Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV

Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.     

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP, and Sephadex G-200 columns. The overall purification was about 1200-fold, with 25% yield. The purified enzyme showed one band on disc gel electrophoresis and two bands on sodium dodecyl sulfate electrophoresis with molecular weights of 62 000 and 66 000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 mumol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze gamma- or beta-endorphin, or dynorphin, but it does hydrolyze neutral and basic aminoacyl beta-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin, and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum, and molecular weight, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M, and the soluble aminopeptidase for enkephalin degradation.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

Purification and properties of rat brain hexokinase

Rat brain hexokinase has been purified to homogeneity as judged by disc-gel electrophoresis, isoelectric focusing, and analytical ultracentrifugation. More than 50% of the initial activity could be obtained in homogeneous form (sp act, 60 units/mg protein) by a simple procedure consisting essentially of two steps: relatively specific solubilization of the enzyme from the mitochondrial membrane by glucose-6-P, followed by DEAE-cellulose column chromatography. The molecular weight is approximately 98,000; this same molecular weight was observed when the denatured enzyme was examined by the SDS-polyacrylamide electrophoretic technique, strongly suggesting that the enzyme consists of a single polypeptide chain. In accord with this view, a single N-terminal amino acid, glycine, has been recovered in 80% yield based on a molecular weight of 98,000. The amino acid composition of the rat brain hexokinase has been determined and found to be very similar to that previously reported for the bovine brain enzyme (Schwartz, G. P., and Basford, R. E. (1967) Biochemistry6, 1070, suggesting extensive sequence homology. A notable feature of the brain hexokinases is a relatively low aromatic amino acid content, as judged by the amino acid composition and the relatively low molar extinction coefficient.     

Purification and properties of rat brain pyruvate kinase

Rat brain pyruvate kinase was purified to near homogeneity by a three-step process involving ammonium sulfate precipitation and phosphocellulose and Blue-Sepharose CL-6B column chromatography. The enzyme migrated on polyacrylamide gel along with a commercial sample of rabbit muscle pyruvate kinase. The enzyme showed a hyperbolic relationship with phosphoenolpyruvate and ADP, with apparent Km's of 0.18 and 0.42 X 10(-3) M, respectively. The enzyme was inhibited by ATP, the effect being more pronounced at unsaturating concentrations of phosphoenolpyruvate. L-Phenylalanine was found to be a strong inhibitor of the enzyme, with the Ki for inhibitor being 0.11 mM. The inhibition by phenylalanine was more pronounced at pH 7.4 than at pH 7.0, and appeared to be competitive with phosphoenolpyruvate. L-Alanine and fructose 1,6-bisphosphate prevented the inhibition of the enzyme by phenylalanine. Ca2+ was found to be a strong inhibitor of the enzyme, and the inhibition was more marked at saturating phosphoenolpyruvate concentrations. The kinetic properties of the purified brain pyruvate kinase suggest that the enzyme may be distinct from the muscle or liver enzymes.     

Neuron-specific aminopeptidase and puromycin-sensitive aminopeptidase in rat brain development

Neuron-specific aminopeptidase (NAP) and the ubiquitous puromycin-sensitive aminopeptidase (PSA) were compared in the rat hippocampus during early development. Hippocampus contains the highest amount of NAP determined by a fast-protein liquid chromatography-aminopeptidase analyzer using Leu -naphthylamide as substrate. Both enzymes were found in the hippocampus in all ages. NAP was lower in immature rat; the 19th embryonic-day fetus contained the least. It increased steeply during the prenatal through the early postnatal period, 9-fold by the first month. The rate of increase diminished subsequently, increasing 20% in the second month and 13% in the third. The age-dependent increase in NAP activity was parallel to its protein expression as determined by Western blot. The specific molecular activity (hydrolytic activity/NAP antigenicity) in newborn, 15-day-old, and 30-day-old rats were 1.00, 0.88, and 1.00, respectively. The PSA developmental profile without linear increase in activity was distinct from NAP. PSA activity was higher than NAP in decreasing order, 100–4 times, during the same development span. Similarly, different growth profiles for NAP and PSA were also found in the primary culture of developing cerebellar granule cells. Puromycin (1–5 M) blocked neurite outgrowth and caused apoptosis by nonantibiotic effects. Our data suggest that the synaptosome-enriched NAP plays a role in neuron growth, differentiation, and information programming.     

