Determination of lipid-bound sulfate by ion chromatography and its application to quantification of sulfolipids from kidneys of various mammalian species

A variety of procedures have been developed for determining the sulfate ester content of various biomolecules. Ion chromatography (IC), that is, quantitation of ionic substances by ion conductimetry after separation by anion-exchange chromatography, has been increasingly utilized for the determination of inorganic sulfate in clinical and environmental samples. We adopted suppressed-mode IC to the determination of lipid- or glycolipid-bound sulfate released by acid hydrolysis and found that it has the advantage of increased precision for wide concentration ranges (30 pmol to approximately micromol) and lack of interference from other lipids. To minimize deterioration of the separation column, the lipophilic constituents in the acid hydrolysate were removed by a two-phase partition system of chloroform-methanol-water. The inorganic sulfate was quantitatively extracted into the aqueous phase by replacing water with an alkaline buffer. By this method, the concentration of sulfolipids was determined in the kidney of mammals with various body mass. Sulfolipids were more concentrated in the kidney of smaller animals, which have higher maximum urine concentrating activity per gram of body mass, supporting the hypothesis of the function of sulfolipids as an ion barrier on the luminal surface of renal tubules.     

Acetate, lactate, propionate, and isobutyrate as electron donors for iron and sulfate reduction in Arctic marine sediments, Svalbard

The contribution of volatile fatty acids (VFA) as e(-)-donors for anaerobic terminal oxidation of organic carbon through iron and sulfate reduction was studied in Arctic fjord sediment. Dissolved inorganic carbon, Fe(2+), VFA concentrations, and sulfate reduction were monitored in slurries from the oxidized (0-2 cm) and the reduced (5-9 cm) zone. In the 0-2 cm layer, 2/3 of the mineralization could be attributed to sulfate reduction and 1/3 to iron reduction. In the 5-9 cm layer, sulfate reduction was the sole mineralization process. Acetate and lactate turnover rates were measured by radiotracer. Inhibition of sulfate reduction with selenate resulted in the accumulation of acetate, propionate, and isobutyrate. The acetate turnover rates determined by radiotracer and accumulation after inhibition were similar. VFA turnover accounted for 21% and 52% of the mineralization through sulfate reduction in the 0-2 and 5-9 cm layer, respectively. Acetate and lactate turnover in the inhibited 0-2 cm slurry was attributed to iron reduction and accounted for 10% and 2% of the iron reduction. Therefore, 88% and 79% of the iron and sulfate reduction in the 0-2 cm layer, respectively, must be fueled by alternative e(-)-donors. The accumulation of VFA in the selenate-inhibited 0-2 cm slurry did not enhance iron reduction, indicating that iron reducers were not limited by VFA availability.     

Turnover of heparan sulfate proteoglycans from substratum adhesion sites of murine fibroblasts

Substratum adhesion sites from murine Balb/c SVT2 fibroblasts are enriched in heparan sulfate proteoglycans which have been implicated in mediating adhesion of these cells to a fibronectin-adsorbed tissue culture substratum. Most of the heparan sulfate isolated from newly formed adhesion sites is found covalently attached to protein as proteoglycan while a significant portion of heparan sulfate from older sites has been identified as a single-chain species. This observation suggests that there may be catabolism of the heparan sulfate proteoglycan during the "maturation" of these adhesion sites at the cell's undersurface. Zwittergent 3-12 selectively extracts the single-chain class of heparan sulfate from either newly formed or "mature" adhesion sites while leaving the proteoglycan firmly bound in these sites. In an effort to further characterize the metabolism of these proteoglycans, substratum adhesion sites were isolated at various times after the cells had been pulse-radiolabeled using radioactive sulfate and subsequently chased. Greater than 80% of the sulfate-radiolabeled material is lost from the substratum-attached material within 24-48 h. Characterization of both the Zwittergent-soluble and -resistant heparan sulfate indicated that there was an initial accumulation followed by a rapid loss of a portion of the radiolabeled heparan sulfate as the single-chain Zwittergent-soluble class. However, most of the heparan sulfate proteoglycan was lost from the adhesion sites following approximately a 4-h time lag during the chase period without going through a smaller molecular weight intermediate. The turnover properties of the heparan sulfate proteoglycan in the EGTA-detachable cells were different from those in the substratum-attached fraction of the cell. The significance of these two different mechanisms of turnover of heparan sulfate proteoglycan in adhesion sites is discussed in relation to the role of this proteoglycan in mediating adhesion processes.     

