Properties and kinetics of a neutral beta-galactosidase from rabbit kidney

A neutral beta-galactosidase has been purified by concanavalin A-Sepharose affinity chromatography, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and hydroxylapatite chromatography. The enzyme was purified 126-fold with a yield of about 21%. This form has a neutral optimal pH (7.5) and it is located in the cytosolic fraction. It shows a wide pH stability from pH 4.5 to 8.0, but it is very unstable at low pH values. Its isoelectric point is 4.9 and this value does not change on neuraminidase treatment. The estimated molecular weight was 47 000. The neutral form shows beta-D-galactosidase, beta-D-fucosidase and beta-D-glucosidase activities, all of them associated in a single peak in all the purification steps. p-Nitrophenyl beta-D-galactosides, p-nitrophenyl beta-D-fucosides and p-nitrophenyl beta-D-glucosides competed fully for a common active site in mixed-substrate experiments. Using gamma-D-galactonolactone as competitive inhibitor the Ki values were always coincident for the three activities. The effect of NaCl, methyl mannoside and some sugars (fucose, galactose and glucose) was studied.     

Purification and some properties of tartrate-sensitive acid phosphatase from rabbit kidney cortex.

Two forms of tartrate-sensitive acid phosphatases (EC 3.1.3.2) were purified from rabbit kidney cortex by a multiple-column-chromatography method. The basic form constituted 90% of the enzyme and migrated as a single band of protein on polyacrylamide-gel electrophoresis. The proteins contaminating the acidic form did not exceed 5% of the total protein. The specific activity towards p-nitrophenyl phosphate was 12 mumol/min per mg for the basic form and 0.7 mumol/min per mg for the acidic form. The basic form of the enzyme differs from the acidic form in its heat-stability, Km values, inhibition rates by tartrate and fluoride and substrate specificities. Relative to p-nitrophenyl phosphate hydrolysis rate, the acidic form hydrolysed a variety of physiological monophosphate esters, whereas the basic form hydrolysed only CMP and phosphoenolpyruvate. Bacterial neuraminidases had no effect on the activity and mobility of the acidic form on polyacrylamide-gel electrophoresis. Both forms have the same molecular weight (101000 +/- 4000) and are probably composed of two identical subunits. The question whether the two forms of the enzyme are different proteins or whether one is a modified form of the other is discussed.     

Heterogeneity of intermediate filament proteins from rabbit spinal cord

Large amounts of a neurofilament-enriched fraction may be prepared from spinal cord homogenates by a simple, three-step procedure. This involves flotation of filament-containing axon fragments, extraction with Triton X-100, and washing by sedimentation through a sucrose density gradient. The material obtained by this procedure includes both large mats of individual 10-nm filaments and tightly packed bundles of filaments. SDS-gel electrophoresis of these fractions indicates that the fractions are formed of four polypeptides: the three which are generally considered to form neurofilaments (P200, P150, and P68) and another, with a molecular weight of about 50,000 daltons (P50), which is thought to be derived from fibrous astrocytes. Analysis of these filament fractions on two-dimensional gels indicates heterogeneity among each of the different molecular weight classes. The largest polypeptide of neurofilaments, P200, focuses at several spots in the pH gradient. P68 and P150 are more acidic: each appears as a pair of overlapping spots. P50 resolves into a complex of spots of about the same molecular weight but with different isoelectric points. Heterogeneity is not unique to these filament polypeptides but appears to be a characteristic of all fibrous proteins of the nervous system.     

