Renal glomerular collagen synthesis in streptozotocin diabetes Reversal of increased basement membrane synthesis with insulin therapy

The effect of diabetes and insulin on basement membrane synthesis in vitro be renal glomeruli obtained from normal and diabetic rats was determined. Four groups of experimental animals were used: age-matched controls; streptozotocin-diabetic; and streptozotocin-diabetic treated with insulin for half or all of the duration of diabetes. Isolated glomeruli were incubated with [¹⁴C]-lysine and the radioactive lysine and hydroxylysine in glomerular proteins were measured. [¹⁴C]Lysine incorporation and hydroxy[¹⁴H]lysine synthesis were elevated in diabetic glomeruli. Progressive diminution in ¹⁴C-labelled protein and hydroxy[¹⁴C]lysine formation was observed in incubations containing glomeruli from insulin-treated diabetic rats, with greater reversal toward normal following longer periods of exogenous insulin administration. Basement membrane synthesis, determined by the appearance of labelled hydroxylysine in membranes obtained from sonicated glomeruli, was increased in diabetic preparations. Reversal of these changes toward normal values was observed in glomeruli from rats treated with insulin immediately following induction of diabetes. The results indicate that basement membrane synthesis is increased in renal glomeruli from streptozotocin-diabetic rats, and that this process is restored toward normal with continuous insulin therapy.     

Metabolism of [14C] arachidonic acid in the isolated rabbit spleen

Infusion of [¹⁴C] arachidonic acid (AA) into the isolated, Tyrode perfused rabbit spleen resulted in the release of a substance into the venous effluent with the musculotropic activity and chromatographic properties of prostaglandin (PG)E₂. Smaller amounts of radioactive materials with the chromatographic properties of PGF_2α, 6-keto-PGF_1α, and PGD₂ were also released. The radiolabeled material released in largest amounts from the spleen was identified as PGE₂ on the basis of: 1) Co-chromatography with PGE₂ in three solvent systems, 2) Conversion of the radioactive material and of authentic [³H] PGE₂ to similar products by treatment with sodium borohydride and with potassium hydroxide, and 3) Stability of the musculotropic activity in Tyrode solution at 37°C. Release of the major and minor radioactive products was inhibited by pretreatment of the spleen with either indomethacin or 5,8,11,14-eicosatetraynoic acid.     

Metabolism of [C] arachidonic acid in the isolated rabbit spleen

Infusion of [¹⁴C] arachidonic acid (AA) into the isolated, Tyrode perfused rabbit spleen resulted in the release of a substance into the venous effluent with the musculotropic activity and chromatographic properties of prostaglandin (PG)E₂. Smaller amounts of radioactive materials with the chromatographic properties of PGF_2α, 6-keto-PGF_1α, and PGD₂ were also released. The radiolabeled material released in largest amounts from the spleen was identified as PGE₂ on the basis of: 1) Co-chromatography with PGE₂ in three solvent systems, 2) Conversion of the radioactive material and of authentic [³H] PGE₂ to similar products by treatment with sodium borohydride and with potassium hydroxide, and 3) Stability of the musculotropic activity in Tyrode solution at 37°C. Release of the major and minor radioactive products was inhibited by pretreatment of the spleen with either indomethacin or 5,8,11,14-eicosatetraynoic acid.     

