Isolation from bovine brain of a fraction containing capillaries and a fraction containing membrane fragments of the choroid plexus

Combined differential and density gradient centrifugation was used for the isolation of a capillary-rich fraction from the cerebral cortex and a brush border containing fraction from the bovine choroid plexus. The activities of γ-glutamyl transpeptidase and several other marker enzymes were monitored during the fractionation procedure. Electron microscopic examination showed a membrane-rich fraction in the choroid plexus high in γ-glutamyl transpeptidase and 5'-nucleotidase activities. From the brain cortex, a capillary-rich fraction was obtained which was high in γ-glutamyl transpeptidase and alkaline phosphatase activities. A histochemical examination showed γ-glutamyl transpeptidase activity localized in the capillary walls.     

Comparative studies on membranes from bovine choroid plexus and rat kidney cortex.

Comparative subcellular fractionation studies on rat kidney and bovine choroid plexus using differential centrifugation and free flow electropheresis were undertaken because of the morphological and functional similarities of the epithelial cells of both tissues. The activities of three enzymes commonly used as markers for brush border membranes in kidney were measured in fractions of each tissue. γ-Glutamyl transpeptidase, alkaline phosphatase, and 5'-nucleotidase copurified in membrane fractions of renal cortex collected by differential centrifugation. Application of a similar fractionation procedure to choroid plexus gave relatively similar results, except for alkaline phosphatase, the yield of which was substantially reduced in a fraction enriched with two marker enzymes. Further fractionation of γ-glutamyl transpeptidase and alkaline phosphatase activities in these membrane fractions was achieved using free flow electropheresis. The two enzymes from kidney exhibited discrete peaks with a small separation, while the electropheretic pattern of γ-glutamyl transpeptidase from choroid plexus was biphasic. Alkaline phosphatase was observed to migrate with the more basic γ-glutamyl transpeptidase peak.     

Membrane Populations of Bovine Choroid Plexus: Separation by Density Gradient Centrifugation in Modified Colloidal Silica

Abstract: The choroid plexus is intimately involved in the production and regulation of the cerebrospinal fluid. Populations of surface membranes from this epithelial tissue were separated by density gradient centrifugation by use of modified colloidal silica (Percoll). A fraction of heavy microsomes (P₃) containing plasma membranes was prepared by differential centrifugation. Membranes in fraction P₃ were mixed with a given concentration of Percoll and density gradients generated during centrifugation. When fraction P₃ was mixed with 20% (v/v) Percoll and centrifuged at 20,000 r.p.m. for 1 h in a 50.2 Ti fixed-angle rotor, membranes containing alkaline phosphatase (AP) were found at a density of 1.037 g/cm³ while those containing NaK ATPase were found at 1.047 g/cm³. With more shallow density gradients using 12% and 14% Percoll, a broad shoulder of AP activity became manifest at densities greater than 1.060 g/cm³ suggesting multiple populations of membranes containing AP. Membranes containing AP could also be separated from membranes containing γ-glutamyl transpeptidase (γ-GTP); this separation was most pronounced in 12% Percoll. The activity of γ-GTP could not be separated from activity of NaK ATPase. Total protein was distributed broadly throughout the gradients. Studies have been undertaken to compare the behavior of choroidal membranes in Percoll gradients with that of renal membranes because the biochemical anatomy of the kidney has been extensively studied. In contrast to choroidal membranes, renal membranes with NaK ATPase activity were found to have densities lower than those membranes with AP. Thus, the distribution of membrane-bound enzymes from kidney in a Percoll gradient was exactly the opposite of that observed for these same enzymes from choroid plexus. In addition, unlike the γ-GTP activity of choroid plexus, γ-GTP from kidney could be separated from the activities of both alkaline phosphatase and NaK ATPase. These marked differences in membrane populations between choroid plexus and kidney as defined by Percoll density gradient centrifugation analyses are presumably reflective of differences in the functions of the two epithelial tissues.     

gamma-Glutamyl transpeptidase in Hydra littoralis.

$\text{Hydra}$$\text{littoralis}$ exhibits high γ-glutamyl transpeptidase activity, i.e., about 12% of the activity (determined with glutathione) of rat kidney. Histochemical studies show that the enzyme is located mainly in the gastric and sub-hypostome regions; the enzyme is also present in the tentacles and basal disc. These results and the presence of other enzymes of the γ-glutamyl cycle suggest that the cycle plays a role in the metabolism of glutathione in hydras and that γ-glutamyl transpeptidase may function in their digestive and absorptive processes and possibly also in the behavioral response to glutathione.     

Prostaglandin D synthase in microvessels from the cat cerebral cortex

Microvessels, a mixture composed predominantly of small arterioles and capillaries (7–80μ diameter), were isolated from the rat cerebral cortex by selective nylon sieving and glass bead elutriation. The morphology and purity of the microvessel and cerebral cortex filtrate (virtually free of vascular contamination) were monitored by light microscopy and by the activity of several enzymes: γ-glutamyl transpeptidase, GSH-transferase, prostacyclin synthase and PGD synthase. Prostacyclin and PGD synthesizing activities as well as γ-glutamyl transpeptidase activity were localized to the microvessels of the rat cerebral cortex whereas GSH-S-transferase was restricted to the non-vascular filtrate function. The characteristics of the PGD synthase were similar to those of the purified enzyme previously described for the rat brain. The microvessel (MV) PGD synthase was localized to the cytosol fraction of the microvessels and did not require reduced glutathione for activity. The enzyme was inhibitd by pre-incubation with p-hydroxymercuribenzoate (lmM) or N-ethylmaleimide (lmM). The MV PGD synthase saturated at 15–20μM PGH₂, exhibited an apparent K_M of 9.6μM, and a pH optimum of 8.0–8.1. These findings suggest roles for both prostacyclin and PGD synthesis by the rat cerebral vasculature in the autoregulation of cerebral blood flow and/or function. These studies also indicate that the major source of PGI₂ and PGD₂ synthesis by rat brain homogenates is the microvasculature.     

Isolation of anthglutin, an inhibitor of gamma-glutamyl transpeptidase from Penicillum oxalicum.

Anthglutin, a new inhibitor of γ-glutamyl transpeptidase, has been isolated from the cultured medium of Penicillium oxalicum and its structure established as l-γ-l-glutamyl-2-(2-carboxyphenyl)hydrazine. The isolation of anthglutin was achieved by ion-exchange chromatography. Anthglutin inhibited γ-glutamyl transpeptidase specifically and the kinetic analysis of the inhibition showed that anthglutin inhibited the enzyme competitively with regard to the glutamyl donor, γ-glutamyl-p-nitroanilide, and noncompetitively with regard to the glutamyl acceptor, glycylglycine. K₁ values were 5.7 μm for the hog kidney enzyme, 18.3 μm for the human kidney enzyme, 13.6 μm for the human liver soluble enzyme, and 10.2 μm for the bound enzyme. After oral administration of [¹⁴C]methionine and anthglutin to rats, no effect of anthglutin was observed on the absorption of methionine in the intestine.     

NIACIN AND NIACINAMIDE ACCUMULATION BY RABBIT BRAIN SLICES AND CHOROID PLEXUS IN VITRO

The transport into and release of¹⁴C-labeled niacin and niacinamide from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated ¹⁴C by specific, energy-dependent mechanisms when [¹⁴C]niacinamide was added to the incubation medium. The saturable accumulation velocities, which were linear for 30 min, depended, in part, on incorporation of the [¹⁴C]niacinamide into NAD. The X_T and Y_max for ¹⁴C accumulation with [¹⁴C]niacinamide in the medium by brain slices and choroid plexus were 0.80 μM and 1.45 μmolkg^?1 (30 min)^?1, and 0.23 μM and 18.6 μmol kg^?1 (30 min)^?1 respectively. In vitro, the choroid plexus, unlike brain slices, vigorously concentrated ¹⁴C by a separate, specific energy-dependent process when ¹⁴C niacin was added to the incubation medium. The saturable accumulation velocity, which was linear for 30 min, depended completely on the metabolism of [¹⁴C]niacin. The K_T and Y_max for¹⁴C accumulation by choroid plexus with [¹⁴C]niacin in the medium were 18.1 μM and 439 μmol kg^?1 (30 min)^?1 respectively. Whether preincubated in [¹⁴C]niacin or [¹⁴C]niacinamide, choroid plexus released predominantly [¹⁴C]niacinamide.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VITRO STUDIES

