embryo femurs in a medium without exogenous inorganic phosphate, but containing calcium and GPI-bALP substrates, served as in vitro model of mineral formation. The mineralization process was initiated by the formation of inorganic phosphate, arising from the hydrolysis of a substrate by GPI-bALP. Several mineralization media containing different substrates were analysed after an incubation time ranging from 1.5 h to 144 h. The measurements of Ca/Pi ratio and infrared spectra permitted us to follow the presence of amorphous and noncrystalline structures, while the analysis of X-ray diffraction data allowed us to obtain the stoichiometry of crystals. The hydrolysis of phosphocreatine, glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-bisphosphate by GPI-bALP produced hydroxyapatite in a manner similar to that of beta-glycerophosphate. Several distinct steps in the mineral formation were observed. Amorphous calcium phosphate was present at the onset of the mineral formation, then poorly formed hydroxyapatite crystalline structures were observed, followed by the presence of hydroxyapatite crystals after 6-12 h incubation time. However, the hydrolysis of either ATP or ADP, catalysed by GPI-bALP in calcium-containing medium, did not lead to the formation of any hydroxyapatite crystals, even after 144 h incubation time, when hydrolysis of both nucleotides was completed. In contrast, the hydrolysis of AMP by GPI-bALP led to the appearance of hydroxyapatite crystals after 12 h incubation time. The hydroxyapatite formation depends not only on the ability of GPI-bALP to hydrolyze the organic phosphate but also on the nature of substrates affecting the nucleation process or producing inhibitors of the mineralization.     

Interaction of calf intestinal alkaline phosphatase with 8-anilinonaphthalene-1-sulphonate

Calf intestinal alkaline phosphatase is inhibited by 8-anilinonaphthalene-1-sulphonate (ANS). The inhibition is uncompetitive but non-linear. Hill plots of the inhibition data have slopes of 1.4-1.8 suggestive of positive cooperativity. Fluorescence titration revealed that 2 molecules of ANS bind per molecule of enzyme with no evidence of cooperativity. The Kd for ANS obtained by fluorescence was 1.8 X 10(-6) mol/l but the approximate Ki for inhibition was 1 X 10(-3) mol/l. Thus, the fluorescence and kinetic experiments appear to monitor different events.     

Induction of plasma membrane alkaline phosphatase in rat liver

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.     

Biochemical and genetic analysis of a mutant with altered alkaline phosphatase activity in Dictyostelium discoideum

Alkaline phosphatase is one of several enzymes that accumulate in a temporally regulated sequence during the development of Dictyostelium discoideum. These enzymes can be used to monitor specific gene expression; moreover, isolation and analysis of mutations in the structural gene(s) can serve to indicate some of the essential steps in programmed synthesis and morphogenesis. A mutation (alpA) which affects the activity and substrate affinity of alkaline phosphatase was isolated in D discoideum using a procedure for screening large numbers of clones. Alkaline phosphatase activity at all stages of vegetative growth and development was altered by the mutation. Several physical properties of the enzyme from growing cells and developed cells were compared and found to be indistinguishable. It is likely that a single enzyme is responsible for the majority of alkaline phosphatase activity in growth and development. The mutation is coexpressed in diploids heterozygous for alpA and maps to linkage group III. One of the haploid segregants isolated from these diploids carries convenient markers on each of the six defined linkage groups and can be used for linkage analysis of other genetic loci.     

Interaction of D-beta-hydroxybutyrate apodehydrogenase with phospholipids.

The interaction of a soluble homogeneous preparation of D-beta-hydroxybutyrate apodehydrogenase with phospholipid was studied in terms of restoration of enzymic activity and complex formation. The purified apoenzyme, which is devoid of lipid, is inactive. It is reactivated specifically by the addition of lecithin or mixtures of phospholipids containing lecithin. Mitochondrial phospholipid, i.e. the mixture of phospholipids in mitochondria, reactivates with the highest specific activity (approximately 100 micromol of DPN reduced/min/mg at 37 degrees and with the greatest efficiency (2.5 to 4 mol of lecithin/mol of enzyme subunit). Each of the lecithins of varying chain length and unsaturation reactivated the enzyme, albeit to differing extents and efficiencies. In general, lecithins containing unsaturated fatty acid moieties reactivated better than those containing the comparable saturated lipid. Optimal reactivation can be obtained for the various lecithins when they are microdispersed together with phosphatidylethanolamine. When the lecithins are added microdispersed together with both phosphatidylethanolamine and cardiolipin, maximal efficiency is obtained. Also, PC6:0 and 8:0 reactivate as soluble molecules, so that a phospholipid bilayer is not necessary to reactivate the enzyme. Complex formation was studied using gel exclusion chromatography. It can be shown that each of the phospholipids which reactivate combines with the apoenzyme. Mitochondrial phospholipid, which reactivates the best, binds most effectively; PC8:0, which reactivates with poor efficiency, can be shown to bind with low affinity, and negligible binding occurs at concentrations which do not reactivate the enzyme. Since the apoenzyme is apparently homogeneous and devoid of phospholipid or detergents, it would appear that reactivation does not involve reversal of inhibition such as by removal of a regulatory subunit or detergent from the catalytic subunit. Rather, we conclude that phospholipid is a necessary and integral portion of this enzyme whose active form is a phospholipid-protein complex. The apoenzyme also forms a complex with phosphatidylethanolamine and/or cardiolipin, which do not reactivate enzymic activity. Salt dissociates such complexes in contrast with the lecithin-apoenzyme complex. Binding of phospholipid is a necessary but not sufficient requisite for enzymic activity. The same energies of activation are obtained from Arrhenius plots for the membrane-bound enzyme and for the purified soluble enzyme reactivated with mitochondrial phospholipid or different lecithins. This observation is compatible with the view that the purified enzyme has not been adversely modified in the isolation. Furthermore, essentially the same energies of activation were obtained for saturated lecithins below their transition temperatures and for unsaturated lecithins above their transition temperatures. Hence, there is no indication that a lipid phase transition occurs to influence the activity of this enzyme.     

Decoding signals for membrane protein assembly using alkaline phosphatase fusions.

We have used genetic methods to investigate the role of the different domains of a bacterial cytoplasmic membrane protein, MalF, in determining its topology. This was done by analyzing the effects of MalF topology of deleting various domains of the protein using MalF-alkaline phosphatase fusion proteins. Our results show that the cytoplasmic domains of the protein are the pre-eminent topogenic signals. These domains contain information that determines their cytoplasmic location and, thus, the orientation of the membrane spanning segments surrounding them. Periplasmic domains do not appear to have equivalent information specifying their location and membrane spanning segments do not contain information defining their orientation in the membrane. The strength of cytoplasmic domains as topogenic signals varies, correlated with the density of positively charged amino acids within them.     

Synthesis of phospholipids containing photoactivatable carbene precursors in the headgroups and their crosslinking with membrane proteins

Many integral membrane enzymes require for their activity interactions with the polar headgroups of phospholipids, in addition to the hydrophobic interactions within the lipid bilayer. The interactions with the polar headgroups may have preferential or absolute specificity. To study such interactions, phospholipids have been synthesized which carry photoactivable moieties in their headgroups. Three types of phospholipids, PL-I, PL-II and PL-III, were synthesized. The synthetic phospholipids, PL-I and PL-II were able to reconstitute enzymatic activity of the membrane proteins which were studied. Covalent crosslinking between these phospholipids and the membrane proteins was demonstrated after photolysis of the reconstituted phospholipid-protein complexes.     

An in vitro production of bone specific alkaline phosphatase

Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadaveric bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented.