An ecto-nucleotide pyrophosphatase is one of the main enzymes involved in the extracellular metabolism of ATP in rat C6 glioma

The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.     

Purification and characterization of Clostridium difficile glutamate dehydrogenase

Recombinant Clostridium difficile glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, EC 1.4.1.2) was purified 177-fold to electrophoretic homogeneity with a 62% recovery through a four-step procedure involving gel filtration and ion-exchange and dye affinity chromatography. The approximate molecular weights of the native enzyme by gel filtration and subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with a hexameric structure for the purified enzyme. The enzyme-catalyzed glutamate oxidation was an NAD-dependent sequential process in which NADP could not be substituted as coenzyme. Several dinucleotide analogs of NAD structurally altered in either the pyridine or the purine moiety were observed to function as coenzymes when substituted for NAD. Nicotinamide mononucleotide did not serve as a coenzyme for glutamate oxidation. Product inhibition by NADH was competitive with respect to NAD. In deadend inhibition studies, adenosine diphosphoribose was shown to be an effective coenzyme-competitive inhibitor.     

Study of the interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA

Glyceraldehyde-3-phosphate dehydrogenase binds to homologous and heterologous single-stranded but not double-stranded DNA. Binding to RNA, poly(A) and poly(dA-dT) has also been observed. Enzyme binding to these nucleic acids leads to the formation of an insoluble complex which can be sedimented at low speed. The interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA is strongly inhibited by NAD and NADH but not by NADP. Adenine nucleotides, which inhibit the dehydrogenase activity by competing with NAD for its binding site (Yang, S.T. and Deal, W.C., Jr. (1969) Biochemistry 8, 2806--2813), also inhibit enzyme binding to DNA, whereas glyceraldehyde-3-phosphate and inorganic phosphate are non-inhibitory. These results suggest that DNA interacts through the NAD binding sites of glyceraldehyde-3-phosphate dehydrogenase. In accordance with this idea, it was found that DNA also binds to lactate dehydrogenase, an enzyme containing a similar dinucleotide binding domain, and that this binding is inhibited by NADH. A study of the base specificity of the DNA-glyceraldehyde-3-phosphate dehydrogenase interaction using dinucleoside monophosphates shows that inhibition of DNA binding by the dinucleotides requires the presence of a 3'-terminal adenosine and is greater when the 5'-terminus contains a pyrimidine instead of a purine. These results suggest that the dinucleotides bind at the NAD site of the dehydrogenase and that the enzyme would interact preferentially with PypA dinucleotides present in the nucleic acid.     

Sigma-binding site ligands inhibit K+ currents in rat locus coeruleus neurons in vitro

The effect of triphenyltin on the activity of membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated. Triphenyltin inhibits the hydrolysis of chromatophore membrane-bound pyrophosphatase in a pH-dependent pattern, being maximal at pH 9-10. At basic pH values, the inhibition produced by this organotin on membrane-bound pyrophosphatase is very similar to that produced on the chromatophore H+ATPase (I50 = 14.4 and 10 microM, respectively). Detergent-solubilized membrane-bound pyrophosphatase is also inhibited by triphenyltin, but the cytoplasmic enzyme of R. rubrum is inhibited only slightly. The inhibitory effect of triphenyltin on membrane-bound pyrophosphatase is the same with Mg-PPi or Zn-PPi, and is dependent on the chromatophore membrane concentration. Triphenyltin modified mainly the Vmax of the enzyme, and only slightly its Km. Free Mg2+ does not reverse the inhibition. Reducing agents prevent triphenyltin inhibition of the membrane-bound pyrophosphatase, but their effect is due to an alteration of the inhibitor, and not to a modification of thiol groups of the enzyme. The most likely site for triphenyltin inhibition in chromatophore membrane-bound pyrophosphatase is a component either within or closely associated with the membrane.     