Abstract— The transport into and release of tritium labeled vitamin B₆ ([³H]B₆) from rabbit brain slices and isolated choroid plexuses were studied. In vitro, both brain slices and choroid plexus concentrated [³H]B₆ by an energy dependent uptake system when [³H]pyridoxine (PIN) was added to the incubation medium. Most of the [³H] within the tissues was phosphorylated [³H]B₆. In each tissue, the nonphosphorylated vitamers inhibited the uptake of [³H]PIN from the medium significantly more than the phosphorylated vitamers. The concentrations of the nonphosphorylated B₆ vitamers necessary to inhibit brain and choroid plexus uptake of [³H]PIN from the medium by 50% were approx 0.4 μm and 5–10μm respectively after a 30 min incubation. Both brain slices and choroid plexus readily released (46 and 56% respectively in 30 min) previously accumulated [³H]B₆ into artificial CSF. However, brain slices released only nonphosphorylated [³H]B₆, whereas the choroid plexus released predominantly phosphorylated [³H]B₆. Addition of unlabeled PIN to the release media significantly increased the percentage of [³H]B₆ released by both brain slices and choroid plexus. The results of these in vitro studies provide evidence that: (1) both brain slices and chloroid plexus possess specific uptake and release mechanisms for B₆, and (2) these mechanisms tend to regulate intracellular B₆ levels. These studies also suggest that the choroid plexus serves as a locus for the transfer of B₆ from blood to CSF and is the source of most of the phosphorylated B₆ in CSF.     

Modification of the acceptor binding site of gamma-glutamyl transpeptidase by the diazonium derivatives of p-aminohippurate and p-aminobenzoate

Hippurate and maleate have been shown to bind to the aminoacylglycine (acceptor) binding site of γ-glutamyl transpeptidase, thereby stimulating the hydrolysis of γ-glutamyl compounds at the expense of transpeptidation (Thompson, G. A., and Meister, A. (1979) J. Biol. Chem.254, 2956–2960; Thompson, G. A., and Meister, A. (1980) J. Biol. Chem.255, 2109–2113). It has now been found that a number of benzoate derivatives also bind and modulate rat kidney transpeptidase, as indicated by their ability to enhance the rate of inactivation of transpeptidase by the glutamine antagonist l-(αS, 5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Furthermore, rapid loss of transpeptidase activity results upon preincubation of the enzyme with the diazonium derivatives of p-aminohippurate and p-aminobenzoate. The modified enzyme can still hydrolyze γ-glutamyl substrates but is no longer modulated by hippurate and maleate. Loss of transpeptidase activity was not associated with incorporation of radioactive label from diazotized [¹⁴C]p-aminohippurate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the modified enzyme revealed a nondissociable species, M_r 68,000, shown to result from crosslinking of the two subunits of transpeptidase (M_r 46,000 and 22,000, respectively). The crosslinking of the subunits paralleled the extent of inactivation of transpeptidation activity and both crosslinking and inactivation were prevented by treatment with the diazotized derivatives in the presence of either hippurate or maleate. These and other data indicate that the diazonium derivatives of p-aminohippurate and p-aminobenzoate interact with the acceptor binding site and produce a stable bond between amino acid residues in the vicinity of this site which, thus, appears to be located in the intersubunit contact region.     

gamma-Glutamyl transpeptidase: relation to immunoglobulin A and free secretory component.

