The turnover rate of rabbit urinary Tamm–Horsfall glycoprotein

1. The turnover rate of urinary Tamm-Horsfall glycoprotein in rabbits was determined by two different methods. The first involved measurement of the pool size of the glycoprotein in rabbit kidney and the daily urinary excretion rate by a radioimmunoassay from which the turnover rate was calculated. 2. The second method made use of the incorporation in vivo of Na(2) (14)CO(3) and sodium [(14)C]acetate. After a single intramuscular injection of one of these compounds, urine collections were made every 24h and the glycoprotein was isolated and its specific radioactivity was determined. 3. Incorporation of the label into urinary HCO(3) (-), urea and plasma fibrinogen was also examined. The specific radio-activities of the O-acetyl, sialic acid, aspartic acid and glutamic acid residues isolated from the Tamm-Horsfall glycoprotein were compared and their half-lives were compared with that of the intact glycoprotein. The two methods gave results in quite close agreement and indicated a half-life for the glycoprotein of approx. 9h. 4. An attempt was made to localize the glycoprotein within the kidney and within the cell. It is present throughout the kidney, but was not detected in the brush-border fraction isolated from the proximal tubules. From differential cell-centrifugation studies, the glycoprotein seemed to be predominantly present in the soluble fraction (100000g supernatant). This suggests that it is either largely a soluble cytoplasmic component or is very loosely bound to a membrane, being readily released under the gentlest homogenization procedure. 5. The half-life of Tamm-Horsfall glycoprotein in human kidney was found by the radioimmunoassay method to be approx. 16h. The similarity between the composition of Tamm-Horsfall glycoprotein and human erythropoietin is discussed.     

Subunits of Tamm–Horsfall glycoprotein

1. The effect of 6m-guanidine hydrochloride on the urinary glycoprotein described by Tamm & Horsfall (1952) is to produce a homogeneous subunit of molecular weight approx. 100000. 2. Complete reduction of the disulphide bonds of this subunit does not decrease the molecular weight, suggesting that all disulphide bonds are intrachain. 3. Comparison of sedimentation and viscometric behaviour of unreduced and reduced material in 6m-guanidine hydrochloride is consistent with reduction causing an opening-up of intrachain disulphide bonds to give a more asymmetric molecule.     

Tamm–Horsfall urinary glycoprotein. The chemical composition

1. A revised amino acid and carbohydrate composition of human Tamm-Horsfall glycoprotein is presented. 2. No significant differences were obtained in the amino acid composition of Tamm-Horsfall glycoprotein isolated from patients with cystic fibrosis. 3. The glycoprotein was shown to possess a high half-cystine content of 1 per 11-12 amino acid residues, which has been confirmed by performic acid oxidation and S-alkylation with iodoacetate and iodoacetamide. No thiol groups were detected in the glycoprotein. 4. Treatment of the glycoprotein with 0.5m-sodium hydroxide at 4 degrees C for 2 days did not release heterosaccharide material, which suggests that the predominant carbohydrate-protein linkages present are not of the O-glycosidic type. 5. No N-terminal amino acid was detected in the glycoprotein.     

Tamm–Horsfall urinary glycoprotein. The subunit structure

1. Subunit molecular weights of 76000-82000 were obtained for native and alkylated Tamm-Horsfall glycoprotein by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. 2. A further estimate of the subunit molecular weight of 79000+/-4000 was obtained by disc gel electrophoresis in sodium dodecyl sulphate. 3. A minimum value of the chemical molecular weight of 79000+/-6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 4. Similar values were obtained for the subunit molecular weight of Tamm-Horsfall glycoprotein from patients with cystic fibrosis. 5. On ultracentrifugation both in 1.0% sodium dodecyl sulphate and in 70% formic acid, Tamm-Horsfall glycoprotein sedimented as a single component, slightly faster than serum albumin. 6. On reduction of the disulphide bonds the same subunit molecular weight was obtained, which suggested that these bonds are intrachain.     

The development of a radioimmunoassay for Tamm–Horsfall glycoprotein in serum

The Tamm–Horsfall glycoprotein prepared by salt precipitation from urine was found to comprise a heterogeneous collection of aggregates. These could be disaggregated with 8m-urea, following which chromatography on a column of Bio-Gel A.15m yielded a homogeneous glycoprotein of mol.wt. 73000 together with several unidentified impurities. Gel filtration of normal plasma showed the glycoprotein to exist predominantly in a form that is eluted identically with the purified preparation. In one case, material of higher molecular weight was also detected. The purified glycoprotein was used to develop a rapid specific radioimmunoassay for its measurement in human serum or plasma by the use of the Tamm–Horsfall glycoprotein, labelled with ¹²⁵I by the chloramine-t method as the tracer, an antiserum raised in rabbits, and separation of the bound and free fractions by a second antibody covalently linked to magnetizable particles. Parallelism was demonstrated between the standard preparation and samples. Recovery of added standard to serum varied between 99 and 109%. Total assay time was less than 4h with an intra-assay and inter-assay coefficient of variation of less than 10%. There were no significant differences in the ranges covered with regard to either age or sex, and no circadian rhythm was observed in normal subjects. A physiological range of 70–540ng/ml was established based on serum samples from 95 subjects with normal renal function, as defined by their serum creatinine and urea concentrations. No Tamm–Horsfall glycoprotein was detected in the serum of six anephric patients.     

