Influence of ring- and side-chain substituents on the transamination of aromatic amino acids by two multispecific aspartate-aromatic aminotransferase isozymes purified from bushbean shoots

The breadth of substrate specificity shown by the multispecific aspartate-aromatic aminotransferase of bushbean (Phaseolus vulgaris) has been investigated by testing the ability of two cytosolic isozymes (I and II), purified from shoot tissue, to catalyse transamination reactions between a range of ring- and sidechain-substituted aromatic amino acids and 2-oxoglutarate. Ring-substituted phenylalanines were the most reactive substrates whereas ring-substitution in tyrosine or tryptophan resulted in transamination rates lower than those observed with the parent amino acids. All side chain-substituted analogues were found to be totally inactive. The highest activity shown by any ring-substituted phenylalanine was observed with the 4-amino- compound, followed closely by the 4-hydroxy- and 4-halogen-compounds. In contrast, 4-nitrophenylalanine was completely inactive. These trends were consistent for both isozymes I and II, but only isozyme II showed greatly enhanced activity over that found with the parent amino acid when certain ring-substituted analogues were tested. The varying capacity of the bushbean isozymes to utilize the present range of substituted amino acids is compared with previous reports on the substrate specificity shown by aspartate and aromatic aminotransferases isolated from mammalian and microbial systems. A model for the mechanism of activation observed with bushbean isozyme II in the presence of certain 4-substituted aromatic amino acids is proposed, based on current understanding of the nature of the active site of animal aspartate aminotransferases.     

Functional significance of aromatic amino acid: aromatic keto acid aminotransferases in rat brain and liver: competition for tryptophan between aminotransferases and tryptophan hydroxylase in vitro.

Using low concentrations of substrates and cofactors, a comparison was made of the relative rates by which aminotransferases catalysed transaminations between aromatic amino acids and aromatic or aliphatic keto acids. Tryptophan aminotransferase in homogenates of rat midbrain and liver transaminated phenylpyruvate at a rate 70 to 150-fold greater than the rate with α-ketoglutarate at low concentrations of substrates. Phenylalanine aminotransferase in liver and midbrain also was more active with aromatic keto acids than with aliphatic keto acids. However, tyrosine aminotransferase in dialysed homogenates of midbrain transaminated α-ketoglutarate and phenylpyruvate at approximately equal rates. Fresh homogenates of midbrain contained an inhibitor which markedly decreased tyrosine aminotransferase activity with α-ketoglutarate but not with phenylpyruvate. Tyrosine aminotransferase in homogenates of rat liver transaminated α-ketoglutarate and phenylpyruvate at equal rates below 10 μM keto acid, but above 10 μM, transamination of α-ketoglutarate was favoured. With homogenates of liver, transamination of α-ketoglutarate, but not phenylpyruvate, by tyrosine was increased 650% by exogenous pyridoxal phosphate. Since tryptophan aminotransferase in the brain may compete with tryptophan hydroxylase for available tryptophan, a comparison was made of the relative activities of tryptophan hydroxylase and tryptophan aminotransferase. At concentrations above 7.5 μM phenylpyruvate, transamination was 8 to 17-fold greater than the rate of hydroxylation of 50 μM tryptophan.     

Metabolism of 2-oxoacid analogues of leucine,valine and phenylalanine by heart muscle,brain and kidney of the rat

[1-¹⁴C]-Labelled 2-oxoacid analogues of leucine, valine and phenylalanine were used to study the metabolism of these 2-oxoacids in the brain, kidney and heart muscle of rats. By following the ¹⁴CO₂ release during 30–60 min of incubation at 37°C the decarboxylation rate was determined and measurement of the ¹⁴C-incorporation into the corresponding amino acid yielded the transamination rate. From these rates, decarboxylation/transamination ratios could be calculated which are indicative for the metabolic fate of the 2-oxoacid in the various organs. The results obtained show that all three tissues are capable of utilizing the 2-oxoacid analogues of leucine, valine and phenylalanine, however, to a different extent: kidney > heart muscle > brain. The decarboxylation/transamination ratios reveal that the branched-chain 2-oxoacids are predominantly decarboxylated in kidney and heart muscle while in brain they are mainly transaminated. The ratios calculated for phenylpyruvate in all tissues are within 0.19 and 0.36, indicating that this 2-oxoacid is preferentially transaminated. The results are discussed with respect to possible dietary alterations of enzymes involved in 2-oxoacid metabolism in order to improve transamination of these compounds.     

Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L.

A comparative study is presented of the activities of enzymes of glycine and serine metabolism in leaves, germinated cotyledons and root apices of pea (Pisum sativum L.). Data are given for aminotransferase activities with glyoxylate, hydroxypyruvate and pyruvate, for enzymes associated with serine synthesis from 3-phosphoglycerate and for glycine decarboxylase and serine hydroxymethyltransferase. Aminotransferase activities differ between the tissues in that, firstly, appreciable transamination of serine, hydroxypyruvate and asparagine occurs only in leaf extracts and, secondly, glyoxylate is transaminated more actively than pyruvate in leaf extracts, whereas the converse is true of extracts of cotyledons and root apices. Alanine is the most active amino-group donor to both glyoxylate and hydroxypyruvate. 3-Phosphoglycerate dehydrogenase and glutamate: O-phosphohydroxypyruvate aminotransferase have comparable activities in all three tissues, except germinated cotyledons, in which the aminotransferase appears to be undetectable. Glycollate oxidase is virtually undetectable in the non-photosynthetic tissues and in these tissues the activity of glycerate dehydrogenase is much lower than that of 3-phosphoglycerate dehydrogenase. Glycine decarboxylase activity in leaves, measured in the presence of oxaloacetate, is equal to about 30–40% of the measured rate of CO₂ fixation and is therefore adequate to account for the expected rate of photorespiration. The activity of glycine decarboxylase in the non-photosynthetic tissues is calculated to be about 2–5% of the activity in leaves and has the characteristics of a pyridoxal-and tetrahydrofolate-dependent mitochondrial reaction; it is stimulated by oxaloacetate, although not by ADP. In leaves, the measured activity of serine hydroxymethyltransferase is somewhat lower than that of glycine decarboxylase, whereas in root apices it is substantially higher. Differential centrifugation of extracts of root apices suggests that an appreciable proportion of serine hydroxymethyltransferase activity is associated with the plastids.Abbreviation GOGAT l-Glutamine:2-oxoglutarate aminotransferase     

Characterization of two multispecific aspartate aminotransferase isozymes purified from bushbean shoots capable of transaminating aromatic amino acids and severalDL-chloro-phenylalanines

Two electrophoretically distinct isozymes ofL-phenylalanine aminotransferase (Enz I, Enz II) purified from a total soluble shoot extract of bushbean have been characterized. The M_rs of Enz I and Enz II were 100 000 and 110 000, respectively. Both isozymes showed pH optima of 8.5. Enz I was able to use either 2-oxoglutarate (2-OG) or oxaloacetate (OAA) equally as a keto acid substrate whenL-phenylalanine was the amino donor, while Enz II preferred 2-OG. Neither isozyme was able to use glyoxylate or pyruvate in the presence ofL-phenylalanine. When tested with a range of protein amino acids, both Enz I and Enz II showed the highest rate of transamination withL-aspartate, indicating that both isozymes wereL-aspartate aminotransferases capable of also showingL-aromatic aminotransferase activity.L-Phenylalanine aminotransferase activity relative toL-aspartate aminotransferase activity was found to be 0.6 % for Enz I and 3.3% for Enz II. Lineweaver-Burk plots of kinetic data gave apparent K_m values (mM) for Enz I of 2.3 (L-Asp), 55.0 (L-Phe) and 9.0 (2-OG) and for Enz II, 2.8 (L-Asp), 320.0 (L-Phe) and 8.2 (2-OG). The values were confirmed by treatment of the data by Hill plots. When tested with a series of 12 ring-substitutedDL-chlorophenylalanines, Enz I was active only with the 3-chloro- and 4-chloro-compounds, while Enz II was active with all three monochloro-compounds as well as with the 2,4-, 2,6- and 3,4-dichlorophenylalanines. The activity of Enz II with 4-chlorophenylalanine was very high, 222 % higher than that observed withDL-phenylalanine. Enz I was completely inhibited by 1.0 mM Ca²⁺ while Enz II was unaffected by this cation, which suggested different subcellular locations for each isozyme. Cell fractionation studies indicated, however, that both Enz I and Enz II were cytoplasmic. Different isozymes of this multispecific aspartate—aromatic aminotransferase were found in the chloroplasts and mitochondria of bushbean shoots.     