The experiments reported show that bovine γ-glutamyl transpeptidase can be separated from free secretory component. An ion-exchange Chromatographic procedure was developed to analyze the incubation mixtures of the enzyme with glutathione or S-(2-acetamido)-glutathione and glycylglycine. Using this system or the γ-glutamyl p-nitroanilide assay, no significant transpeptidase activity could be detected in the free secretory component-containing fractions of DEAE-cellulose chromatography. Gel filtration on Biogel A-5M showed that the bovine whey transpeptidase chromatographed in the void volume suggesting an aggregate of a minimum molecular weight of about 5 × 10⁶. The transpeptidase could be separated from all immunoglobulins in bovine whey and human colostrum by a combination of agarose gel filtration and immunoadsorption. Concentrated samples of human and sheep saliva showed normal amounts of secretory component, but no detectable γ-glutamyl transpeptidase activity. These experiments show that (1) the transpeptidase and secretory component are two different proteins, and (2) the transpeptidase is present in bovine and human milk as a high molecular weight aggregate which does not include any of the immunoglobulins.     

Isolation from bovine brain of a fraction containing capillaries and a fraction containing membrane fragments of the choroid plexus.

Combined differential and density gradient centrifugation was used for the isolation of a capillary-rich fraction from the cerebral cortex and a brush border containing fraction from the bovine choroid plexus. The activities of gamma-glutamyl transpeptidase and several other marker enzymes were monitored during the fractionation procedure. Electron microscopic examination showed a membrane-rich fraction in the choroid plexus high in the gamma-glutamyl transpeptidase and 5'-nucleotidase activities. From the brain cortex, a capillary-rich fraction was obtained which was high in gamma-glutamyl transpeptidase and alkaline phosphatase activities. A histochemical examination showed gamma-glutamyl transpeptidase activity localized in the capillary walls.     

NIACIN AND NIACINAMIDE TRANSPORT IN THE CENTRAL NERVOUS SYSTEM. IN VIVO STUDIES

Abstract– The concentration ol niacinamide in plasma and CSF was 0.5 and 0.7 μm respectively. The, mechanisms by which niacin and niacinamide, which are not synthesized in brain, enter brain, CSF and choroid plexus were investigated by injecting [¹⁴C]niacin or [¹⁴C]niacinamide intravenously and intraventricularly. [¹⁴C]Niacin or [¹⁴C]niacinamide, with or without unlabeled niacin or niacinamide, were infused intravenously at a constant rate into conscious rabbits. At 3 h, [¹⁴C]niacinamide, but not [¹⁴C]niacin, readily entered CSF, choroid plexus and brain. The addition of 4.1 mmol/kg niacinamide to the infusate markedly depressed the relative entry of [¹⁴C]niacinamide into choroid plexus and brain but not into CSF. After intraventricular injection, [¹⁴C]niacin was rapidly cleared from CSF and readily entered brain and choroid plexus. The addition of unlabeled niacin to the intraventricular injectate decreased the clearance of [¹⁴C]niacin from CSF and the entry of [¹⁴C]niacin into choroid plexus and brain. Unlike niacin, carrier niacinamide (82 μmol) in the injectate did not depress the extremely rapid clearance of intraventricularly injected [¹⁴C]niacinamide from CSF but did decrease the entry of [¹⁴C]niacinamide into brain. These results show that the control of entry and exit of niacinamide and niacin is the mechanism, at least in part, by which total niacin and NAD levels in brain cells are regulated. In the case of niacinamide which readily passes between CSF and plasma, the regulation of entry of niacinamide into brain cells by a high affinity accumulation system is an integral part of the homeostatic system. In the case of niacin, penetration into CSF and the extracellular space of brain from plasma as well as regulation of entry into brain cells by a saturable accumulation system are two distinct parts of the homeostatic system. In vivo, niacin that enters the central nervous system is converted to the principal plasma vitamer, niacinamide, in its free or bound forms such as NAD.     

gamma-Glutamyl transpeptidase in Hydra.

γ-Glutamyl transpeptidase (EC 2.3.2.2) activity is described in the coelenterate, $\text{Hydra}$$\text{attenuata}$, using the substrate γ-glutamyl-p-nitroanilide. The properties of the γ-glutamyl donor required for binding to the transpeptidase were investigated by measuring the ability of GSH analogs to inhibit the release of p-nitroaniline. Whereas no binding was observed when the γ-glutamyl moiety was altered, analogs with substitution in the Cys residue were capable of binding to the enzyme. A specificity for the Gly residue was indicated because analogs containing Leu or Tyr in place of Gly exhibited decreased binding capacities for the hydra transpeptidase. A comparison of these data with those obtained using the same analogs in the GSH induced feeding response bioassay shows that γ-glutamyl transpeptidase activity and the GSH receptor for the hydra feeding response have different specificities.     

