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.     

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.     

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.     

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.     

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 properties of glutamyl aminopeptidase from chicken egg-white

Hydrolytic activities characteristic for different aminopeptidases were detected in the egg-white of unfertilized chicken eggs, and one aminopeptidase was isolated in an electrophoretically homogeneous form. The isolated aminopeptidase preferentially hydrolyzed bonds of alpha-glutamyl residue at the NH(2)-end of synthetic substrates and peptides. The enzyme is a dimer with an M(r) of 320,000 and pI of 4.2. Its optimal pH and temperature are 7.6 and 60 degrees C, respectively. EDTA, amastatin, and N-bromosuccinimide are inhibitors, while Ca2++ and Mn2+ are activators of the enzyme Ca2+ also stabilizes the enzyme. According to the observed properties, the isolated chicken egg-white aminopeptidase belongs to the glutamyl aminopeptidases.     

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 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 dipeptidyl peptidase IV from human urine

Dipeptidyl peptidase IV (DPP-IV) (EC 3.4.14.5) has been purified from normal human urine using immunoaffinity chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular mass of urinary DPP-IV was estimated to be 280 kDa by gel filtration. The Km value for the hydrolysis of (Gly-L-Pro)-4-methylcoumaryl-7-amide tosylate was calculated to be 1.25 +/- 0.25 X 10(-4) M; the pH optimum and pI were 8.7 and 6.5, respectively. These properties were the same as those of renal DPP-IV. Both renal and this urinary DPP-IV were inhibited by diisopropylfluorophosphate and activated by phospholipid. From these results we suppose that urinary DPP-IV may be derived from the kidney rather than from the blood     

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.     

Rabbit intestinal aminopeptidase N. Purification and molecular properties

The detergent and protease forms of rabbit intestinal aminopeptidase N were purfied for chemical investigations and future specific immunological labeling of the enzyme in situ. The purification of the detergent form required a special technique called 'reverse immunoabsorbant chromatography'. The specific activity of the detergent form finally obtained was identical to that of the protease form. A significant charge micro heterogeneity persisted in the most purified preparations, due probably to a certain level of variability in the sugar moiety. The major proteolytic cleavage which occurred at the hydrophilic-hydrophobic junction of the detergent form during its conversion into the protease form was well defined. But additional splittings probably in C-terminal region of the molecules led to several protease forms differing by their size. The molecular weight assigned to the peptide liberated during the above conversion was overestimated due to preferential detergent binding to hydrophobic structures. The correct value, estimated by a new isotopic dilution method, was 3800 (36-38 residues) for the peptide originating from the rabbit enzyme. The real anchor plunging into the membrane core is possibly still shorter. Comparative N-terminal residue determinations in the detergent form, the protease form and the peptide difinitely confirmed that the enzyme is anchored to the bursh border membrane by its N-terminal region.