Metabolic Fate of Sarcosyl-14C-glycine in Normal Young Rats

The metabolic fate of the non-physiological synthetic dipeptide, sarcosyl-[2-¹⁴C]glycine, was investigated in normal young rats in vivo and in vitro compared to that of seryl-[2-¹⁴C]glycine as a typical example of common physiological dipeptides. The radioactive dipeptides were synthesized from sarcosine or L-serine and [2-¹⁴C]glycine in our laboratory. When radioactive sarcosylglycine was given intraperitoneally, about 30% of the dose was excreted in the urine, and more than 90% of the urinary radioactivity was present in the sarcosylglycine fraction. The recovery of radioactivity in the the expired carbon dioxide and body protein was 8 and 50% of the dose, respectively, during a 24-h period. When the labeled serylglycine was given, the recovery of radioactivity in the urine was 8% of the dose, and 15% in the expired carbon dioxide. In slices of the kidney, liver and small intestine from normal rats, serylglycine was rapidly and almost completely hydrolyzed, and a large amount of free glycine was released. However, sarcosylglycine was hardly hydrolyzed to the corresponding amino acids in the liver and small intestine, and only slightly in the kidney. These results suggest that a considerable amount of sarcosylglycine given intraperitoneally was rapidly excreted into the urine of normal young rats, reflecting less sensitivity to hydrolysis by tissue peptidase(s) when compared to serylglycine.     

Regulation of pathways of glucose metabolism in kidney. The effect of experimental diabetes on the activity of the pentose phosphate pathway and the glucuronate-xylulose pathway.

Measurements have been made of the activities of enzymes of the pentose phosphate pathway, the glucuronate-xylulose pathway, hexokinase and phosphofructokinase in kidney of diabetic and normal rats. The activities of these enzymes keep pace with kidney growth, remaining constant per gram tissue but showing a marked increase on the basis of total activity per 100 g body wt. The formation of ¹⁴CO₂ from [1-¹⁴C]glucose and [6-¹⁴C]glucose by kidney slices from diabetic rats was decreased to approximately half the control value; evidence was obtained for an equivalent dilution of the glucose 6-phosphate pool. Correction of the ¹⁴CO₂ yields for the change in specific activity of glucose 6-phosphate yielded values consistent with the enzyme profile. Calculations from specific yields of ¹⁴CO₂ provided evidence for an increased flux of glucose via the pentose phosphate pathway in the kidney in diabetes. The results are discussed in relation to kidney hypertrophy in diabetes and the requirement for ribose 5-phosphate and NADPH for biosynthetic reactions and in relation to the thickening of the basement membrane in diabetes. These results are in accord with the concept of glucose overutilization by non-insulin-requiring tissues.     

Characterization of [14C]apiogalacturonans synthesized in a cell-free system from Lemna minor.

Radioactive polysaccharide was synthesized when uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (containing some uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate)) was incubated with a particulate enzyme preparation from Lemna minor. Characterization experiments established that the product: (i) was insoluble in methanol and water, (ii) contained d-[U-¹⁴C]apiose (75%) and d-[U-¹⁴C]xylose (25%), and (iii) was soluble in 1% ammonium oxalate. The material solubilized by ammonium oxalate (solubilized product): (i) was separated into five fractions by column chromatography with diethylaminoethyl-Sephadex (DEAE-Sephadex), (ii) contained [U-¹⁴C]apiobiose side chains that were removed by hydrolysis at pH 4, and (iii) was degraded by fungal pectinase. Both d-[U-¹⁴C]apiose residues of the [U-¹⁴C]apiobiose side chains were synthesized in vivo since radioactivity was distributed equally between the two residues. The presence of uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) during synthesis of radioactive polysaccharide resulted in: (i) an increase in the incorporation of radioactive d-[U-¹⁴C]apiose into solubilized product, (ii) an increase in the ratio of d-[U-¹⁴C]apiose to d-[U-¹⁴C]xylose present in solubilized product, (iii) an increase in the amount of [U-¹⁴C]apiobiose plus d-[U-¹⁴C]apiose released from the solubilized product by hydrolysis at pH 4, and (iv) a tighter binding of the solubilized product to DEAE-Sephadex. These results show that apiogalacturonans similar to or the same as those synthesized by the intact plant were synthesized in the particulate enzyme preparation isolated from L. minor. [¹⁴C]Apiogalacturonans completely free of d-[U-^l4C]xylose were not isolated. The [¹⁴C]apiogalacturonan with the least d-[U-¹⁴C]xylose still had 4.8% of its radioactivity present in d-[U-¹⁴C]xylose. The possibility remains that d-xylose is a normal constituent of the apiogalacturonans of the cell wall of L. minor.     