Effects of ions on adenosine binding and enzyme activity of purified S-adenosylhomocysteine hydrolase from bovine kidney

The present investigation was undertaken to determine the effect of various ions on the characteristics of S-adenosylhomocysteine (SAH) hydrolase from bovine kidney. The binding sites of [3H]-adenosine to purified SAH hydrolase were not influenced by phosphate, magnesium, potassium, sodium, chloride or calcium ions at physiological cytosolic concentrations. To test whether NAD+ in the SAH hydrolase is essential for adenosine binding, we prepared the apoenzyme by removing NAD+ with ammonium sulfate. The resulting apoenzyme did not exhibit any [3H]-adenosine binding. Since the apoenzyme was enzymatically inactive, it is suggested that adenosine binds to the active site and not to an allosteric site of the intact enzyme. The kinetics of the hydrolysis and the synthesis of SAH catalyzed by the enzyme SAH hydrolase were measured in the presence and absence of phosphate and magnesium. Phosphate increased the Vmax for both synthesis and hydrolysis. However, only the affinity of adenosine for SAH synthesis was significantly enhanced from 10.1+/-1.3 microM to 5.4+/-0.5 microM by phosphate. This effect was already maximal at a phosphate concentration of 1 mM. All other tested ions were without effect on the enzyme activity. Our results show that phosphate at physiological concentrations shifts the thermodynamic equilibrium of SAH hydrolase in the direction of SAH synthesis. These findings imply that SAH-sensitive transmethylation reactions are inhibited during renal hypoxia when intracellular levels of phosphate, adenosine, and SAH are elevated.     

The gene, ialA, associated with the invasion of human erythrocytes by Bartonella bacilliformis, designates a nudix hydrolase active on dinucleoside 5'-polyphosphates

ialA, one of two genes associated with the invasion of human red blood cells by Bartonella bacilliformis, the causative agent of several diseases, has been cloned and expressed in Escherichia coli. The protein, IalA, contains an amino acid array characteristic of a family of enzymes, the Nudix hydrolases, active on a variety of nucleoside diphosphate derivatives. IalA has been purified, identified, and characterized as an enzyme catalyzing the hydrolysis of members of a class of signaling nucleotides, the dinucleoside polyphosphates, with its highest activity on adenosine 5'-tetraphospho-5'-adenosine (Ap4A), but also hydrolyzing Ap5A, Ap6A, Gp4G, and Gp5G. In each case, a pyrophosphate linkage is cleaved yielding a nucleoside triphosphate and the remaining nucleotide moiety.     

Interactions of calcium and magnesium with the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae

The activity of the mitochondrial inorganic pyrophosphatase from Saccharomyces cerevisiae was measured in the presence of increasing concentrations of magnesium and calcium. Calcium pyrophosphate (dissociation constant Kd = 1.9 microM) inhibited pyrophosphatase by competition with magnesium pyrophosphate (Kd = 50 microM). The small movements of calcium detected in mitochondria from yeast may be physiologically significant for the control of inorganic pyrophosphatase activity and the concentration of pyrophosphate in the matrix of yeast mitochondria.     

Purification and characterization of inorganic pyrophosphatase from Methanobacterium thermoautotrophicum (strain delta H)

Inorganic pyrophosphatase (EC 3.6.1.1.) has been isolated from the archaebacterium Methanobacterium thermoautotrophicum (strain delta H). The enzyme was purified 850-fold in three steps to electrophoretic homogeneity. The soluble pyrophosphatase consists of four identical subunits: the molecular mass of the native enzyme estimated by gel filtration was approx. 100 kDa and denaturing polyacrylamide gel electrophoresis gave a single band of 25 kDa. The enzyme also may occur as an active dimer formed by dissociation of the tetramer. The pyrophosphate showed an optimal activity at 70 degrees C and a pH of 7.7 (at 60 degrees C) and was not influenced by dithiothreitol, sodium dithionite or potassium chloride. The enzyme was very specific for pyrophosphate (PPi) and Mg2+. Magnesium could be partially replaced by Co2+ (15%). The reaction was inhibited for 60% by 1 mM Mn2+ in the presence of 24 mM Mg2+. In addition, the enzyme was inhibited by potassium fluoride (50% at 0.9 mM). Kinetic analysis revealed positive co-operativity for both Mg2+ and PPi with Hill coefficients of 3.3 and 2.0, respectively. Under the experimental conditions at which the enzyme was present as its dimer, the apparent Km of PPi and magnesium were determined and were approx. 0.16 mM and 4.9 mM, respectively; Vmax was estimated at about 570 U/mg.     

Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 photoreduced 2,4-dinitrophenol to 2-amino-4-nitrophenol by a nitrophenol reductase activity which was induced in the presence of nitrophenols and was repressed in ammonium-grown cells. The enzyme was located in the cytosol, required NAD(P)H as an electron donor, and used several nitrophenol derivatives as alternative substrates. The nitrophenol reductase was purified to electrophoretic homogeneity by a simple method. The enzyme was composed of two 27-kDa subunits, was inhibited by metal chelators, mercurial compounds, and Cu2+, and contained flavin mononucleotide and possibly nonheme iron as prosthetic groups. Purified enzyme also exhibited NAD(P)H diaphorase activity which used tetrazolium salt as an electron acceptor.     

The sequencing expression, purification, and steady-state kinetic analysis of quinolinate phosphoribosyl transferase from Escherichia coli

The nadC gene from Escherichia coli was isolated and sequenced. The gene was then cloned into an expression vector and, following transformation, the resulting bacteria were able to produce quinolinate phosphoribosyl transferase as about 2% of the soluble protein. The enzyme was purified in five steps leading to a homogeneous preparation. The enzyme reaction shows an ordered binding mechanism where the magnesium ion complex of 5-phosphoribosyl-1-pyrophosphate binds first followed by quinolinic acid. The products are pyrophosphate CO2, and nicotinate mononucleotide. Product inhibition studies show that nicotinate mononucleotide is a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate while pyrophosphate is noncompetitive with respect to both 5-phosphoribosyl-1-pyrophosphate and quinolinic acid. Phthalic acid and fructose-1,6-bisphosphate were used as dead-end inhibitors. Phthalate was competitive with respect to quinolinic acid but uncompetitive with respect to 5-phosphoribosyl-1-pyrophosphate. Fructose-1,6-bisphosphate was a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate and noncompetitive with respect to quinolinic acid. The Km values for the substrates are 15.6 microM for 5-phosphoribosyl-1-pyrophosphate and 6.4 microM for quinolinic acid.     

Arteriosclerosis obliterans that was improved by LDL apheresis

An enzyme hydrolyzing flavine-adenine dinucleotide (FAD) to flavine mononucleotide (FMN) and adenosine monophosphate (AMP) was purified about 460-fold over the isolated lysosomal membranes with 9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and the absence of SDS. Purification procedures included: preparation of crude lysosomal membranes, solubilization with Triton X-100, WGA-Sepharose, Con A-Sepharose, hydroxylapatite chromatography, gel filtration with Superdex 200, DEAE ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass of the purified enzyme, estimated by gel filtration with Superdex 200, was approximately 560 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular weight of 140,000. The pH optimum for FAD hydrolysis was 8.5 with an apparent Km of 0.1 mM and the isoelectric point was pH 7.3. The activity was inhibited by o-phenanthroline, EDTA, DTT, and NEM and was slightly stimulated by Zn ion, but was not affected by Ca or Mg ions. The purified FADase contained N-linked complex type oligosaccharide chains lacking neuraminic acids. The NH2 terminal 21 amino acid residues of the purified FADase were Ser-Pro-Cys-Val-Cys-Asp-Pro-Val-Val-Val-Cys-Lys-Val-Val-Pro-Cys-Thr-Leu- Ala-Leu .     