The biochemical basis for growth inhibition by L-phenylalanine in Neisseria gonorrhoeae

Clinical isolates of Neisseria gonorrhoeae are commonly subject to growth inhibition by phenylpyruvate or by L-phenylalanine. A blockade of tyrosine biosynthesis is indicated since inhibition is reversed by either L-tyrosine or 4-hydroxyphenylpyruvate. Phenylalanine-resistant (PheR) and phenylalanine-sensitive (PheS) isolates both have a single 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase that is partially inhibited by L-phenylalanine (80%). However, PheS and PheR isolates differ in that the ratio of phenylpyruvate aminotransferase to 4-hydroxyphenylpyruvate aminotransferase is distinctly greater in PheS isolates than in PheR isolates. A mechanism for growth inhibition is proposed in which phenylalanine exerts two interactive effects. (i) Phenylalanine decreases precursor flow to 4-hydroxyphenylpyruvate through its controlling effect upon DAHP synthase; and (ii) phenylalanine is largely transaminated to phenylpyruvate, which saturates both aminotransferases, preventing transamination of an already limited supply of 4-hydroxyphenylpyruvate to L-tyrosine.     

TRANSAMINATIONS BETWEEN AMINO ACIDS AND KETO ACIDS ELEVATED IN PHENYLKETONURIA AND MAPLE SYRUP URINE DISEASE

Abstract— The transamination between amino acids and aliphatic and aromatic keto acids has been investigated in homogenates of human and rat brain. Tryptophan, phenylalanine and 3,4-dihydroxyphenylalanine (DOPA) at concentrations of 3.6 min and below trans-aminated aromatic keto acids more rapidly than α-ketoglutarate; lower K_m values were found for tryptophan and phenylalanine in the presence of the aromatic keto acid. Rat brain and liver arninotransferases exhibited similar affinities for tryptophan in the presence of different keto acids. Branched chain keto acids were also acceptors of the amino groups of tryptophan and DOPA. In brain homogenates α-ketoglutarate and p -hydroxyphenyl-pyruvate were transaminated by tyrosine and 5-hydroxytryptophan at about equal rates, whereas a-ketoglutarate was transaminated more rapidly with aliphatic amino acids. At concentrations of 1.6 m DOPA and 0.8 mM p -hydroxyphenylpyruvate, transamination was 6-fold greater than the rate of formation of dopamine. The dihydroxyphenylpyruvate formed during arninotransfer from DOPA by brain tissue was not readily decarboxylated, whereas 65–70 per cent of the indolepyruvate formed from tryptophan was decarboxylated. We suggest that an increased rate or degree of transamination between tryptophan and aromatic and branched chain keto acids may explain the increased excretion of non-hydroxylated indolic acids in phenylketonuria and'maple syrup urine'disease, respectively. Increased aminotransfers from tryptophan and DOPA may reduce the amounts of precursors available for the synthesis of serotonin and catecholamines, both of which are at low levels in the sera of untreated phenylketonurics.     

Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis

In all organisms synthesising phenylalanine and/or tyrosine via arogenate, a prephenate aminotransferase is required for the transamination of prephenate into arogenate. The identity of the gene encoding this enzyme in the organisms where this activity occurs is still unknown. Glutamate/aspartate-prephenate aminotransferase (PAT) is thus the last homeless enzyme in the aromatic amino acids pathway. We report on the purification, mass spectrometry identification and biochemical characterization of Arabidopsis thaliana prephenate aminotransferase. Our data revealed that this activity is housed by the prokaryotic-type plastidic aspartate aminotransferase (At2g22250). This represents the first identification of a gene encoding PAT.     