Glycogen synthesis in the fungus Neurospora crassa

An enzymic activity, obtained from Neurospora crassa, catalyzing the incorporation of [¹⁴C]glucose from ADP-[¹⁴C]glucose into a glucan of the glycogen type, is described. The properties of the ADPglucose: glycogen glucosyltransferase as compared with those of the already known UDP glucose: glycogen glucosyltransferase were studied. The radioactive products obtained with UDP-[¹⁴C]glucose or ADP-[¹⁴C]glucose released all the radioactivity as maltose after α or β amylase treatment. Glucose 6-phosphate stimulated the synthetase when UDP-[¹⁴C]glucose was the substrate but the stimulation was much greater with ADP-[¹⁴C]glucose as glucosyl donor. Glucose 6-phosphate plus EGTA gave maximal stimulation. The system was completely dependent on the presence of a ‘primer’ of the α 1 → 4 glucan type.     

The influence of insulin on the metabolism of glucosamine in the isolated rat diaphragm muscle

1. The influence of insulin on the metabolism of [1-¹⁴C]glucosamine by diaphragm muscle from normal rats and rats rendered diabetic with streptozotocin has been studied. 2. The glucosamine was converted into glucosamine 1-phosphate, glucosamine 6-phosphate, glycogen, lactate and small amounts of other unidentified intermediates. 3. Insulin increased the incorporation of ¹⁴C into glycogen in both the normal and diabetic muscle, but did not increase the formation of the glucosamine phosphate esters. 4. The ¹⁴C content in the glycogen was present partly as glucose and partly as glucosamine; there was significantly more [¹⁴C]glucose in the glycogen of the diabetic muscle than in that of the normal muscle.     

A radioactive assay for acetylcholinesterase using anion-exchange disk

A radioactive assay for acetylcholinesterase is described. The assay is based on the separation of [¹⁴C]acetate from [¹⁴C]acetylcholine by differential adsorption of the former on DEAE anion-exchange disks. The procedure is simple and sensitive and eliminates the use of ion-exchange resin columns or organic extractions. Moreover, when unpurified enzyme preparations are assayed, linear steady-state kinetics can be observed with this method as contrasted to the nonlinear colorimetric method using acetylthiocholine and dithiobisnitrobenzoate. This method also permits the detection in biological samples of low levels of acetylcholinesterase activity, which is not detectable by the colorimetric method. Using the present radioactive method, cellular levels of acetylcholinesterase have been surveyed in N4TG1 neuroblastoma cells, NG108-15 neuroblastoma x glioma hybrid cells, H9c2 myoblasts, and 3T3-L1 and 3T3-C2 fibroblasts.     

An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis

1. [¹⁴C]Malonyl-CoA was incorporated into isoprenoids by cell-free yeast preparations, by preparations from pigeon and rat liver, and by Hevea brasiliensis latex. 2. In agreement with previous reports the incorporation of acetyl-CoA into isoprenoids was not inhibited by avidin and was not stimulated by HCO₃⁻. In a cell-free yeast preparation addition of HCO₃⁻ stimulated the formation of fatty acids from acetyl-CoA and decreased the incorporation into unsaponifiable lipids. 3. The labelling patterns of β-hydroxy-β-methylglutaryl-CoA formed from [2-¹⁴C]- and [1,3-¹⁴C]-malonyl-CoA in rat and pigeon liver preparations were those that would be expected if malonyl-CoA underwent decarboxylation to acetyl-CoA before incorporation. 4. The labelling pattern of ergosterol formed by cell-free yeast preparations from [2-¹⁴C]malonyl-CoA was also consistent with decarboxylation of malonyl-CoA before incorporation. 5. The incorporation of [2-¹⁴C]malonyl-CoA into mevalonate by rat liver preparations was related to the malonyl-CoA decarboxylase activity present in the preparation.     