A rapid, enzymatic assay for the measurement of inorganic pyrophosphate in animal tissues

A simple, rapid enzymatic assay for the determination of inorganic pyrophosphate in tissue and plasma has been developed using the enzyme pyrophosphate--fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) which was purified from extracts of Propionibacterium shermanii. The enzyme phosphorylates fructose-6-phosphate to produce fructose-1,6-bisphosphate using inorganic pyrophosphate as the phosphate donor. The utilization of inorganic pyrophosphate is measured by coupling the production of fructose-1,6-bisphosphate with the oxidation of NADH using fructose-bisphosphate aldolase (EC 4.1.2.13), triosephosphate isomerase (EC 5.3.1.1), and glycerol-3-phosphate dehydrogenase (NAD+)(EC 1.1.1.8). The assay is completed in less than 5 min and is not affected by any of the components of tissue or plasma extracts. The recovery of pyrophosphate added to frozen tissue powder was 97 +/- 1% (n = 4). In this assay the change in absorbance is linearly related to the concentration of inorganic pyrophosphate over the curvette concentration range of 0.1 microM to 0.1 mM.     

Clinical evaluation of failure to thrive in older people

Deoxyhypusine synthase catalyzes the NAD+-dependent formation of deoxyhypusine in the eIF-5A precursor protein by transferring the 4-aminobutyl moiety of spermidine. This enzyme has recently been shown to be essential for cell viability and growth of yeast [Sasaki, K., Abid, M.R., and Miyazaki, M. (1996) FEBS Lett. 384, 151 154]. We have purified and characterized the enzyme from the yeast Saccharomyces carlsbergensis. The yeast and recombinant enzymes had a specific activity of 1.21 to 1.26 pmol per min per pmol of protein, and recognized both the eIF-5A precursor proteins almost equally as judged from their similar K(m) and V(max) values. Size exclusion chromatography and SDS-PAGE indicated that the active form of the enzyme is a homotetramer consisting of 43-kDa subunits. The enzyme showed a strict specificity for its substrates, NAD+, spermidine and eIF-5A precursor protein. Among all the substrates tested, only NAD+ showed a protective effect against heat inactivation of the enzyme suggesting that NAD+ initiates some conformational change in the enzyme. NADH exhibited a strong non-competitive inhibition (product inhibition). Unexpectedly, FAD, FMN, and riboflavin showed a moderate competitive inhibition. The competitive inhibition by diamines was maximal with compounds resembling spermidine in carbon chain length. 1,3-Diaminopropane inhibited the enzyme strongly in a competitive manner (product inhibition). On the other hand, putrescine did not inhibit the enzyme or act as a substrate. A polyclonal antibody raised against the yeast recombinant enzyme specifically inhibited deoxyhypusine synthase activity. The cross-reactivity (by Western blotting) of this antibody with the crude extracts varied depending on the source, indicating species specificity.     

Purification and characterization of a soluble form of mammalian adenylyl cyclase

An engineered, soluble form of mammalian adenylyl cyclase has been expressed in Escherichia coli and purified by three chromatographic steps. The enzyme utilizes one molecule of ATP to synthesize one molecule of cyclic AMP and pyrophosphate at a maximal specific activity of 12.8 micromol/min/mg, corresponding to a turnover number of 720 min-1. Although devoid of membrane spans, the enzyme displays all of the regulatory properties that are common to mammalian adenylyl cyclases. It is activated synergistically by Gsalpha and forskolin and is inhibited by adenosine (P-site) analogs with kinetic patterns that are identical to those displayed by the native enzymes. The purified enzyme is also inhibited directly by the G protein betagamma subunit complex. After adenovirus-mediated expression in adenylyl cyclase-deficient HC-1 cells, the enzyme can be stimulated synergistically by Gs-coupled receptors and forskolin.     