Aminotransferases in the bivalve mollusc Mytilus edulis L. and short term effects of crude oil in brackish water

Transaminases are among the crucial enzymes in amino acid metabolism, which in aquatic organisms is known to be affected by exposure to oil hydrocarbons. The transamination reactions in Mytilus edulis L. were studied to estimate their adequacy to indicate short term oil exposure in mussels. The transamination reactions were measured using paper chromatography and spectrophotometry. A high degree of transamination was observed between 2 oxoglutarate and alanine, aspartate and ornithine. A slight degree of transamination was shown with methionine, leucine, isoleucine, phenylalanine, serine, tryptophan, threonine, tyrosine and valine. No transamination was observed between 2 oxoglutarate and glycine, arginine, histidine, lysine, proline, citrulline and alanine. The effect of the water accommodated fraction WAF of crude oil on selected transaminase reactions was measured. The highest changes during the WAF exposure were mostly observed in the gills and mantle. Alanine aminotransferase EC 2.6.1.2 activity in the mantle was, at its highest, 55 over the control. Aspartate aminotransferase EC 2.6.1.1 activity increased in the gills by 52 . For ornithine transamination, in the gills the highest increase was by 75 and in the mantle by 50 . The metabolic pathways involved in the alterations of aminotransferase activities are discussed. It is concluded that ornithine transamination in gills is a potential indicator for short term crude oil exposure in Mytilus edulis. More studies are needed to evaluate the effects of other organic pollutants on ornithine transamination.     

Chemical synthesis of 4,5-dioxovaleric acid and its nonenzymatic transamination to 5-aminolevulinic acid

4,5-Dioxovaleric acid (DOVA) was synthesized from 5-bromolevulinic acid via formation of the pyridinium bromide of 5-bromolevulinic acid, followed by nitrone formation with p-nitrosodimethylaniline, and hydrolysis of the nitrone to yield DOVA. Partial purification of DOVA was obtained by passage of the reaction mixture through a cation exchange column. DOVA was identified by paper electrophoresis and by a specific fluorometric assay. DOVA was nonenzymatically transaminated to 5-aminolevulinic acid (ALA) with glycine serving as the amino donor. Other compounds tested were less effective amino donors. Glyoxylic acid was identified as a reaction product by paper electrophoresis and a specific calorimetric test. ALA was identified by paper electrophoresis, paper chromatography of a pyrrole derivative, reaction with Ehrlich reagent, and by its enzymatic conversion by a barley extract to porphobilinogen and uroporphyrin. The nonenzymatic transamination was inhibited by Tris and was stimulated by high pH. The existence of this nonenzymatic activity is discussed in relation to previous reports of dova transaminase activity in cell extracts.     

Two mechanisms produce tissue-specific inhibition of fatty acid oxidation by oxfenicine.

Oxfenicine [S-2-(4-hydroxyphenyl)glycine] is transaminated in heart and liver to 4-hydroxyphenylglyoxylate, an inhibitor of fatty acid oxidation shown in this study to act at the level of carnitine palmitoyltransferase I (EC 2.3.1.21). Oxfenicine was an effective inhibitor of fatty acid oxidation in heart, but not in liver. Tissue specificity of oxfenicine inhibition of fatty acid oxidation was due to greater oxfenicine transaminase activity in heart and to greater sensitivity of heart carnitine palmitoyltransferase I to inhibition by 4-hydroxyphenylglyoxylate [I50 (concentration giving 50% inhibition) of 11 and 510 microM for the enzymes of heart and liver mitochondria, respectively]. Branched-chain-amino-acid aminotransferase (isoenzyme I, EC 2.6.1.42) was responsible for the transamination of oxfenicine in heart. A positive correlation was found between the capacity of various tissues to transaminate oxfenicine and the known content of branched-chain-amino-acid aminotransferase in these tissues. Out of three observed liver oxfenicine aminotransferase activities, one may correspond to asparagine aminotransferase, but the major activity could not be identified by partial purification and characterization. As reported previously for malonyl-CoA inhibition of carnitine palmitoyltransferase I, 4-hydroxyphenylglyoxylate inhibition of this enzyme was found to be very pH-dependent. In striking contrast with the kinetics of malonyl-CoA inhibition, 4-hydroxyphenylglyoxylate inhibition was not affected by oleoyl-CoA concentration, but was partially reversed by increasing carnitine concentrations.     

