Comparison of the size and physical properties of gamma-glutamyltranspeptidase purified from rat kidney following solubilization with papain or with Triton X-100.

gamma-Glutamyltranspeptidase is associated with the brush border membrane of kidney proximal straight tubule cells. It can be solubilized qualitatively by treatment with papain or Triton X-100. Neither procedure affects its catalytic activity but the two resulting forms of the enzyme differ considerably in their physical properties. The papain-solubilized transpeptidase is soluble in aqueous buffers and was purified 430-fold. It has an s20,w of 4.9 S, a Stokes radius of 36 A, and a calculated molecular weight of 69,000. It appears homogeneous by sedimentation equilibrium centrifugation (Mr=66,700). In contrast, the Triton-solubilized transpeptidase is soluble only in the presence of detergents and was purifed 300-fold. This form of the enzyme has a Stokes radius of 70 A but an s20,w of only 4.15 S. Aggregation of the enzyme just below the critical micelle concentration of Triton X-100 and its ability to bind 1.16 mg of Triton X-100-protein complex was calculated to be 169,000, but the glycoprotein portion of the complex is 52% of the total mass (87,000). The mass of Triton X-100 (82,000) is consistent with its reported micelle molecular weight. Treatment of the Triton-purified transpeptidase with papain or bromelain results in a form of the enzyme identical in all respects with the papain-purified enzyme. Both the Triton- and papain-purified transpeptidase exhibit two protein bands on sodium lauryl sulfate-polyacrylamide gel electrophoresis. The smaller subunits of the two forms appear identical (Mr=27,000), while the larger subunits of the Triton- and papain-purified enzyme have apparent molecular weights of 54,000 and 51,000, respectively. These data suggest that a peptide (3,000 to 19,000) in the larger subunit of gamma-glutamyltranspeptidase is responsible for its binding to Triton micelles and probably for holding the enzyme in the brush border membrane.     

Reconstitution of lipid vesicles associated with HVJ (Sendai virus) sikes. Purification and some properties of vesicles containing nontoxic fragment A of diphtheria toxin

A mixture of HVJ (Sendai virus) spike proteins, the nontoxic fragment A of diphtheria toxin, lecithin, and cholesterol was solubilized in sucrose solution containing a nonionic neutral detergent. The liposomal vesicles which formed on removal of the detergent by dialysis were purified by gel filtration and centrifugation on a sucrose gradient. The resulting purified vesicles had hemagglutinating activity, hemolytic activity and, after solubilization, the enzymic activity of fragment A. The vesicles had no cell fusion activity. Electron microscopy showed that both the outside and inside of membranes of the vesicles were associated with the spikes. When the vesicles were freeze-fractured, no large aggregates of particles were seen on either face. Such fragment A-containing lipid vesicles (liposomes) with HVJ spikes bound to mamalian cell membrane and released their fragment A into the cytoplasm causing cell death. Neither fragment A-containing liposomes without spikes nor empty liposomes with spikes were toxic.     

The activity of gamma-glutamyltranspeptidase in regenerating rat liver

gamma-Glutamyltranspeptidase is expressed at low levels in the liver of the male Fischer 344 rat where it exhibits 15-fold purification and 33% recovery in isolated plasma membranes. While the activity of the enzyme is unaltered in regenerating liver 24 h after partial hepatectomy, it increases steadily thereafter over a period of one week. Seven days after partial hepatectomy the enzyme is maximally activated: 5.6-fold in liver homogenates and 5.3-fold in isolated liver plasma membranes. The enzyme declines in activity over the next fourteen days and is expressed at normal levels three weeks after partial hepatectomy. These results demonstrate that the activity of gamma-glutamyltranspeptidase increases in regenerating liver but that the increase is out of phase with the proliferative response.     

Phosphate-independent glutaminase from rat kidney. Partial purification and identity with gamma-glutamyltranspeptidase.

Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a 200-fold purified preparation of this glutaminase. The purified enzyme also hydrolyzes gamma-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or gamma-glutamyhydroxamate, which is partially relieved by increasing concentrations of maleate. Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the gamma-glutamyltranspeptidase reaction. The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate0independent glutaminase and gamma-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities. Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity. In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells. The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase. Because of the localization of this activity on the brush border membrane, these resuts are consistent with the possibility that the physiological conditions induced by metabolic acidosis could convert this enzyme from a broad specificity transpeptidase to a glutaminase. Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

Biosynthesis and degradation of gamma-glutamyltranspeptidase of rat kidney

gamma-Glutamyltranspeptidase (gamma GTP) of rat kidney is an intrinsic glycoprotein bound to the plasma membrane and composed of two nonidentical subunits and an amino-terminal portion of the heavy subunit anchors the enzyme to the membrane. The mechanisms of biosynthesis, post-translational processing and degradation of the enzyme were studied using mono-specific antibody raised to gamma-glutamyltranspeptidase purified from rat kidney. The following results were obtained. Double isotope labeling in vivo showed that gamma-glutamyltranspeptidase is synthesized as a precursor form with a single polypeptide chain of 78,000 daltons, and then processed post-translationally by limited proteolysis, resulting in two subunits of 50,000 and 23,000 daltons. Incorporation of [3H]leucine or [35S]methionine into the precursor form increased until 60 min after their intravenous injection, and a pulse-chase experiment showed that the half life of the precursor form was 53 min. [3H]Fucose and [3H]glucosamine could also be incorporated into the precursor form, showing that glycosylation of the enzyme occurs at the stage of the precursor form. Rat kidney labeled with [3H]fucose was subjected to subcellular fractionation. The Golgi fraction contained the glycosylated precursor form and a small amount of subunits, and the plasma membrane fraction contained mostly subunits with a significant amount of precursor, suggesting that post-translational processing of the precursor occurs on the plasma membrane. The apparent half lives of the native enzyme and the heavy and light subunits were all estimated as 4.3 +/- 0.5 days by labeling with [3H]leucine or [3H]fucose. gamma-Glutamyltranspeptidase has a different turnover rate from aminopeptidase M, which is located in the microvillus membrane close to gamma-glutamyltranspeptidase.     

Membrane interactions of rat intestinal alkaline phosphatase: role of polar head groups

Lipid-protein interactions with purified membranous intestinal alkaline phosphatase have been studied by using rat intestine. The enzyme was incorporated equally well into neutral lecithin and anionic liposomes, including those made from phosphatidic acid alone. It could not be solubilized with chaotropic salts nor by phospholipases C and D from either native membranes or phospholipid vesicles. Detergents effected nearly complete release of enzyme from the vesicles. Phosphatase activity was lost upon treatment with phospholipase D alone. The activity was restored with free choline, or choline containing phospholipids, but not by the addition of other phospholipids or amines. The catalytic activity was also lower when the enzyme was bound to a phosphatidylcholine vesicle containing additional phosphatidic acid. Neither phosphatidylserine nor phosphatidylinositol addition altered enzyme activity. These results show that the enzyme binds to the membrane by a primary hydrophobic interaction with membrane phospholipids without requiring the polar head group and that the enzyme activity is affected via a secondary interaction with choline. We suggest that choline protects the active site of brush border alkaline phosphatase from inhibition by endogenous membrane phosphate groups.     

Phospholipase D Stimulates Release of Nascent Secretory Vesicles from the trans-Golgi Network

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.     

Isolation and characterization of monopinocytotic vesicles containing polyomavirus from the cytoplasm of infected mouse kidney cells.