Incorporation of 35S-sulfate and 3H-glucosamine into heparan and chondroitin sulfates during the cell cycle of B16-F10 cells

Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of 35S-sulfate and 3H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M.     

Human placental chorionic renin: production, purification and characterization

Native human renin, produced from the culture of human chorionic trophoblasts, has been purified to homogeneity on a milligram scale using a five-step purification scheme. The chorion cells secrete 50-200 milliGoldblatt Units of trypsin-activatable prorenin per ml into the medium. The pro-enzyme is partially purified by ammonium sulfate fractionation and chromatographies on QAE-Sephadex and cibracon blue-agarose. Following conversion of prorenin to the active enzyme by porcine trypsin, the renin is purified to homogeneity by affinity chromatography and gel filtration. Chorionic prorenin has a molecular weight of 43,000; the active enzyme 40,000. Both proteins exist as a single polypeptide chain as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions. The average specific activity of six different preparations was found to be 1072 Goldblatt Units/mg. The amino acid composition and N-terminal sequence of the active enzyme has been determined and is identical to the human kidney enzyme. Microheterogeneity of chorionic renin was demonstrated by isoelectrofocusing analysis. The physical characterization of chorionic renin is compared with that reported for the human kidney enzyme.     

Regulation of mitochondrial protein turnover by thyroid hormone(s)

1. The effect of thyroidectomy on turnover rates of liver, kidney and brain mitochondrial proteins was examined. 2. In the euthyroid state, liver and kidney mitochondria show a synchronous turnover with all protein components showing more or less identical half-lives compared with the whole mitochondria. The brain mitochondrial proteins show asynchronous turnover, the soluble proteins having shorter half-lives. 3. Mitochondrial DNA (m-DNA) of liver and kidney has half-lives comparable with that of whole mitochondria from these tissues. 4. Thyroidectomy results in increased half-lives of liver and kidney mitochondria, with no apparent change in the half-life of brain mitochondria. 5. A detailed investigation of the turnover rates of several protein components revealed a significant decrease in the turnover rates of mitochondrial insoluble proteins from the three tissues under study. 6. The turnover rates of m-DNA of liver and kidney show a parallel decrease. 7. Thus it is apparent that thyroid hormone(s) may have a regulatory role in maintaining the synchrony of turnover of liver and kidney mitochondria in the euthyroid state. Turnover of brain mitochondria may perhaps be regulated by some other factor(s) in addition to thyroid hormone(s). 8. It seems likely that during mitochondrial turnover m-DNA and insoluble proteins may constitute a major unit. 9. The mitochondrial protein contents of the three tissues are not affected by thyroidectomy. 10. No correlation was seen between the turnover rate of mitochondria and cathepsin activity in any of the tissues under study in normal or thyroidectomized animals. 11. On the other hand, mitochondrial proteinase activity shows good correlation with the turnover rates of mitochondria in normal animals, and a parallel decrease in activity comparable with the decreased rates of turnover is observed after thyroidectomy. 12. It is concluded that mitochondrial proteinase activity may play a significant role in their protein turnover.     

Characterizing the non-reducing end structure of heparan sulfate

The reducing end of heparan sulfate has been known for a long time, but information on the non-reducing end has been lacking. Recent studies indicate that the non-reducing end of heparan sulfate might be the place where fibroblast growth factor signaling complex forms. The non-reducing end also changes with heparanase digestion and, thus, might serve as a marker for tumor pathology. Using high performance liquid chromatography-coupled mass spectrometry, we have identified and characterized the non-reducing end of bovine kidney heparan sulfate. We find that the non-reducing end region is highly sulfated and starts with a glucuronic acid (GlcA) residue. The likely sequence of the non-reducing end hexasaccharides is GlcA-GlcNS6S-UA+/-2S-GlcNS+/-6S-Ido2S-GlcNS+/-6S (where GlcNS is N-sulfate-D-glucosamine, S is sulfate, UA is uronic acid, and Ido is iduronic acid). Our data suggests that the non-reducing end of bovine kidney heparan sulfate is not trimmed by heparanase and is capable of supporting fibroblast growth factor signaling complex formation.     