Diamine Transamination Catalyzed by ω-Amino Acid : Pyruvate Aminotransferase of Pseudomonas sp. F–126

ω-Amino acid: pyruvate aminotransferase of Pseudomonas sp. F–126 catalyzes a transamination between various diamines and pyruvate, an exclusive amino acceptor. Based on a stoichiometric studies it was shown that one of the two amino groups of 1,2-diaminoethane, putrescine and cadaverine transaminated to pyruvate. The transamination between putrescine and pyruvate seemed to proceed by a ping-pong bi bi mechanism. Michaelis constants for putrescine and pyruvate were calculated to be 76.9 and 6.25 mm, respectively.     

Synthesis of chiral arogenic acid via immobilized microbial proteins

Although l-(8S)-arogenate has been recognized as a potential precursor of l-phenylalanine or l-tyrosine biosynthesis for only a few years, it is widely distributed in nature. The biochemical formation of arogenate has involved its isolation from the culture supernatant of a mutant strain of Neurospora crassa, a lengthy procedure of 20-day duration. We now report an improved approach using immobilized crude enzyme extracts from a cyanobacterium. The starting materials, chorismic acid or prephenic acid, are readily available, and overall yields ranging from 40 to 60% are obtained. The whole procedure takes only 1 day. Crude, unfractionated enzyme extracts from Synechocystis sp. ATCC 29108 are immobilized on a phenoxyacetyl cellulose solid support. The hydrophobic binding of the extract proteins did not denature chorismate mutase or prephenate aminotransferase, the enzymes catalyzing the conversion of chorismate to prephenate and prephenate to arogenate, respectively. This microbial system was ideally suited for preparation of arogenate, since other enzyme activities which might compete for prephenate or chorismate as substrates, or which might further metabolize arogenate, were absent or inactive under the conditions used. In addition to the substrates prephenate or chorismate, pyridoxal-5′-phosphate (the coenzyme required for transamination), as well as leucine (amino donor for transamination of prephenate), was added. The reaction product, arogenate, was separated from the starting materials by preparative thin-layer chromatography.     

Aminotransferases in the bivalve mollusc Mytilus edulis L. and short term effects of crude oil in brackish water

Transaminases are among the crucial enzymes in amino acid metabolism, which in aquatic organisms is known to be affected by exposure to oil hydrocarbons. The transamination reactions in Mytilus edulis L. were studied to estimate their adequacy to indicate short term oil exposure in mussels. The transamination reactions were measured using paper chromatography and spectrophotometry. A high degree of transamination was observed between 2 oxoglutarate and alanine, aspartate and ornithine. A slight degree of transamination was shown with methionine, leucine, isoleucine, phenylalanine, serine, tryptophan, threonine, tyrosine and valine. No transamination was observed between 2 oxoglutarate and glycine, arginine, histidine, lysine, proline, citrulline and alanine. The effect of the water accommodated fraction WAF of crude oil on selected transaminase reactions was measured. The highest changes during the WAF exposure were mostly observed in the gills and mantle. Alanine aminotransferase EC 2.6.1.2 activity in the mantle was, at its highest, 55 over the control. Aspartate aminotransferase EC 2.6.1.1 activity increased in the gills by 52 . For ornithine transamination, in the gills the highest increase was by 75 and in the mantle by 50 . The metabolic pathways involved in the alterations of aminotransferase activities are discussed. It is concluded that ornithine transamination in gills is a potential indicator for short term crude oil exposure in Mytilus edulis. More studies are needed to evaluate the effects of other organic pollutants on ornithine transamination.     