Monopinocytotic vesicles containing polyomavirus were isolated from the cytoplasm of mouse kidney cells infected with polyomavirus using sucrose density gradients. Nonenclosed, membrane-associated virions released by the action of neuraminidase separated from vesicle-enclosed virions in the sucrose gradient. Marker enzyme assays indicated the derivation of the vesicle membrane from the plasma membrane of the cell. The 125I-labeled virus enclosed in the vesicle sedimented more slowly in the gradient and was not observed unless infection and endocytosis had occurred. Detergent treatment of virion-containing vesicles caused the release of polyomavirus with sedimentation properties similar to those of purified polyoma virions. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of virion proteins from vesicles containing virions demonstrated patterns of proteins similar to those of purified intact virions. Electron microscopy confirmed the presence of single intact virions inside vesicles. The study of these monopinocytotic virion-containing vesicles represents a further step in elucidating the early events of polyomavirus infection.     

Structural changes in membranes of large unilamellar vesicles after binding of sodium cholate

The interaction of the bile salt cholate with unilamellar vesicles was studied. At low cholate content, equilibrium binding measurements with egg yolk lecithin membranes suggest that cholate binds to the outer vesicle leaflet. At increasing concentrations, further bile salt binding to the membrane is hampered. Before the onset of membrane solubilization, diphenylhexatriene fluorescence anisotropy decreases to a shallow minimum. It then increases to the initial value in the cholate concentration range of membrane solubilization. At still higher cholate concentrations, a drop in fluorescence anisotropy indicates the transformation of mixed disk micelles into spherical micelles. Perturbation of the vesicle membranes at molar ratios of bound cholate/lecithin exceeding 0.15 leads to a transient release of oligosaccharides from intravesicular space. The cholate concentrations required to induce the release depend on the size of the entrapped sugars. Cholesterol stabilizes the membrane, whereas, in spite of enhanced membrane order, sphingomyelin destabilizes the membrane against cholate. Freeze-fracture electron microscopy and phosphorus-31 nuclear magnetic resonance (31P NMR) also reflect a change in membrane structure at maximal cholate binding to the vesicles. In 31P NMR spectra, superimposed on the anisotropic line typically found in phospholipid bilayers, an isotropic peak was found. This signal is most probably due to the formation of smaller vesicles after addition of cholate. The results were discussed with respect to bile salt/membrane interactions in the liver cell. It is concluded that vesicular bile salt transport in the cytoplasm is unlikely and that cholate binding is restricted to the outer leaflet of the canalicular part of the plasma membrane.     

Purification and characterization of a cortical secretory vesicle membrane fraction

A membrane fraction has been prepared by sucrose density gradient fractionation of purified cortical secretory vesicles from the eggs of the sea urchin Strongylocentrotus purpuratus. The purified cortical vesicle membrane fraction has a phospholipid to protein ratio of 1.76 and exhibits a morphology typical of biological membranes as seen by electron microscopy. The protein composition of the purified membranes was analyzed by SDS-polyacrylamide gel electrophoresis and shown to be distinct from that of eggs, cell surface complex, cortical vesicles, fertilization product, and yolk platelets. Alkaline extraction (pH 11.0) of peripheral membrane proteins increased the phospholipid to protein ratio to 2.55 and removed several polypeptides. Immunoblot analysis of the isolated cortical vesicle membrane fraction revealed low levels of contamination with two major cortical vesicle content proteins. Fractions enriched in egg plasma membranes and yolk platelet membranes also have been isolated and compared with the cortical vesicle membranes by SDS-polyacrylamide gel electrophoresis. The protein compositions of the three membrane fractions were found to contain very little overlap, indicating that the cortical vesicle membrane preparation is relatively free of contamination from these likely noncortical vesicle sources of membrane. Both the plasma membrane and cortical vesicle membrane samples were found by immunoblotting to contain actin.     