Purification and properties of human intestine alanine aminopeptidase

Human intestinal alanine aminopeptidase has been purified to greater than 90% homogeneity. The enzyme was released from mucosal cell membranes by Triton X-100 treatment. The native enzyme had a molecular weight of 206,000 in dilute buffer and 108,000 in the presence of sodium dodecyl sulfate. The enzyme was inhibited by chelators suggesting the presence of a metal ion in the enzyme. The most potent chelator inhibitor tested, o-phenanthroline, gave mixed kinetics (Ki = 67 micro M). Activity was restored by removal of the chelator. The enzyme was inhibited competitively by amino acids having hydrophobic side chains such as L-phenylalanine (Ki = 0.67 mM). Puromycin and methicillin also inhibited the enzyme in the competitive (Ki = 12.5 micro M) and noncompetitive (Ki = 4.6 mM) manner, respectively. Kinetic analysis of several amino acid beta-naphthylamides as substrates demonstrated the preference for substrates having hydrophobic or basic amino terminal residues with no beta-branching. L-Methionyl-beta-naphthylamide was the most tightly bound with L-alanyl-beta-naphthylamide was the most rapidly hydrolyzed.     

Separation and characterization of a dipeptidyl aminopeptidase that degrades enkephalins from monkey brain

A dipeptidyl aminopeptidase (a membrane-bound enzyme) which cleaved Met-enkephalin and released dipeptide (Tyr-Gly) was partially purified from monkey brain. A fraction containing both exoaminopeptidase and dipeptidyl aminopeptidase activity was obtained from DE-52 cellulose column chromatography. The dipeptidyl aminopeptidase activity in this fraction was not inhibited by addition of bestatin (300 μg/ml), while the exoaminopeptidase was strongly inhibited. Both enzymes were separated by AH-Sepharose 4B column chromatography. The molecular weight of the dipeptidyl aminopeptidase was calculated about 110,000. The enzyme activity was inhibited by addition of diisopropylfluorophosphate (DFP) or o-phenanthroline.     

Purification and characterization of aminopeptidase B from goat brain

Aminopeptidase B was purified from goat brain with a purification fold of ～280 and a yield of 2.7%. The enzyme revealed a single band on both native acrylamide gel and SDS-PAGE thereby confirming apparent homogeneous preparation and its monomeric nature. The enzyme exhibited a molecular mass of 80.2 kDa and 79.7 kDa on Sephadex G-200 and SDS-PAGE respectively. The pH optimum was 7.4 and the enzyme was stable between pH 6.0 and 9.0. l-Arg-βNA was the most rapidly hydrolyzed substrate followed by Lys-βNA. The K_m value with Arg-βNA was found to be 0.1 mM. Metal chelating and –SH reactive agents strongly inhibited the enzyme activity. 1,10-Phenanthroline exhibited mixed type of inhibition with a K_i of 5 × 10^?5 M. The enzyme was highly sensitive to urea. Metal ions like Ni²⁺, Cd²⁺, Fe²⁺and Hg²⁺ inhibited the enzyme, whereas Co²⁺, Zn²⁺, Mn²⁺and Sn²⁺ slightly activated the enzyme.     

Mammalian lens dipeptidyl aminopeptidase III

An intracellular exopeptidase identified as dipeptidyl aminopeptidase III (DAP III) was found to be abundant in the bovine lens. The enzyme contained in aqueous extracts exhibited a marked preference, compared to other dipeptidyl-β-naphthylamides, for the release of Arg-Arg from Arg-Arg-2-NNap at the optimum pH 9.0 and 37°. The K_m for this substrate was estimated to be 2.83 × 10^?5M. Lens DAP III was inhibited by EDTA, p-chloromercuriphenyl sulfonate, and puromycin. Lens aminopeptidase activities measured at pH 7.5 on the β-naphthylamides of leucine, alanine, and arginine, included for comparison, suggested that not only is leucine aminopeptidase abundant, but also other aminopeptidases that appear to include alanine aminopeptidase and aminopeptidase B.     

Purification and properties of rat brain pyruvate carboxylase.

Rat brain pyruvate carboxylase was purified 2000-fold and some of its properties and kinetic parameters were investigated. The use of (NH4)2SO4 gradient solubilization on a Celite column and precipitation with polyethylene glycol permitted purification to an estimated 20% purity. Except for a few subtle kinetic differences this enzyme is indistinguishable from rat liver pyruvate carboxylase.     