Rabbit Tamm–Horsfall urinary glycoprotein. Chemical composition and subunit structure

1. Tamm-Horsfall glycoprotein from rabbit urine has been isolated and characterized. The homogeneity of the preparation has been established by a variety of procedures including disc gel electrophoresis and ultracentrifugation in aqueous solution, sodium dodecyl sulphate and formic acid. 2. The chemical composition has been determined and a carbohydrate content of approx. 31% was obtained. The relative contents of the amino acids were shown to be very similar to those in human Tamm-Horsfall glycoprotein. A trace of lipid was also detected. 3. Leucine was identified as the only N-terminal amino acid. 4. The subunit structure was investigated in the presence of sodium dodecyl sulphate by gel filtration and disc gel electrophoresis. These studies indicated that the subunit possessed a molecular weight of approx. 84000+/-6000. A similar value was obtained after reduction and S-alkylation of the glycoprotein indicating that the disulphide bonds were all intrachain. 5. A minimum value for the chemical molecular weight of 85000+/-6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 6. The immunological properties of the glycoprotein were studied. Cross reactivity was demonstrated between human Tamm-Horsfall glycoprotein and a guinea-pig anti-rabbit Tamm-Horsfall antiserum.     

Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm–Horsfall glycoprotein in adult hamster kidney

1. Tamm-Horsfall glycoprotein was isolated from hamster urine and antiserum against it was produced in rabbits. Immunoglobulin G was isolated from the antiserum. 2. Indirect methods of immunofluorescence staining were applied to kidney sections previously fixed by both perfusion and immersion methods. Tamm-Horsfall glycoprotein was identified associated with only the cells of the ascending limb of the loop of Henle and the distal convoluted tubule. Maculae densae were free of the glycoprotein. 3. Indirect immunoperoxidase procedures with light microscopy were applied to kidney sections. The results extended those found by immunofluorescence by showing that the glycoprotein is largely associated with the plasma membrane of the cells. Macula densa cells were shown to be free of the glycoprotein, although the luminal surface of the remaining cells in the transverse section of the nephron at that region was shown to contain it. 4. A variety of immuno-electron-microscopic techniques were applied to sections previously fixed in a number of ways. Providing periodate/lysine/paraformaldehyde was used as the fixative, the glycoprotein was often seen to be present not only on the luminal surface of the cells of the thick ascending limb of the loop of Henle and of the distal convoluted tubule, but also on the basal plasma membrane, including the infoldings. 5. It is generally accepted that the hyperosmolarity in the medulla of the kidney results from passage of Cl(-) ions with their accompanying Na(+) ions across the single cell layer of the lumen of the thick ascending limb of the loop of Henle, a region of the nephron with relatively high impermeability to water. We suggest that Tamm-Horsfall glycoprotein operates as a barrier to decrease the passage of water molecules by trapping the latter at the membrane of the cells. Our hypothesis requires the glycoprotein on the basal plasma membrane also.     

Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Partial $\text{in}\text{vitro}$ sialylation of biantennary and triantennary glycopeptides of α₁-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution ¹H-NMR spectroscopic analysis of the isolated products enabled the assignment of the $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→3)}\text{Man}$ branch as the most preferred substrate site for sialic acid attachment. The $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→6)}\text{Man}$ branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal $\text{N}$-acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.     