Characterization of right-side-out membrane vesicles rich in (Na,K)-ATPase and isolated from dog kidney outer medulla

When purified on a sucrose gradient, basolateral membranes from dog kidney outer medulla are found to be very rich in (Na,K)-ATPase; about 50% of the membrane protein is comprised of this enzyme. (Na,K)-ATPase activity is activated 3- to 5-fold by detergent treatment, and this has been previously attributed to the impermeable vesicular nature of the membranes. Porcine trypsin inactivates only that fraction of (Na,K)-ATPase activity seen without detergent, consistent with a right-side-out orientation of membrane vesicles; the trypsin sensitivity and detergent activation of [3H]ouabain binding in the presence of Na+ + Mg2+ + ATP or Mg2+ + Pi are also consistent with this hypothesis. Using nearly isosmotic Hypaque density gradient centrifugation a population of impermeable right-side-out membrane vesicles (H1) is separated from a leaky population (H2). (Na,K)-ATPase activity in the H1 population is 20-fold activated by detergent and insensitive to porcine trypsin. The vesicle volume is 2.4 microliters/mg, and monovalent cations passively equilibrate with the intravesicular volume on a time scale of 5-30 min. Very rapid ouabain sensitive 22Na efflux from the vesicles is observed when ATP is photolytically released from intravesicular caged ATP.     

Purification and functional reconstitution of the voltage-sensitive sodium channel from rabbit T-tubular membranes

The voltage-sensitive sodium channel has been purified from rabbit T-tubular membranes and reconstituted into defined phospholipid vesicles. Membranes enriched in T-tubular elements (specific [3H]nitrendipine binding = 41 +/- 9 pmol/mg of protein, n = 7) were isolated from fast skeletal muscle. After solubilization with Nonidet P-40, the sodium channel protein was purified to greater than 95% of theoretical homogeneity based on the specific activity of [3H]saxitoxin binding. Two subunits of Mr approximately 260,000 and 38,000 were found; these bands co-distributed with the peak of [3H]saxitoxin binding on sucrose gradients. The purified protein was reconstituted into egg phosphatidylcholine vesicles and retained the ability to gate specific 22Na+ influx in response to activation by batrachotoxin or veratridine. All activated fluxes were blocked by saxitoxin and tetrodotoxin. On sucrose gradients, the distribution of protein capable of functional channel activity paralleled the distribution of specific [3H]saxitoxin binding and of the Mr 260,000 and 38,000 components. The cation selectivity for the reconstituted, batrachotoxin-activated channel was Na+ greater than K+ greater than Rb+ greater than Cs+, with flux ratios of 1:0.13:0.02:0.008. Nine of 25 monoclonal antibodies raised against the rat sarcolemmal sodium channel cross-reacted with the rabbit T-tubular sodium channel in a solid-phase radioimmunoassay. Six of these antibodies showed specific binding to immunoblot transfers of T-tubular membrane proteins. Each labeled a single band at Mr approximately 260,000 corresponding in mobility to the large subunit of the sodium channel.     

Transmembrane orientation of the mannose 6-phosphate receptor in isolated clathrin-coated vesicles

The mannose 6-phosphate (Man-6-P) receptor is an integral membrane glycoprotein which mediates intracellular transport and receptor-mediated endocytosis of lysosomal proteins. Clathrin-coated vesicles, which have been shown to be significantly involved in these processes, have also been shown to be a major subcellular site of the receptor. In order to define the orientation of the Man-6-P receptor within the coated vesicle membrane, highly purified preparations of coated vesicles were prepared from bovine brain employing D2O/sucrose gradient centrifugation and Sephacryl S-1000 column chromatography. Using [35S]methionine-labeled lysosomal enzymes secreted by Chinese hamster ovary cells as receptor ligand, significant binding activity was detected only upon permeabilization of the coated vesicle membranes with detergent. Prior treatment of intact vesicles with proteinase K resulted in similar binding activity upon permeabilization. However, examination of the receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with rabbit anti-receptor serum revealed that proteinase K treatment of intact vesicles reduced the size of the receptor by 12,000 daltons. A similar decrease in size was obtained when the vesicles were treated with carboxypeptidase Y. These results suggest that the Man-6-P receptor is a transmembrane protein with its lysosomal enzyme binding site oriented toward the lumen of the coated vesicle and its C-terminal end exposed to the exterior or cytoplasmic portion of the vesicle membrane.     