Phosphorylation of glycerol and dihydroxyacetone in Acetobacter xylinum and its possible regulatory role

Extracts of Acetobacter xylinum catalyze the phosphorylation of glycerol and dihydroxyacetone (DHA) by adenosine 5'-triphosphate (ATP) to form, respectively, L-alpha-glycerophosphate and DHA phosphate. The ability to promote phosphorylation of glycerol and DHA was higher in glycerol-grown cells than in glucose- or succinate-grown cells. The activity of glycerol kinase in extracts is compatible with the overall rate of glycerol oxidation in vivo. The glycerol-DHA kinase has been purified 210-fold from extracts, and its molecular weight was determined to be 50,000 by gel filtration. The glycerol kinase to DHA kinase activity ratio remained essentially constant at 1.6 at all stages of purification. The optimal pH for both reactions was 8.4 to 9.2. Reaction rates with the purified enzyme were hyperbolic functions of glycerol, DHA, and ATP. The Km for glycerol is 0.5 mM and that for DHA is 5 mM; both are independent of the ATP concentration. The Km for ATP in both kinase reactions is 0.5 mM and is independent of glycerol and DHA concentrations. Glycerol and DHA are competitive substrates with Ki values equal to their respective Km values as substrates. D-Glyceraldehyde and l-Glyceraldehyde were not phosphorylated and did not inhibit the enzyme. Among the nucleotide triphosphates tested, only ATP was active as the phosphoryl group donor. Fructose diphosphate (FDP) inhibited both kinase activities competitively with respect to ATP (Ki= 0.02 mM) and noncompetitively with respect to glycerol and DHA. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) inhibited both enzymic activities competitively with respect to ATP (Ki (ADP) = 0.4 mM; Ki (AMP) =0.25 mM). A. xylinum cells with a high FDP content did not grow on glycerol. Depletion of cellular FDP by starvation enabled rapid growth on glycerol. It is concluded that a single enzyme from A. xylinum is responsible for the phosphorylation of both glycerol and DHA. This as well as the sensitivity of the enzyme to inhibition by FDP and AMP suggest that it has a regulatory role in glycerol metabolism.     

Removal of 5'-terminal m7G from eukaryotic mRNAs by potato nucleotide pyrophosphatase and its effect on translation

The procedure for isolation of nucleotide pyrophosphatase (E.C. 3.6.1.9.) from potato has been modified to yield an endonuclease-free preparation purified 2300-fold. The enzyme was used for specific cleavage of pyrophosphate linkages in the 5'-terminal cap (m7GpppN) of several eukaryotic messenger RNAs. Enzymatic removal of 5'-terminal pm7G from reovirus, rabbit globin and Artemia salina mRNAs resulted in an almost complete loss (greater than 80%) of their template activities in a cell-free protein synthesizing system from wheat germ. Incubation with nucleotide pyrophosphatase did not decrease the translation of phage f2 RNA in an Escherichia coli cell-free system.     

CD38 and ADP-ribosyl cyclase catalyze the synthesis of a dimeric ADP-ribose that potentiates the calcium-mobilizing activity of cyclic ADP-ribose

CD38, a lymphocyte differentiation antigen, is also a bifunctional enzyme catalyzing the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and its hydrolysis to ADP-ribose (ADPR). An additional enzymatic activity of CD38 shared by monofunctional ADP-ribosyl cyclase from Aplysia californica is the exchange of the base group of NAD+ (nicotinamide) with various nucleophiles. Both human CD38 (either recombinant or purified from erythrocyte membranes) and Aplysia cyclase were found to catalyze the exchange of ADPR with the nicotinamide group of NAD+ leading to the formation of a dimeric ADPR ((ADPR)2). The dimeric structure of the enzymatic product, which was generated by recombinant CD38 and by CD38(+) Namalwa cells from as low as 10 microM NAD+, was demonstrated using specific enzyme treatments (dinucleotide pyrophosphatase and 5'-nucleotidase) and mass spectrometry analyses of the resulting products. The linkage between the two ADPR units of (ADPR)2 was identified as that between the N1 of the adenine nucleus of one ADPR unit and the anomeric carbon of the terminal ribose of the second ADPR molecule by enzymatic analyses and by comparison with patterns of cADPR cleavage with Me2SO:tert-butoxide. Although (ADPR)2 itself did not release Ca2+ from sea urchin egg microsomal vesicles, it specifically potentiated the Ca2+-releasing activity of subthreshold concentrations of cADPR. Therefore, (ADPR)2 is a new product of CD38 that amplifies the Ca2+-mobilizing activity of cADPR.     