Protein localization of SLC26A2 (DTDST) in rat kidney

The SLC26 family represents a group of integral membrane anion transport proteins. Mutations in one member of this protein family, SLC26A2 (DTDST or diastrophic dysplasia sulfate transporter), result in various chondrodysplasias due to undersulfation of proteoglycans in chondrocytes, a major site of DTDST protein expression. DTDST mRNA has been detected in the kidney, but protein expression has not been characterized. Our objective for this study was to determine the protein localization of this sulfate transporter in the kidney. We used immunofluorescence (IMF) techniques with an anti-DTDST monoclonal antibody to examine kidneys harvested from adult rats. Double labeling was performed with antibodies directed against megalin, which is found in the microvillus membrane and coated pits of the proximal tubule. IMF analysis indicated that DTDST protein expression was limited to the microvillus membrane of proximal tubule cells in the renal cortex but absent in glomeruli and other nephron segments. DTDST was also detected in isolated rat kidney proximal tubule microvillus membranes by Western blot analysis, confirming the immunofluorescent localization of the DTDST transporter to this nephron segment. The functional role of the DTDST protein in the kidney is unknown, but it may play a role in proximal tubule sulfate transport.     

The turnover number for band 3-mediated sulfate transport in phosphatidylcholine bilayers

The anion transport system of the human erythrocyte membrane was reconstituted in unilamellar phosphatidylcholine vesicles, and a vesicle subpopulation of a narrow size distribution was isolated from the sample by gel filtration. In this subpopulation, the turnover number of the transport protein (the band 3 protein) for sulfate transport was determined. It was found that, in the reconstituted system, the protein transports sulfate 5-10 times faster than in the human erythrocyte membrane.     

The purification and characterization of human kidney L-arginine:glycine amidinotransferase

Human kidney L-arginine:glycine amidinotransferase (transamidinase) has been purified to a homogeneous state as defined by native and sodium dodecyl sulfate gel electrophoresis and by ultracentrifugation (sedimentation equilibrium) experiments. The four steps in the isolation procedure were chromatography with DEAE-cellulose, gel filtration with Sephadex G-150, chromatography with phenyl Sepharose, and high-pressure liquid chromatography with hydroxylapatite. The final product represented a 90-fold purification of the enzyme. Human kidney transamidinase is a dimer with a molecular mass of 89,000 Da and subunit masses of 44,000 Da. The Km for arginine and glycine were both 2.5 mM and the Vmax was 0.5 mumol ornithine/min/mg protein. The ultraviolet absorption spectrum, specific activity, and isoelectric points were determined for human kidney transamidinase. Multiple forms of the enzyme were obtained by isoelectric focusing. Human kidney transamidinase cross-reacted with polyclonal antibodies raised to rat kidney transamidinase. All of the properties of human kidney transamidinase that we have examined were similar to those of rat kidney transamidinase. A close evolutionary relationship between the rat and human kidney transamidinase is suggested.     

In vivo biosynthesis and turnover of 35S-labeled glomerular basement membrane

Renal glomerular basement membrane was labeled in vivo by the injection of tracer amounts of radioactive sulfate into normal adult rats. The biosynthesis and turnover of [³⁵S]glycosaminoglycans in purified basement membrane was determined from the specific activity of ³⁵S in pronase digests of basement membranes isolated 1–7 days after injection. Peak radioactive labeling occurred 24 h after injection following which the specific activity of basement membrane sulfate, expressed as cpm/μg uronic acid, progressively declined over the ensuing period of study. The biologic half-life of radioactive sulfate in basement membrane was estimated at about 7 days, which is within the range previously reported for [³⁵S]glycosaminoglycans in whole renal cortex. The findings indicate that ³⁵S-labeled components of glomerular basement membrane have a relatively rapid turnover.     

Urinary excretion of glycosaminoglycans and albumin in experimental diabetes mellitus

Diabetes mellitus was induced in one group of rats by a single injection of streptozotocin. The glycemia, the body weight, and the blood systolic pressure were measured every week, and the 24 h urine volume and urinary excretions of creatinine, albumin and glycosaminoglycans were measured every 2 weeks. At the end of the experiment (12 weeks) the weight and the glycosaminoglycan composition of the kidneys were determined. All the diabetic animals were hyperglycemic, hypertense, and did not gain weight during all the experimental period. Albuminuria appeared from the second week on. Rat urine was shown to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate, and the glycosaminoglycan excretion decreased in all diabetic animals. The onset of the change in glyco-samino-glycan excretion rate was a very early event, appearing in the second week after diabetes induction. The main glycosaminoglycan found in normal rat kidney was heparan sulfate and, in contrast to the urine, the total kidney glycosaminoglycans increased in diabetic kidney, due to chondroitin sulfate and dermatan sulfate accumulation. The heparan sulfate concentration (per tissue dry weight) did not change. Our results suggest that quantification of urinary glycosaminoglycans may be a useful tool for the early diagnosis of diabetic nephropathy.     

Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits.

L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.     