Transport of the large-neutral amino acids by the gamma-glutamyl cycle: a proposal.

The γ-glutamyl cycle has been proposed by Meister (1973) as one possible mechanism for the mediation of amino acid transport. The high energy requirement of the pathway, the very low specificity of γ-glutamyl transpeptidase and the inability to account for trans membrane stimulation of amino acid entry are but three criticisms of this hypothesis. It is proposed that the various objections can be overcome by postulating that the soluble form of γ-glutamyl transpeptidase transfers the γ-glutamyl moiety from gluthathione to glutamine (in the case of brain) and that the membrane sequestered form of this enzyme catalyzes the exchange of the γ-glutamyl group between γ-glutamyl glutamine and an entering neutral amino acid. The released glutamine leaves the cell. The γ-glutamyl amino acid then passes into the cytoplasm where it is acted upon by either γ-glutamyl cyclotransferase or the soluble γ-glutamyl transpeptidase which transfers the γ-glutamyl group to another molecule of glutamine. It is postulated that access to the membrane-bound enzyme is dependent on the relative lipophilia of the entering large-neutral amino acids. The available data support this mechanism. By regeneration of γ-glutamyl glutamine, a low expenditure of energy is required for the transport process. Specificity of transpeptidation is attained by the constraints of access to the membrane bound enzyme site.     

Gamma-glutamyl transpeptidase of rat seminal vesicles; effect of orchidectomy and hormone administration on the transpeptidase in relation to its possible role in secretory activity.

γ-Glutamyl transpeptidase was prepared from rat seminal vesicles by two methods and was found to be similar to rat kidney γ-glutamyl transpeptidase with respect to substrate specificity, stimulation of “glutaminase” activity by maleate, and apparent molecular weight. Histochemical studies demonstrated that γ-glutamyl transpeptidase is concentrated in the secretory epithelium of the seminal vesicle. Like the epithelium itself, the enzyme responds to the presence or absence of testosterone. The content and specific activities of γ-glutamyl transpeptidase and γ-glutamyl cyclotransferase in rat seminal vesicles are low in orchidectomized animals, an effect which is reversed by administration of testosterone but accentuated by estradiol administration. These enzymes may be involved in the secretory functions of the seminal vesicles.     

Immobilization of γ-glutamyl transpeptidase,a membrane enzyme,in gel beads via liposome entrapment

Bovine kidney γ-glutamyl transpeptidase, a membrane enzyme, was immobilized in gel beads by application of the method of Wallstén et al. (Biochim. Biophys. Acta, 982, 47–52, 1989). The gel beads were equilibrated with a dispersion of the enzyme, phospholipids, and cholate and subsequently dialyzed against a buffer for reconstitution and immobilization of enzyme-bound liposomes in the pores of the beads. From the standpoints of the immobilized contents of protein and phospholipids and of the reactivity of γ-glutamyl transpeptidase, a dialysis buffer of Tris-HCl (pH 7.5), a phospholipid concentration of 45 mg/ml in the enzyme-phospholipid-cholate dispersion, and the use of Sepharose CL-6B as the support gel were found to be most appropriate for the immobilization of γ-glutamyl transpeptidase, γ-Glutamyl transpeptidase was activated and stabilized by reconstitution in liposomes. In operation with a packed bed reactor, liposome-bound γ-glutamyl transpeptidase immobilized in Sepharose CL-6B exhibited relatively stable and constant activity for 12 h. In addition, it was found that enzyme substrates were able to pass through the pores of the gel beads to interact with the enzyme present on the outer surface of the liposome membrane in the gel beads. These results thus indicated that a novel support made up of liposomes and Sepharose CL-6B would permit efficient immobilization of lipid-requiring and/or membrane enzymes.     