Lipid-mediated glycosylation of a white matter membrane glycoprotein with an apparent molecular weight of 24,000.

White matter membrane preparations from pig brain catalyze the transfer of [¹⁴C]mannose from exogenous [¹⁴C]mannosylphosphoryldolichol into an endogenous oligosaccharide lipid. Under the same incubation conditions label is also incorporated into endogenous membrane glycoproteins. The enzymatic labeling of both classes of endogenous acceptors is stimulated by the addition of Ca²⁺. Several enzymatic properties of the mannosyltransferase activity responsible for the transfer of mannose from mannosylphosphoryldolichol into the oligosaccharide lipid intermediate have been examined. The [Man-¹⁴C] oligosaccharide lipid synthesized by this in vitro system has the solubility, hydrolytic and chromatographic characteristics of a pyrophosphate-linked oligosaccharide derivative of dolichol. The free [Man-¹⁴C]oligosaccharide liberated from the carrier lipid by mild acid treatment is estimated to contain 8 glycose units. All of the [¹⁴C]mannosyl units in the [Man-¹⁴C]oligosaccharide derived from exogenous [¹⁴C]mannosylphosphoryldolichol are released as free [¹⁴C]mannose by an α-mannosi-dase. No [¹⁴C]mannose is released during incubation with a β-mannosidase. The presence of an N,N′-diacetylchitobiose unit at the reducing end of the lipid-bound [Man-¹⁴C]oligosaccharide is indicated by its susceptibility to digestion by endo-β-N-acetylglucosaminidase H. Pronase digestion of the enzymatically labeled [Man-¹⁴C]glycoprotein yields a single [Man-¹⁴C]gly-copeptide fraction on Bio-Gel P-6 that appears to be slightly larger than the free [Man-¹⁴C]oligosac-charide released from the carrier lipid by mild acid hydrolysis. The [Man-¹⁴C]glycopeptide is cleaved by endo-β-N-acetylglucosaminidase H, and the neutral [Man-¹⁴C]oligosaccharide product appears to be identical to the product formed when the lipid-bound [Man-¹⁴C]oligosaccharide is degraded by the endoglycosidase. The glycopeptide linkage in the [Man-¹⁴C]glycoprotein is stable to mild alkali treatment. These results are consistent with the dolichol-linked [Man-¹⁴C]oligosaccharide, mannosy-lated via exogenous [¹⁴C]mannosylphosphoryldoiichol, being subsequently transferred en bloc from dolichyl pyrophosphate to asparagine residues in endogenous membrane polypeptide acceptors. SDS-polyacrylamide gel electrophoresis of the [Man-¹⁴C]glycoprotein, labeled when white matter membranes are incubated with [¹⁴C]mannosylphosphoryldolichol. revealed a major labeled polypeptide with an apparent mol wt of 24,000. A minor labeled membrane glycoprotein is also seen, having an apparent mol wt of 105,000.     

Lipolytic cleavage of dolichyl oleate catalyzed by calf brain membranes

Calf brain membranes catalyze the lipolytic cleavage of dolichyl [¹⁴C]oleate added as an aqueous dispersion in Triton X-100. The enzymatic release of [¹⁴C]oleate from the dolichyl ester is not affected by divalent cations or EDTA, but the lipase activity is inhibited by iodoacetamide and pHMB. The amount of [¹⁴C]oleate released is dependent on the time of incubation, the amount of membrane protein added and the concentration of the radiolabeled lipid substrate. Dolichyl ester hydrolase activity exhibits a pH optimum of 7.5, distinguishing this lipase activity from cholesteryl ester hydrolase (5.0–5.5) and triolein hydrolase (5.0) activity associated with the same membrane preparations. The enzymatic hydrolysis of dolichyl [¹⁴C]oleate is also partially inhibited by oleate and free dolichol, possibly by end-product inhibition.     