Comparison of Acetate Turnover in Methanogenic and Sulfate-Reducing Sediments by Radiolabeling and Stable Isotope Labeling and by Use of Specific Inhibitors: Evidence for Isotopic Exchange

Acetate turnover in the methanogenic freshwater anoxic sediments of Lake Vechten, The Netherlands, and in anoxic sediments from the Tamar Estuary, United Kingdom, and the Grosser Jasmunder Bodden, Germany, the latter two dominated by sulfate reduction, was determined. Stable isotopes and radioisotopes, inhibitors (chloroform and fluoroacetate), and methane flux were used to provide independent estimates of acetate turnover. Pore water acetate pool sizes were determined by gas chromatography with a flame ionization detector, and stable isotope-labeled acetate was determined by gas chromatography-mass spectrometry. The appearance of acetates with a different isotope labeling pattern from that initially added demonstrated that isotopic exchange occurred during methanogenic acetate metabolism. The predominant exchange processes were (i) D-H exchange in the methyl group and (ii) (sup13)C-(sup12)C exchange at the carboxyl carbon. These exchanges are most probably caused by the activity of the enzyme complex carbon monoxide dehydrogenase and subsequent methyl group dehydrogenation by tetrahydromethanopterine or a related enzyme. The methyl carbon was not subject to exchange during transformation to methane, and hence acetate with the methyl carbon labeled will provide the most reliable estimate of acetate turnover to methane. Acetate turnover rate estimates with these labels were consistent with independent estimates of acetate turnover (acetate accumulation after inhibition and methane flux). Turnover rates from either radioisotope- or stable isotope-labeled methyl carbon isotopes are, however, dependent on accurate determination of the acetate pool size. The additions of large amounts of stable isotope-labeled acetate elevate the acetate pool size, stimulating acetate consumption and causing deviation from steady-state kinetics. This can, however, be overcome by the application of a non-steady-state model. Isotopic exchange in sediments dominated by sulfate reduction was minimal.     

Hormonal regulation of sodium/sulfate co-transport in renal epithelial cells

Serum sulfate concentrations are elevated in infants, young children, and pregnant women due, at least in part, to increased renal sulfate reabsorption. Little is known about the effects of hormones, particularly those involved in growth, development, and pregnancy, on renal sulfate reabsorption. The objective of this investigation was to examine the effects of growth hormone (GH), insulin-like growth factor 1 (IGF-1), progesterone (PG), and 17beta-estradiol (EST) on renal sodium/sulfate co-transport. 35S-sulfate uptake was determined in Madin-Darby canine kidney (MDCK)/NaSi-1 cells (MDCK cells that have been stably transfected with rat sodium/sulfate co-transporter (NaSi-1) cDNA) and in opossum kidney (OK) cells. NaSi-1 mRNA was determined by RT-PCR and protein levels by ELISA. GH (0.1 nM) significantly increased the sodium/sulfate co-transport in MDCK/NaSi-1 cells up to 35%. IGF-1 induced a concentration-related stimulation of the sodium/sulfate co-transport with a maximal response observed at 1000 nM (59% increase). Sodium-dependent sulfate uptake was significantly increased when cells were preincubated with 10 nM PG, 10 nM EST, or 10 nM PG/10 nM EST up to 41%, 46%, or 39%, respectively. OK cells exhibited endogenous sodium-dependent sulfate transport; significantly increased sodium/sulfate co-transport was also observed in OK cells that were preincubated with GH, IGF-1, and PG/EST, although not with EST alone. The NaSi-1 mRNA and NaSi-1 protein levels were significantly increased in MDCK/NaSi-1 cells treated with 0.1 nM GH, 100 nM IGF-1, 10 nM PG, and/or 10 nM EST compared with control. These results suggest that the increased renal sulfate reabsorption that occurs in neonates, young and pregnant humans, and animals could be mediated by the increased steady-state levels of NaSi-1 mRNA produced by the higher plasma concentrations of GH, IGF-1, or PG/EST.     