Purification and properties of esterases characteristic of adult rat brain

1. A method for the partial purification of an esterase fraction, present in the brain of the adult but not the newborn rat, is described. A 54-fold purification was achieved in three steps. 2. When subjected to starch-gel electrophoresis, the purified fraction resolved into three bands of esterase activity. Two of these bands migrate close together and faster than other esterases in the brain. These two esterases are inhibited by p-hydroxymercuribenzoate but not by di-isopropyl phosphorofluoridate. The third band is di-isopropyl phosphorofluoridate-sensitive and migrates just behind the two leading esterases. 3. After treatment with di-isopropyl phosphorofluoridate, to obviate the effects of the di-isopropyl phosphorofluoridate-sensitive esterase, the enzyme preparation hydrolyses alpha-naphthyl acetate, alpha-naphthyl propionate and alpha-naphthyl butyrate, but not cholesteryl acetate. The V(max.) for the naphthyl esters decreased with increase in chain length of the acyl group. The acetate ester is hydrolysed 34 times as fast as the butyrate and about seven times as fast as the propionate derivative. The K(m) values for these three esters, measured at pH7.2 and 37 degrees , are 2.8x10(-4)m, 3.1x10(-4)m and 7.3x10(-5)m for the acetate, propionate and butyrate derivatives respectively. 4. The Hofstee (1952) plots for the kinetic data show a single line, indicating that the two most-rapidly migrating esterases, although electrophoretically separable, are not kinetically distinguishable in the substrate ranges examined.     

Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain.

1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.     

Purification and properties of myo-inositol-1-phosphatase from rat brain

myo-Inositol-1-phosphatase [EC 3.1.3.25] was purified from a cytosolic fraction of rat brain. The purified enzyme appeared homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 29,000. The molecular weight of the native enzyme was 55,000 as determined by molecular sieve chromatography. These values indicated that the native enzyme was composed of two identical subunits. The isoelectric point of the enzyme was 4.6. The enzyme hydrolyzed inositol-1-phosphate, 2'-AMP, 2'-GMP, beta-glycerophosphate, and alpha-glycerophosphate; the ratio of the reaction rates was 100 : 84 : 73 : 64 : 32. The Km values for inositol-1-phosphate, 2'-AMP, and beta-glycerophosphate were 1.2 X 10(-4) M, 1.9 X 10(-4) M, and 7.7 X 10(-4) M, respectively. Mn2+ and Ca2+ were strong competitive inhibitors against Mg2+, with Ki values of 3 microM and 20 microM, respectively. This result suggests that myo-inositol-1-phosphatase might be regulated by intracellular Ca2+ and/or Mn2+. Li+, which is known to show a therapeutic effect on manic-depressive disease and also to prolong the intrinsic periods of circadian rhythms in various organisms, was a potent uncompetitive inhibitor and inhibited 50% of the activity at 1 mM. The possibility that myo-inositol-1-phosphatase and inositol phospholipid metabolism are involved in circadian rhythm oscillation is discussed in terms of Li actions.     

Purification and properties of rat brain succinic semialdehyde dehydrogenase.

Succinic semialdehyde dehydrogenase from rat brain has been purified to electrophoretic homogeneity. It has a molecular weight of about 140, 000 and is composed of two apparently identical subunits. The reaction catalized by the pure protein is entirely dependent on endogenous --SH groups. The Kim (limits) for NAD and succinic semialdehyde are 2 X 10(-5) M and 1 X 10(-4) M respectively at the optimum pH of 8.6. Inhibition studies show that the reaction mechanism is a compulsory ordered on where NAD binds first followed by succinic semialdehyde.     

Purification and some properties of ATP-citrate lyase from rat brain

ATP-citrate lyase has been purified from rat brain by a new procedure which yields an enzyme of specific activity of 21 U/mg protein (37 °C) (2050-fold purification). Purity (by sodium dodecyl sulfate-gel electrophoresis) of the preparation was comparable to that of rat liver ATP-citrate lyase of similar specific activity. Both brain and liver ATP-citrate lyase have the same electrophoretic mobility, as well as the same immunoreactivity against specific rabbit anti-rat liver ATP-citrate lyase antibody. These data indicate that rat brain ATP-citrate lyase is similar or identical to that present in rat liver. Intraperitoneally injected ³²P_i was incorporated into the structural phosphate of ATP-citrate lyase in rat liver but not into the rat brain enzyme.