The substrate specificity of acid α-glucosidase from rabbit muscle

1. Acid alpha-glucosidase was purified 3500-fold from rabbit muscle. 2. The enzyme was activated by cations, the degree of activation varying with the substrate. Enzyme action on glycogen was most strongly activated and activation was apparently of a non-competitive type. With rabbit liver glycogen as substrate, the relative V(max.) increased 15-fold, accompanied by an increase in K(m) from 8.3 to 68.6mm-chain end over the cation range 2-200mm-Na(+) at pH4.5. Action on maltose was only moderately activated (1.3-fold, non-competitively) and action on maltotriose was marginally and competitively inhibited. 3. The pH optimum at 2mm-Na(+) was 4.5 (maltose) and 5.1 (glycogen). Cation activation of enzyme action on glycogen was markedly pH-dependent. At 200mm-Na(+), the pH optimum was 4.8 and activity was maximally stimulated in the range pH4.5-3.3. 4. Glucosidase action on maltosaccharides was associated with pronounced substrate inhibition at concentrations exceeding 5mm. Of the maltosaccharides tested, the enzyme showed a preference for p-nitrophenyl alpha-maltoside (K(m) 1.2mm) and maltotriose (K(m) 1.8mm). The extrapolated K(m) for enzyme action on maltose was 3.7mm. 5. The macromolecular polysaccharide substrate glycogen differed from linear maltosaccharide substrates in the kinetics of its interaction with the enzyme. Activity was markedly dependent on pH, cation concentration and polysaccharide structure. There was no substrate inhibition. 6. The enzyme exhibited constitutive alpha-1,6-glucanohydrolase activity. The K(m) for panose was 20mm. 7. The enzyme catalysed the total conversion of glycogen into glucose. The hydrolysis of alpha-1,6-linkages was apparently rate-limiting during the hydrolysis of glycogen. 8. Enzyme action on glycogen and maltose released the alpha-anomer of d-glucose. 9. The results are discussed in terms of the physiological role of acid alpha-glucosidase in lysosomal glycogen catabolism.     

The mechanism of the periodate–thiobarbituric acid reaction of sialic acids

1. The chromogen formation from N-acetylneuraminic acid in the periodate-thiobarbituric acid reaction was investigated. Measurement of periodate consumption showed an uptake of approx. 3moles/mole of substrate in neutral as well as in strongly acidic solution. Therefore the chromogen beta-formylpyruvic acid is not a direct product of the periodate oxidation; it is presumed to be formed from the true oxidation product, a hexos-5-uluronic acid, by aldol splitting during the reaction in hot acidic solution with thiobarbituric acid. 2. Methyl (methyl beta-l-threo-hexos-4-enepyranosid)uronate, an analogue of the pre-chromogen, has been shown to yield with thiobarbituric acid in acidic solution a pigment exhibiting an identical absorption spectrum and showing the same behaviour on paper chromatography as the pigment obtained from N-acetylneuraminic acid in the periodate-thiobarbituric acid assay. 3. The substitution at C-2 of methoxyneuraminic acid does not inhibit the periodate-thiobarbituric acid reaction. In neutral solution methoxyneuraminic acid is oxidized by periodate to a substance that reacts readily with thiobarbituric acid in acidic solution. When periodate oxidation is attempted in acidic solution, protonation of the amino group protects this group against oxidation, rendering methoxyneuraminic acid negative in the assay systems of Warren (1959a,b) and Aminoff (1959, 1961).     

Immunological detection of nitrosative stress-mediated modified Tamm–Horsfall glycoprotein (THP) in calcium oxalate stone formers

Abstract

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in hyperoxaluric condition has been proved experimentally. This may result in the formation of the cytotoxic metabolite peroxynitrite, which is capable of causing lipid peroxidation and protein modification. The presence of nitrotyrosine in proteins has been associated with several pathological conditions. The present study investigated the presence of nitrotyrosine in the stone formers Tamm–Horsfall glycoprotein (THP). In vitro nitration of control THP was carried out using peroxynitrite. New Zealand white rabbits were immunized with peroxynitrated THP at 15-day intervals. Antisera collected following the third immunization were assayed for antibody titres using solid-phase ELISA. Antibodies were purified by affinity chromatography. The carbonyl content of control, stone formers and nitrated THP were determined. Western blotting was carried with control, stone formers and nitrated THPs. Immunodiffusion studies demonstrated cross-reaction with nitrated bovine serum albumin. Significant amounts (p<0.001) of carbonyl content were present in both stone formers and nitrated THPs. Western blot analysis confirmed the presence of nitrated amino acid 3-nitrotyrosine in stone formers, which could bring about structural and functional modifications of THP in hyperoxaluric patients. A cross-reaction with nitrated bovine serum albumin confirms that the raised antibody has certain paratopes similar to the epitope of nitrated protein molecules. Detection of 3-nitrotyrosine in stone formers THP indicates that it is one of the key factors influencing the conversion of THP to a structurally and immunologically altered form during calcium oxalate stone formation.     

The maltase, glucoamylase and transglucosylase activities of acid α-glucosidase from rabbit muscle

1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.     