A high-performance liquid chromatography assay for measuring integrated biphenyl metabolism by intact cells: its use with rat liver and human liver and kidney

A rapid, sensitive high-performance liquid chromatography assay with fluorescence detection for measuring biphenyl metabolism by intact cells has been developed. The assay does not require organic solvent extraction or enzymatic digestion for the measurement of hydroxybiphenyl conjugates. The lower limit of detectability for 4-hydroxybiphenyl is 5 pmol injected. Rat hepatocytes incubated with biphenyl form predominantly 4-hydroxybiphenyl sulfate with lesser amounts of 4-hydroxybiphenyl glucuronide and free hydroxybiphenyls, and small amounts of 3-hydroxybiphenyl sulfate and 3-hydroxybiphenyl glucuronide. Slices of fresh human liver incubated with biphenyl form predominantly 4-hydroxybiphenyl glucuronide with some free hydroxybiphenyl and small amounts of 4-hydroxybiphenyl sulfate. 4-Hydroxybiphenyl glucuronide formation by human liver shows a lag time that is not abolished by preincubating the liver without substrate. Human kidney slices incubated with biphenyl form 4-hydroxybiphenyl glucuronide and 4-hydroxybiphenyl sulfate at rates less than one-tenth those seen with human liver. Human kidney slices do not form detectable free hydroxybiphenyl. There is wide intersubject variability in the rates of hydroxybiphenyl metabolite formation by human liver and kidney.     

The physicochemical properties of the cytoplasmic androgen receptor in the kidneys of normal, carrier female (tfm/+) and androgen-insensitive (tfm/y) mice.

The physicochemical properties of the androgen-receptor complex in mouse kidney were determined. The sedimentation coefficient, 7.9S and Stokes radius of 82A, are compatible with an asymmetric protein [frictional ratio f/fo 1.98; axial ratio, assuming a prolate ellipsoid, 20] having a molecular weight of 270,000 daltons. The kidney receptor is a relatively acidic protein of esoelectric point (pI) 4.8 and readily precipitable with protamine sulfate or ammonium sulfate. Studies with protein-specific reagents suggest that both cysteine and tryptophan residues may be necessary for maintaining the functional configuration associated with androgen binding. The kidney receptor can promote the association of testosterone with purified DNA. These properties of the androgen receptor in mouse kidney are remarkably similar to those of male accessory sexual tissue. The receptor detected in carrier female mice (tfm/+ heterozygous for the gene for testicular feminization (tfm), has the same physical properties as that of normal mice. However, due to a decrease in receptor concentration, binding activity is only 69% that of normal. In cytosol from androgen-insensitive mice (tfm/+), a specific androgen receptor cannot be demonstrated by 5 different techniques.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.     

Effect of pregnancy, postnatal growth, and gender on renal sulfate transport.

Serum sulfate concentrations are increased in infants, young children, and pregnant women, compared with adult values. The objective of this investigation was to examine the influences of age, gender, and pregnancy on renal sulfate transport using guinea pigs as an animal model. Membrane vesicles were isolated from the kidney cortex of male animals at four different ages, from male and female adult animals, and from pregnant and nonpregnant female animals. There were no significant differences in marker enzymes for the brush-border membrane (BBM) or basolateral membrane (BLM) among all groups examined. Uptake was determined by a rapid filtration method and membrane fluidity by measuring the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. The Vmax values for Na+ /sulfate co-transport in BBM were significantly increased with decreasing age, whereas the Km for this process was unchanged. The Vmax and Km for Na + /sulfate co-transport in BBM of pregnant animals were significantly higher than the values in the nonpregnant group. Bicarbonate-driven anion exchange of sulfate in BLM was not different among the different age groups. The Vmax for the bicarbonate/sulfate exchange process in BLM was not different between pregnant and nonpregnant groups; however, the Km for this process in BLM of pregnant animals was significantly greater than the value in nonpregnant animals. There were no gender-related differences in sulfate transport in BBM or BLM isolated from adult male and female animals. Renal BBM fluidity was increased with decreasing age and in pregnant animals, suggesting that altered membrane fluidity may represent one possible mechanism to explain the increased sodium/sulfate uptake in young and pregnant animals. The higher Vmax for Na+/sulfate co-transport in young and pregnant animals suggests that there is an increased density of co-transporter protein or an increase in the rate of movement of the carrier protein (i.e., turnover) once loaded with sodium and sulfate. This increased conservation of inorganic sulfate in young and pregnant guinea pigs may be related to the increased demand for sulfated substrates, such as sulfated glycosaminoglycans, during growth and development.     

Phenylalanine hydroxylation in isolated rat kidney tubules

1. Phenylalanine hydroxylation has been demonstrated to occur in isolated rat kidney tubules under physiological conditions. 2. The hydroxylation flux response is hyperbolic with apparent Km and Vmax values of ca 85 microM phenylalanine and 49 nmol tyrosine formed/mg dry wt per hr respectively. 3. Hydroxylation in kidney tubules is substantially less sensitive to effectors of cyclic AMP turnover and Ca2+ mobilization than phenylalanine hydroxylation in isolated liver cells.