Xylan synthetase activity in differentiated xylem cells of sycamore trees (Acer pseudoplatanus)

Particulate enzymic preparations obtained from homogenates of differentiated xylem cells isolated from sycamore trees, catalyzed the formation of a radioactive xylan in the presence of UDP-D-[U-¹⁴C]xylose as substrate. The synthesized xylan was not dialyzable through Visking cellophane tubing. Successive extraction with cold water, hot water and 5% NaOH dissolved respectively 15, 5 and 80% of the radioactive polymer. Complete acid hydrolysis of the water-insoluble polysaccharide synthesized from UDP-D-[U-¹⁴C]xylose released all the radioactivity as xylose. -1,4-Xylodextrins, degree of polymerization 2, 3, 4, 5 and 6, were obtained by partial acid hydrolysis (fuming HCl or 0.1 M HCl) of radioactive xylan. The polymer was hydrolysed to xylose, xylobiose and xylotriose by Driselase which contains 1,4- xylanase activities. Methylation and then hydrolysis of the xylan released two methylated sugars which were identified as di-O-methyl[¹⁴C]xylose and tri-O-methyl-[¹⁴C]xylose, suggesting a 14-linked polymer. The linkage was confirmed by periodate oxidation studies. The apparent K_m value of the synthetase for UDP-D-xylose was 0.4 mM. Xylan synthetase activity was not potentiated in the presence of a detergent. The enzymic activity was stimulated by Mg²⁺ and Mn²⁺ ions, although EDTA in the range of concentrations between 0.01 and 1 mM did not affect the reaction rate. It appears that the xylan synthetase system associated with membranes obtained from differentiated xylem cells of sycamore trees may serve for catalyzing the in vivo synthesis of the xylan main chain during the biogenesis of the plant cell wall.     

Catabolism of 2-methyloctanoic acid and 3beta-hydroxycholest-5-en-26-oic acid

1. 2-Methyl[1-¹⁴C]octanoic acid was synthesized from 2-bromo-octane and ¹⁴CO₂. 2. 2-Methyl[1-¹⁴C]octanoic acid was readily oxidized to propionic acid and carbon dioxide by mitochondrial preparations from liver, less readily oxidized by adrenal and kidney (mitochondria), and only poorly oxidized by heart, spleen and brown fat (mitochondria). 3. 3β-Hydroxy[26-¹⁴C]cholest-5-en-26-oic acid was rapidly oxidized by mammalian-liver mitochondria to propionic acid and carbon dioxide. Caiman-liver and toad-liver mitochondria also oxidized this steroid acid. 4. The oxidation of propionic acid, octanoic acid and palmitic acid by mitochondrial preparations from these various tissues was also studied. 5. Added carnitine did not stimulate 2-methyloctanoic acid oxidation and feebly stimulated 3β-hydroxycholest-5-en-26-oic acid oxidation. 6. The significance of these results is discussed in relation to sterol catabolism in mammals and non-mammalian species.     

The removal of cell surface material by enzymes used to dissociate mammary-gland cells

Summary Treatment of mouse mammary epithelial cells (MMEC) with various enzymes used for dispersing and transferring cells results in extensive digestion of materials on the cell surfaces. MMEC biosynthetially labeled with [³H]fucose, [¹⁴C]fucose and [³H]amino acids or with¹²⁵I by the lactoperoxidase method were exposed to either collagenase plus hyaluronidase, followed by pronase, or to trypsin in concentrations and conditions currently used for cell dispersion. Whereas the latter enzyme preparation solubilized 76% of the trichloroacetic acid precipitable radioactive fucose and 96% of the protein-bound¹²⁵I, collagenase plus hyaluronidase treatment released lesser amounts of each label. Subsequent treatment of the cells with pronase removed additional surface-labeled materials, but the total amounts released were still less than when the trypsin preparation alone was employed. Released cell surface materials were analyzed by gel chromatography. Some of the peaks obtained also were examined by polyacrylamide gel electrophoresis. The labeled materials that remained attached to the MMEC after enzymatic treatment were investigated by these two methods as well. We could show that collagenase plus hyaluronidase solubilized three main glycoprotein components from the cell surface. In addition, we could show that the extensive cell surface damage caused by these two enzyme preparations was due to the high proteolytic activity present in these preparations as judged by their ability to hydrolyze rabbit gamma globulin labeled with¹²⁵I. Even though their membranes were extensively damaged by the enzyme treatments, the dispersed cells could be cultured successfully in vitro and could incorporated fucose into their surfaces in a manner similar to that by intact tissue. Through the use of gel-filtration (cochromatography of [¹⁴C]fucose and [³H]fucose cell surface materials), we could demonstrate the identity of cell surface glycoproteins synthesized by cultured cells and by intact tissue. This work was supported by Grant Nos. CA 11736 and CA 19455 from the National Cancer Institute, and Biomedical Research Support Grant No. RR05467 from the National Institutes of Health, DHEW.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