DL-4-hydroxyphenylglycine catabolism in Pseudomonas putida MW27

Thirteen bacteria were isolated on D-4-hydroxyphenylglycine as sole carbon and energy source. Seven strains transaminated only the D-enantiomer while the other six isolates transaminated both enantiomers of 4-hydroxyphenylglycine. One of the six strains utilizing both enantiomers was characterized as a Pseudomonas putida. This strain, MW27, employed two enantioselective transaminases, to catalyze the initial step in the metabolism of DL-4-hydroxyphenylglycine. The product of the transamination, 4-hydroxyphenylglyoxylate, was further metabolized via 4-hydroxybenzaldehyde and 4-hydroxybenzoate to protocatechuate. Preliminary results indicate that both transaminases are co-ordinately synthesized together with the 4-hydroxyphenylglyoxylate decarboxylase and the NADP⁺-dependent 4-hydroxybenzaldehyde dehydrogenase.     

Transamination of neutral amino acids and 2-keto acids in pancreatic B-cell mitochondria

High aminotransferase activities catalyzing the reactions between L-glutamate and L-glutamine and the aliphatic ketomonocarboxylic acids 2-ketoisocaproate, 2-ketocaproate, and 2-ketoisovalerate were observed in pancreatic B-cell mitochondria. While maximal rates of transamination with L-glutamate were observed in the presence of micromolar concentrations of keto acid, maximal rates of transamination with L-glutamine were recorded only in the presence of millimolar concentrations of keto acid. The insulin secretagogue 2-ketoisocaproate was the most effective transamination partner for L-glutamate, while the insulin secretagogue 2-ketocaproate was the most effective transamination partner for L-glutamine. Since B-cell mitochondria are well supplied with L-glutamate and L-glutamine, 2-ketoglutarate generation in the presence of these two neutral 2-keto acids may be an important prerequisite for their insulin secretory potency. High rates of transamination of 2-ketoglutarate were observed in the pancreatic B-cell mitochondria with the branched-chain amino acids L-leucine and L-valine, but not with L-norleucine. In connection with the ability of L-leucine to activate glutamate dehydrogenase, this high activity of the branched-chain amino acid aminotransferase in pancreatic B-cell mitochondria may provide an explanation for the insulin secretory potency of this amino acid.     

Transamination and oxidation of leucine and valine in rat adipose tissue

Leucine was oxidized by rat adipose tissue at a rate which was not limited by the activity of branched chain amino acid transaminase since high concentrations (10 mM) of [1-14C]leucine and its transamination product, alpha-keto[1-14C]isocaproate, were oxidized at similar rates. Despite the apparent abundance of transaminase activity, however, [1-14C]valine was oxidized at only 10 to 25% of the rate of its transamination product, alpha-keto[1-14C]isovalerate. The net rate at which [1-14C] valine was transaminated by intact tissues was estimated as the sum of the rates of 14CO2 production and alpha-ketoiso[1-14C]valerate release into the medium. Transamination did not limit the rate of valine oxidation since valine was transaminated 3 times as fast as it was oxidized. The rate of valine transamination increased 18-fold when its concentration was raised 100-fold, but the fraction of [1-14C]valine oxidized to 14CO2 remained constant over the range of incubation conditions studied. The oxidation/transamination ratio for leucine was also constant and exceeded the oxidation/transamination ratio for valine unless valine oxidation was stimulated, either by the addition of glucose or leucine. Stimulation of valine oxidation did not increase its transamination but reduced the rate at which alpha-ketoisovalerate was released from the tissue. The faster oxidation of alpha-ketoisocaproate than of alpha-ketoisovalerate may be due to the activation of branched chain alpha-keto acid dehydrogenase by alpha-ketoisocaproate, but the alpha-keto acid oxidation rates do not fully account for the faster transamination of leucine than of valine.     