The effect of acid mucopolysaccharides and acid mucopolysaccharide–proteins on fibril formation from collagen solutions

1. The effects of acid mucopolysaccharides and acid mucopolysaccharide-proteins on the size and rate of formation of fibril aggregates from collagen solutions in pH7.6 buffers were studied by turbidimetric and light-scattering methods. 2. Serum albumin, orosomucoid, methylated cellulose, chondroitin sulphate A and chondroitin sulphate C of molecular weight less than 20000, and hyaluronate of molecular weight less than 40000 did not influence rates of fibril formation. Chondroitin sulphate A, chondroitin sulphate C and hyaluronate of high molecular weight retarded the rate of fibril formation. This effect of high-molecular-weight chondroitin sulphate C decreased with increasing ionic strength. Heparin, though of low molecular weight (13000), was highly effective, as was also heparitin sulphate. The chondroitin sulphate-proteins of very high molecular weight were highly effective, despite the fact that for some preparations the component chondroitin sulphate chains had molecular weights much less than 20000. 3. Agents that had delayed fibril formation were also effective in producing an increase in degree of aggregation of fibrillar collagen, as indicated by dissymmetry changes observed in light-scattering experiments at low collagen concentrations. Methylated cellulose and heparin at 2.5mug./ml. were unusual in decreasing aggregation, but heparin at 0.25mug./ml. increased aggregation. Electron microscopy of gels showed fibrils and fibril aggregates with ;normal' collagen spacing and dimensions consistent with the light-scattering results. 4. The rates of electrical transport of agents and of solvent (electro-osmosis) through collagen gels indicated a contribution of molecular entanglement that increased with increase in molecular size of the agents. Electrostatic binding of heparin to collagen was noted. Binding to collagen during fibril formation was also found for heparitin sulphate and a chondroitin sulphate with extra sulphate groups. 5. Electrostatic binding of acid mucopolysaccharide-proteins to collagen may be an important factor in the organization and functioning of connective tissues at all stages of growth and development. Excluded-volume (molecular-entanglement) effects may also be important. These factors operate simultaneously and interact mutually so that precise assessment of their relative importance is difficult.     

Partial characterization of the sialic acid-free forms of α1-acid glycoprotein from human plasma

Some of the properties of sialic acid-free α₁-acid glycoprotein prepared by mild acid hydrolysis (pH1·6 at 80° for 1hr.) were compared with those of neuraminidasetreated α₁-acid glycoprotein. Chemically, the former contained less fucose (15%) and amide (2%) residues. Physicochemically, it had undergone certain changes primarily pertaining to the secondary structure, so that the specific optical rotation was more negative than that of the latter. A further expression of this change is probably the difference in the pH range of the resolution into two bands on electrophoresis. The resolution of the glycoprotein prepared by mild acid hydrolysis seems to be extended to more acidic pH values both by starch-gel and free moving-boundary electrophoresis. On ultracentrifugation both preparations appeared homogeneous and sedimented with a rate of 3s. Removal of sialyl residues at different pH values, in the range 1–7, showed that 2moles of sialic acid/mole of protein are very strongly bound. The two variants of α₁-acid glycoprotein were isolated from pooled sialic acid-free α₁-acid glycoprotein by preparative starch-gel electrophoresis and from selected blood of normal adults by fractionation by solubility and chromatography. Ultracentrifugal and starch-gel electrophoretic analyses at pH5, with incubation times of 1 or 24hr., demonstrated that no dissociation–association equilibrium (constant sedimentation coefficient and molecular weight) or isomerization (constant apparent electrophoretic mobilities) exist between the two variants. Therefore these variants are not sub-units of native α₁-acid glycoprotein but represent modifications of naturally occurring proteins. Further, it was shown that the difference in the electrophoretic mobilities between the two variants was not due to any difference in amide content. Immunochemically, the two variants share the same determinants.     

The regulatory role of residues 226–232 in phosphoenolpyruvate carboxylase from maize

The regulatory properties of maize phosphoenolpyruvate carboxylase were significantly altered by site-directed mutagenesis of residues 226 through 232. This conserved sequence element, RTDEIRR, is part of a surface loop at the dimer interface. Mutation of individual residues in this sequence caused various kinetic changes, including desensitization of the enzyme to key allosteric effectors or alteration of the K_{0.5 PEP} for the substrate phosphoenolpyruvate. R231A, and especially R232Q, displayed decreased apparent affinity for the activator glucose-6-phosphate. Apparent affinity for the activator glycine was reduced in D228N and R232Q, while the maximum activation caused by glycine was greatly reduced in R226Q and E229A. R226Q and E229A also showed significantly lower sensitivity to the inhibitors malate and aspartate. E229A exhibited a low K_{0.5 PEP}, while the K_{0.5 PEP} of R232Q was significantly higher than that of wild type. Thus these seven residues are critical determinants of the enzyme’s kinetic responses to activators, inhibitors and substrate. The present results support an earlier suggestion that Arg 231 contributes to the binding site of the allosteric activator glucose-6-phosphate, and are consistent with other proposals that the substrate phosphoenolpyruvate allosterically activates the enzyme by binding at or near the glucose-6-phosphate site. The results also suggest that the glycine binding site may be contiguous with the glucose-6-phosphate binding site. Glu 229, which extends from this interface region through the interior of the protein and emerges near the aspartate binding site, may provide a physical link for propagating conformational changes between the allosteric activator and inhibitor binding regions.     

The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida

Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.