A sensitive fluorimetric procedure for the determination of aliphatic epoxides under physiological conditions

Ribose 1-phosphate has been measured in rat tissues by an enzymatic radioactive assay. The sugar phosphate is converted into [¹⁴C]inosine via the two following combined reactions: ribose 1-phosphate + [¹⁴C]adenine ? [¹⁴C]adenosine + phosphate (adenosine phosphorylase); [¹⁴C]adenosine + H₂O → [¹⁴C]inosine + NH₃ (adenosine deaminase). Tissue extracts are incubated in the presence of excess [¹⁴C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of ribose 1-phosphate present in tissue extracts. Liver was found to contain the highest level of ribose 1-phosphate (ca. 800 nmol/g wet wt).     

Remodelling of triacylglycerols in microsomal preparations from developing castor bean (Ricinus communis L.) endosperm

Microsomal preparations from developing castor bean (Ricinus communis L.) endosperm catalyzed remodelling of in-situ-formed triacylglycerol (TAG) species. Castor bean microsomal membranes synthesized [¹⁴C]TAGs from either glycerol 3-phosphate and [¹⁴C]ricinoleoyl-CoA or [¹⁴C]glycerol 3-phosphate and ricinoleoyl-CoA. Upon repelleting and subsequent incubation of the microsomes a redistribution occurred of both the [¹⁴C]glycerol and [¹⁴C]ricinoleoyl moieties of the in-situ-synthesized [¹⁴C]TAGs. Radioactivity was transferred from TAG species with three (3HO-TAG) or two (2HO-TAG)ricinoleoyl groups into species with two or one (HO-TAG) ricinoleoyl groups. Mass analysis of the lipid and fatty acid movements in the membranes showed that a net synthesis of TAGs with no, one and two ricinoleoyl groups occurred at the expense of 3HO-TAG and polar lipids. Thus, the non-hydroxylated acyl groups from polar lipids were used in the remodelling of TAGs. In-vivo feeding of [¹⁴C]ricinoleic acid to slices of castor bean endosperm demonstrated the presence of two radioactive pools of TAGs one in the oil bodies, which was rich in [¹⁴C]3HO-TAG, and one associated with the microsomal membranes, which was dominated by radioactive 1HO-TAG and 2HO-TAG. The microsomal TAG pool was remodelled in vivo in a similar way as in the in-vitro experiments with microsomal membranes. Received: 8 November 1996 / Accepted: 5 February 1997     

Involvement of dolicholmonophosphate in the formation of specific mannosyl-linkages in yeast glycoproteins

A membrane fraction from Saccharomyces cerevisiae catalyzes the transfer of mannosyl residues from GDP-Man partly via dolicholmonophosphate into a heterogenous glycoprotein fraction. The pattern of radioactive products obtained after mannosylation with GDP-[¹⁴C]Man is similar to that obtained with dolicholmonophosphate-[¹⁴C]mannose. In each case more than 70% of the radioactivity can be released by β-elimination. Evidence is presented, that only the mannosyl residue directly linked to protein is incorporated via dolicholmonophosphate.