Catabolism of nutritionally essential amino acids in developing porcine enterocytes

This study was conducted using the piglet model to test the hypothesis that mucosal cells of the neonatal small intestine can degrade nutritionally essential amino acids (EAA). Enterocytes were isolated from the jejunum of 0-, 7-, 14-, and 21-day-old pigs, and incubated for 45 min in Krebs buffer containing plasma concentrations of amino acids and one of the following L-[1-¹⁴C]- or L-[U-¹⁴C]-amino acids plus unlabeled tracees at 0.5, 2, or 5 mM: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. In these cells, branched-chain amino acids (BCAA) were extensively transaminated and 15–50% of decarboxylated branched-chain α-ketoacids (BCKA) were oxidized to CO₂ depending on the age of piglets. BCAA transamination increased but their decarboxylation decreased between 0 and 14 days of age. Addition of 1 and 2 mM α-ketoglutarate to incubation medium dose-dependently stimulated BCAA transamination without affecting their decarboxylation. Western blot analysis revealed that the abundance of mitochondrial BCAA aminotransferase declined but cytosolic BCAA aminotransferase increased between 0 and 14 days of age, with the cytosolic protein being the major isoform in 7- to 21-day-old pigs. BCKA dehydrogenase protein existed primarily as the phosphorylated (inactive) form in enterocytes of newborn pigs and its levels were markedly reduced in older pigs. All measured parameters of BCAA metabolism did not differ between 14- and 21-day-old pigs. In contrast to BCAA, catabolism of methionine and phenylalanine was negligible and that of other EAA was absent in enterocytes from all ages of piglets due to the lack of key enzymes. These results indicate that enterocytes are an important site for substantial degradation of BCAA but not other EAA in the neonatal gut.     

Some Enzymic Activities in the Germinating Oil Palm (Elaeis guineensis) Seedling

In germinating oil palm (Elaeis guineensis var D × P) seedling, an active lipase was present in the shoot but absent from both the kernel and the haustorium. It has an optimum pH of 6.2 and a smaller peak at pH 8.6. The shoot lipase was active against a number of mono-, di-, and triacylglycerols as well as the endogenous lipids present in the shoot, haustorium, and kernel. Activity against related substrates were in the order: trilaurin > dilaurin > monolaurin but monopalmitin > dipalmitin > tripalmitin. The level of the enzyme in the seedling was highest at a relatively early stage of growth (18-21 days) and also higher in dark-grown seedlings. Glyoxylate bypass enzymes (malate synthetase and isocitrate lyase), glutamate-oxaloacetate transaminase, phosphoenolpyruvate carboxykinase and lauroyl-coenzyme A oxidase were located in the haustorium. The levels of the enzymes paralleled seedling development and were slightly higher in light-grown seedlings. Fatty acyl-coenzyme A synthetase activity was very low and was found in both the shoot and haustorium.     

Author index for volume 171

Kinetic analyses of the irreversible inhibition of l-tyrosine and l-phenylalanine transport in Bacillus subtilis by phenylalanine chloromethyl ketone revealed that the inhibition was due to an affinity labeling process. Phenylalanine chloromethyl ketone is a competetive inhibitor of l-tyrosine and l-phenylalanine transport. The K_i values for irreversible inhibition of l-tyrosine and l-phenylalanine transport were 194 and 177 μm, respectively, and the first order rate constants for the alkylation reaction leading to inactivation of transport of l-tyrosine and l-phenylalanine were 0.016 and 0.012 min^?1, respectively. The similarity of these constants are consistent with the involvement of the same functional site for l-phenylalanine and l-tyrosine transport. A second effect of phenylalanine chloromethyl ketone was inhibition of the uptake of neutral, aliphatic amino acids; transport of basic and acidic amino acids was unaffected by it. Since high concentrations of any amino acid did not reduce the inhibitory effects of phenylalanine chloromethyl ketone on transport of neutral, aliphatic amino acids, an independent effect, not due to an affinity labeling process was inferred. A procedure for selective labeling of the l-tyrosine/l-phenylalanine transport system was demonstrated that should be applicable to the introduction of a radioactive label into the transport protein(s).