Effect of sodium deoxycholate on 5'-nucleotidase.

The 5'-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5'-nucleotidase exhibited the same properties as the 5'-nucleotidase in plasma membranes. The 5'-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and Km. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Hydrophobic binding domains of rat intestinal maltase-glucoamylase

Rat intestinal microvillus maltase-glucoamylase was isolated by detergent extraction and purification in the presence of protease inhibitors as previously described and incorporated into phospholipid vesicles. After purification of the vesicles on Sephadex G-50, maltase was labelled with 3-trifluoromethyl-3-(m-[125I]iodophenyl) diazirine ([125I]TID) by photolysis using a water-jacketed mercury vapour lamp with a saturated CuSO4 filter. The labelled enzyme was extracted with acetone, resuspended in 1% Triton X-100, reincorporated into phospholipid vesicles, and digested with activated papain to release the hydrophilic polar head of the enzyme from the vesicle bilayer. Vesicle-bound and free enzyme components were separated on Sepharose 4B. Ninety percent of the enzymatic activity was free, while a similar percentage of radioactive label remained with the vesicles in keeping with the separation of an active polar headpiece from a labelled apolar peptide in the lipid bilayer. The vesicle fractions were subjected to chromatography on Sephadex LH-60 with ethanol--formic acid (7:3) as the eluant. A single radioactive peak (14 kilodaltons (kDa)) was separated from labelled lipid. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the peak showed a radioactive doublet of 26-28 kDa, possibly representing a dimer. No other labelled peptides were found. These results suggest that detergent-solubilized maltase-glucoamylase is inserted into the phospholipid bilayer via an apolar peptide with a minimum molecular mass of 14 kDa. The peptide probably represents a terminal anchor segment of the 145-kDa subunit which is converted to 130 kDa when the membrane-bound enzyme is solubilized by papain.     

Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms

Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of the enzyme to be an amphipathic molecule. The papain treatment of either brush-border membrane vesicles or the purified detergent form of neutral alpha-D-glucosidase released an enzymatic form devoid of these amphipathic properties. Conversely, after trypsin treatment of the "d' form of the enzyme, two enzymatic forms were obtained: the first and major form retained these amphipathic properties; the second form exhibiting the same properties as the papain-released form. Furthermore, only a very small amount of neutral alpha-D-glucosidase can be released after trypsin solubilization of brush-border membrane vesicles, and the released enzyme did not exhibit amphipathic properties. These results were interpreted as meaning that the trypsin attack site on the detergent form of the enzyme had either poor affinity for, or obstructed access to, the proteinase when the enzyme was integrated in native membrane or in Triton X-100 micelles, whereas the proteolytic site of the papain was always accessible.     

Proteoliposome interaction with human erythrocyte membranes. Functional implantation of gamma-glutamyl transpeptidase