Instrumental non invasive diagnosis of arterial circulation disorders of the extremities

1 In citrated platelet-rich plasma, freshly prepared from rabbit blood, the velocity of platelet aggregation was within limits proportional to the log of the concentration of added adenosine diphosphate (ADP). 2 Addition of either adenosine triphosphate (ATP) or its beta,y-methylene analogue inhibited aggregation similarly except that the analogue was about half as potent as ATP. beta,y-Methylene ATP also reversed the optical effects associated with the shape change of platelets very similarly to ATP itself. 3 As beta,y-methylene ATP is not a substrate for nucleoside diphosphokinase, these observations do not support the proposition that inhibition of aggregation by added ATP is due to its utilization by the nucleoside diphosphokinase of platelets.     

Catabolism of Ap4A and Ap5A by rat brain synaptosomes

Adenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) and adenosine 5',5"'-P1,P5-pentaphosphate (Ap5A) are stored in and released from rat brain synaptic terminals. In the present study we investigated the hydrolysis of dinucleotides (Ap4A and Ap5A) in synaptosomes from the cerebral cortex of adult rats. Ap4A and Ap5A, but not Ap3A, were hydrolyzed at pH 7.5 in the presence of 20 mM Tris/HCl, 2.0 mM MgCl2, 10 mM glucose and 225 mM sucrose at 37 degrees C. The disappearance of the substrates measured by FPLC on a mono-Q HR column was both time and protein dependent. Since synaptosome integrity was at least 90% at the end of the assay, hydrolysis probably occurred by the action of an ecto-enzyme. Extracellular actions of adenine dinucleotides at central nervous system terminate due to the existence of ecto-nucleotidases which specifically cleave these dinucleotides. These enzymes in association with an ATP diphosphohydrolase and a 5'-nucleotidase are able to promote the complete hydrolysis of dinucleotides to adenosine in the synaptic cleft.     

Effect of amino acids on purified rat intestinal brush-border membrane aminooligopeptidase

When a hexapeptide, Leu-Trp-Met-Arg-Phe-Ala, or a pentoapeptide, Leu-Trp-Met-Arg-Phe, was incubated in vitro with a purified aminooligopeptidase from rat small intestinal mucosa, the respective C-terminal dipeptides, Phe-Ala and Arg-Phe, were observed to be resistant to hydrolysis. The resistance of these C-terminal dipeptides to hydrolysis was found to be due mainly to the accumulation of inhibitory hydrophobic amino acids liberated in the incubation mixture. The hydrolysis of various peptides by the brush-border membrane peptidase is inhibited to a varying extent by the hydrophobic amino acids L-tryptophan, L-methionine, L-isoleucine, L-leucine, L-tyrosine, and L-phenylalanine, but not the D-form of these amino acids. The inhibition of the hydrolysis of three dipeptides by hydrophobic amino acids showed these amino acids to be competitive inhibitors (same Vmax, the maximal velocity of the enzyme reaction; different Km, the substrate concentration at which the enzyme reaction is half maximal) of one of the dipeptides while exhibiting a mode of inhibition that was not competitive (different Vmax, different Km) with either of the other two dipeptides. These data indicate that the effect of amino acids on the hydrolytic rate of the brush-border membrane aminooligopeptidases must be considered in studies of intestinal hydrolysis and absorption of peptides.