Gene expression of D-amino acid oxidase in rabbit kidney

Although D-amino acid oxidase (DAO) [EC 1.4.3.3] activity in rabbit kidney extract was undetectable, protein immunoreactive toward rabbit anti-pig kidney DAO antiserum and RNAs that hybridized with fragments of human and pig DAO cDNAs were detected distinctly in the rabbit kidney. A cDNA clone, RD22, was isolated from the rabbit kidney cDNA library by hybridization with a fragment of human DAO cDNA. Analysis of the nucleotide sequence revealed a 2,018 nucleotide sequence encoding a protein consisted of 347 amino acids. The number of amino acid residues was identical with those of human and pig DAOs, and the amino acid sequence showed 80 and 83% identity with pig and human DAOs, respectively. RNAs that hybridized with RD22 DNA fragment also existed in rabbit kidney, and their sizes were the same as those of the RNAs detected with the human and pig DAO cDNA fragments. RD22-derived protein was hardly synthesized by an in vitro expression system. However, a cDNA fragment lacking most of the 5'-untranslated region and its mutants containing base changes around the initiation codon did direct protein synthesis. Moreover, the protein derived from the partial cDNA fragment containing a large part of the coding region sequence showed immunoreactivity toward anti-pig DAO antiserum. The results suggest that one of the causes of the very poor synthesis of DAO protein in rabbit kidney is translational suppression in the synthetic process.     

Aggregation-dissociation and stability of acid beta-galactosidase purified from porcine spleen

Sucrose gradient centrifugation of the monomeric form (A1) of porcine spleen beta-galactosidase showed a pH-dependent equilibrium between monomer at neutral pH (pH 7.0) and dimer at acidic pH (pH 5.4-3.0), independent of ionic strength. While the oligomeric form (Ao), which was hardly dissociated under physiological conditions, was dissociated only with some protein denaturing agents into similar catalytic subunit to the A1. Both the A1 and Ao were equally active and stable at acidic pH, in the physiological condition inside lysosome (around pH 4.6).     

Heterogeneity of alpha-L-fucosidase from human kidney during affinity chromatography

During affinity chromatography on N-(epsilon-aminocaproyl)-beta-L-fucopyranosylaminosepharose of the enzyme preparation of alpha-L-fucosidase from human kidney the elution profile of the enzyme revealed two components, which can be designated as alpha-L-fucosidases A and B. In the absence of sodium aside in the buffer mixtures used one of the components (fucosidase A) was retained by an affinity adsorbent, while the other one (fucosidase B) was adsorbed under the same conditions; the latter component was eluted with a solution containing the enzyme inhibitor--L-fucose. Data from the enzyme rechromatography suggest an equilibrium of the fucosidases A and B, which differ in their affinities for the affinity sorbent.     

Purification of G-M-1-ganglioside and ceramide lactoside beta-galactosidase from rabbit brain.

The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.     

Distribution of acid phosphatase, beta-glucuronidase, n-acetyl-beta-d-glucosaminidase and beta-galactosidase in cornea of albino rabbit.

Activities of acid phosphatase, beta-glucuronidase, N-acethyl-beta-D-glucosaminidase and acid beta-galactosidase were investigated histochemically in rabbit corneas. Frozen sections after block fixation in cold 4% formaldehyde with 1% CaCl2 followed by washing in cold physiological saline as well as cold microtome sections of corneas quenched in petroleter chilled with acetone-dry ice mixture, transferred to nonprecooled slides or semipermeable membranes were used. Standard aqueous media were employed in the case of free-floating frozen sections of fixed corneas as well as of cold mictrotome sections (postfixed in cold 4% formaldehyde). Agar media were used in connection with the technic of semipermeable membranes. Gomori method (in the case of acid phosphatase), simultaneous azocoupling methods (substrates derivated of naphthol-AS-BI with hexazonium-p-rosanilin) in the case of acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase and the indigogenic method in the case of acid beta-galactosidase were applied. Enzyme activities in sections of fixed corneas were minimal in comparison with those in cold microtome sections of unfixed material revealed particularly with the technic of semipermeable membranes which is to be preferred. This technic is recommended in studies concerned with lysosomal enzymes in the cornea, particularly in keratocytes. All enzymes investigated were present in corneal epithelium, keratocytes and endothelium. Acid phosphatase displayed the highest activity followed by beta-glucuronidase and acetyl-beta-D-glucosaminidase. The activity of beta-galactosidase was the lowest. For the demonstration of activities in keratocytes sections parallel to the surface are very suitable. In these sections enzyme activities were demonstrated in small granules (apparently lysosomes) present in the central part of their cytoplasm as well as in projections. Diffuse staining was also seen, being the highest in the case of acid phosphatase.     