The transfer of detergent solubilized and purified gamma-glutamyl transpeptidase (gamma-GTase), of hog kidney cortex, from proteoliposomes into human erythrocyte ghost membranes has been studied. The transfer of gamma-glutamyl transpeptidase was observed upon incubation of gamma-GTase incorporated dipalmitoylphosphatidylcholine vesicles with erythrocyte ghost membranes at 37 degrees C for 12 h. The extent of transfer was dependent upon the fluidity of donor proteoliposomes, being more when dipalmitoylphosphatidylcholine proteoliposomes were used compared to dimyristoylphosphatidylcholine, and intermediate values were observed when binary mixtures of DMPC and DPPC were used. Moreover, the transfer of gamma-GTase was facilitated when rigid basic phospholipid proteoliposomes were used as donor. The transfer of gamma-GTase has been observed to be associated with the removal of intrinsic membrane proteins and lipids from erythrocytes, mainly acetylcholinesterase, sphingomyelin, and cholesterol. An enhancement in the fluorescence due to resonance energy transfer was observed when ghost membranes containing fluorescent donor probe were incubated with proteoliposomes containing fluorescent acceptor probe, indicating that fusion but not adsorption of vesicles occurs during the transfer process. However, the inability of entrapped [14C]-sucrose delivery from proteoliposomes into ghost membrane vesicle suggest that fusion per se is not primarily involved in the transfer process. It appears that the transfer of gamma-glutamyl transpeptidase occurs by a collisional transfer process resulting in intermembrane protein transfer. The gamma-glutamyl transpeptidase implanted ghost membranes exhibited the uptake of L-glutamate which was inhibited by serine-borate, an inhibitor of transpeptidase activity. In addition, the uptake of L-glutamate was inhibited by the dipeptide gamma-glutamyl-L-glutamate, thus supporting the proposed role of gamma-glutamyl transpeptidase in the uptake of amino acids in biological membranes.     

Incorporation of bovine enterokinase in reconstituted soybean phospholipid vesicles

Bovine enterokinase was incorporated into vesicles reconstituted from a soybean phospholipid mixture. A thin film hydration procedure (MacDonald, R. I., and MacDonald, R. C. (1975) J. Biol. Chem. 250, 9206-9214) produced vesicles with 40% of the enterokinase activity bound in the membrane. The highest incorporation was observed when cholesterol or dimyristoylphosphatidylethanolamine was added to the soybean phospholipids. Crude and highly purified enterokinase preparations were incorporated to the same extent suggesting that other membrane components were not required for a successful reconstitution. The properties of enterokinase in phospholipid vesicles were compared with those of alkaline phosphatase, which was also added to the reconstitution system, and with the enzyme activities present in vesicles prepared from brush-border membranes. The enzyme activities were not released by solutions of high ionic strength and remained associated with the phospholipid vesicles on gel filtration, ultracentrifugation, and sucrose density centrifugation. Enterokinase and alkaline phosphatase had their active sites exposed to substrate in the brush-border membrane vesicles. In soybean phospholipid vesicles half of the active sites of both enzymes were on the outside, since release of the enzyme with Triton X-100 almost doubled the units of enzyme present. Incubation of the soybean phospholipid and brush-border membrane vesicles with papain released the exposed molecules of enterokinase. The released enzyme molecules were fully active but could not be reincorporated into phospholipid vesicles. This suggests that the structure imbedded in the lipid bilayer was essential for a successful reconstitution. We conclude that the reconstituted soybean phospholipid vesicles are a suitable membrane system for the further study of membrane-bound enterokinase.     

The effect of papain upon proline and sodium transport of rat renal brush-border membrane vesicles

Treatment of renal brush-border membrane vesicles with papain resulted in the removal of the activity of maltase, gamma-glutamyl transpeptidase and leucine aminopeptidase by 85, 50 and 75%, respectively. Stripping of these membrane enzyme activities constituted about 2% of the total membrane proteins and resulted in a widespread diminution in the ability of a variety of amino acids and sugars to be taken up by the membrane vesicles which remained osmotically responsive. Kinetic analysis of the uptake of proline, which was shown previously to be transported by both sodium-dependent and sodium-independent systems, revealed that the Vmax for the sodium-dependent system and Km for the sodium-independent system were halved, but other parameters were not affected indicating that the papain treatment altered sodium-gradient-stimulated entry and the affinity of the sodium-gradient-independent system for proline. Experiments on sodium entry and efflux demonstrate a marked enhancement of flux, so that equilibration of the sodium gradient occurred about 5-times more rapidly than in untreated vesicles. This occurred without any change in the osmotic properties of the vesicle with regard to sodium or amino acid uptake. Studies of fluorescence polarization suggest that incubation with papain does not alter the lipid domains of the membrane.