Localization of gamma-glutamyl transpeptidase in the retinal pigment epithelium and visual receptor cells.

The activities of γ-glutamyl transpeptidase and other enzymes of the γ-glutamyl cycle, a series of reactions that catalyzes the synthesis and utilization of glutathione, were studied in the rabbit retina. Histochemical studies demonstrated that γ-glutamyl transpeptidase is localized in the visual receptor cells and the retinal pigment epithelium. Rat and mouse retinas revealed similar localizations of transpeptidase. These findings are in accord with the view that γ-glutamyl transpeptidase is involved in the transport of amino acids between the retinal pigment epithelium and the avascular visual receptor cells.     

PARTIAL PURIFICATION AND KINETICS OF γ-GLUTAMYL TRANSPEPTIDASE FROM BOVINE CHOROID PLEXUS

Abstract— γ-Glutamyl transpeptidase from bovine choroid plexus has been shown to be a membrane-bound enzyme. Partial purification of the enzyme has been accomplished using detergent extraction and ammonium sulfate fractionation. Important determinants of enzymatic activity with acceptor substrates included chain length, stereoisomerism, and amino acid composition of the acceptors. L-Methionine was the best amino acid substrate and its corresponding peptides L-methionylmethionine and L-methionyl-L-serine were also good γ-glutamyl acceptors. L-Alanine and glycine were poor acceptor substrates; whereas, some peptides containing these amino acids were excellent substrates. Glycylglycine was significantly more effective as a γ-glutamyl acceptor than glycine, triglycine, or tetraglycine. L-Alanylglycine was a superior acceptor to glycine, L-alanine, or L-alanylglycylglycine, while the D-isomer of alanylglycine was only minimally effective as an acceptor substrate. In general glycyl peptides were the best acceptor substrates examined. Our findings that γ-glutamyl transpeptidase could catalyze the transfer of γ-glutamyl groups to glycylglycyl-L-alanine and L-alanylglycylglycine are of special interest, since few examples of tripeptide acceptors for the enzyme have been found. It is suggested that γ-glutamyl transpeptidase might play a role in the inactivation and/or transport of biologically active peptides.     

Riboflavin Homeostasis in the Central Nervous System

Abstract: The mechanisms by which riboflavin, which is not synthesized in mammals, enters and leaves brain, CSF, and choroid plexus were investigated by injecting [¹⁴C]riboflavin intravenously or intraventricularly. Tracer amounts of [¹⁴C]riboflavin with or without FMN were infused intravenously at a constant rate into normal, starved, or probenecid-pretreated rabbits. At 3 h, [¹⁴C]riboflavin readily entered choroid plexus and brain, and, to a much lesser extent, CSF. Over 85% of the [¹⁴C]riboflavin in brain and choroid plexus was present as [¹⁴C]FMN and [¹⁴C]FAD. The addition of 0.2 mmol/kg FMN to the infusate markedly depressed the relative entry of [¹⁴C]riboflavin into brain, choroid plexus, and, less so, CSF, whereas starvation increased the relative entry of [¹⁴C]riboflavin into brain and choroid plexus. After intraventricular injection (2 h), most of the [¹⁴C]riboflavin was extremely rapidly cleared from CSF into blood. Some of the [¹⁴C]riboflavin entered brain, where over 85% of the ¹⁴C was present as [¹⁴C]FMN plus [¹⁴C]FAD. The addition of 1.2³μmol FAD (which was rapidly hydrolyzed to riboflavin) to the injectate decreased the clearance of [¹⁴C]riboflavin from CSF and the phosphorylation of [¹⁴C]riboflavin in brain. Probenecid in the injectate also decreased the clearance of [¹⁴C]riboflavin from CSF. These results show that the control of entry and exit of riboflavin is the mechanism, at least in part, by which total riboflavin levels in brain cells and CSF are regulated. Penetration of riboflavin through the blood-brain barrier, saturable efflux of riboflavin from CSF, and saturable entry of riboflavin into brain cells are three distinct parts of the homeostatic system for total riboflavin in the central nervous system.