Isolation and partial purification of dicarboxylic acid binding protein from luminal-membrane vesicles of rabbit kidney cortex

A specific dicarboxylic acid binding protein was isolated by solubilizing highly purified renal luminal-membrane vesicles with the non-ionic detergent C12E8 , followed by affinity chromatographic procedures. SDS-polyacrylamide gel electrophoresis of the samples containing dicarboxylic acid binding protein showed a single sharp band of an apparent molecular weight of 50 000. After treatment with mercaptoethanol the protein was split in two subunits of apparent molecular weights of 35 000 and 15 000. By analytical ultracentrifugation the minimal molecular weight of the dicarboxylic acid binding protein preparation was calculated to be 54 000. Binding of the radioactive succinate and L-malate to the dicarboxylic acid binding protein preparation as studied by equilibrium dialysis showed saturation phenomenon and was specifically inhibited by addition of D-malate. The dissociation constants for succinate (0.18 mM) and L-malate (0.33 mM) calculated from the binding data agree extremely well with the apparent Km values for these organic acids found in transport studies utilizing intact luminal-membrane vesicles.     

Site of uptake of nonfiltered amino acid in the rabbit kidney

During passage from renal artery to vein, nonfiltered amino acids are known to be extracted by the kidney, an observation generally attributed to their basolateral uptake by tubular epithelium. This attribution is here tested in the rabbit, using the nonmetabolizable analogue cycloleucine as test compound. Uptake of cycloleucine is diffusion limited and could be maximized by lengthening its artery-to-vein transit time by short aortic occlusion. The transient anoxia did not abolish active solute transport in the kidney; the technique permits acute loading of the kidney with, for instance, a nephrotoxicant without excessive exposure of the whole animal. The major portion of cycloleucine taken up by nonfiltering kidneys during occlusion returned to renal venous plasma with a mean delay of 45 sec, as if it had accumulated in the same cellular transport pool through which reabsorbed cycloleucine has to pass. A fraction of the amino acid taken up also reached the tubular lumen. These results support the suggested role of tubule cells in the extraction of amino acids from postglomerular blood.     

The regulation of formation of prostaglandins and arachidonoyl-CoA from arachidonic acid in rabbit kidney medulla microsomes by linoleic acid hydroperoxide

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of linoleic acid (LA) and 13-hydroperoxyoctadecadienoic acid (13-HPODE) on the PG and AA-CoA formation from high and low concentrations of AA (60 and 5 microM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 microM [(14)C]-AA in 0.1M Tris-HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. LA (10-50 microM) reduced only PG formation from both 60 and 5 microM AA. 13-HPODE (10-50 microM) also reduced PG formation from 60 and 5 microM AA, but the inhibitory potency was much stronger than that by LA. Furthermore, 13-HPODE had the potential to increase the AA-CoA formation with a decrease in the PG formation from 5 microM AA. These results suggest that 13-HPODE, but not LA, may shift AA away from COX pathway into ACS pathway under low substrate concentration (near physiological concentration of AA).     

Purification of acid beta-galactosidase and acid neuraminidase from bovine testis: evidence for an enzyme complex

The isolation of an acid neuraminidase from bovine testis is described. Under all experimental conditions this neuraminidase copurifies with acid β-galactosidase, but not with other lysosomal hydrolases. Immunotitration with an antiserum raised against purified human placental β-galactosidase results in the coprecipitation of both enzyme activities. Our data indicate that acid neuraminidase and β-galactosidase are present as an enzyme complex. The possible physiological relevance is discussed.