Metabolism of aminoacyl-p-nitroanilides by rat mammary tissue.

We have examined the metabolism of aminoacyl-p-nitroanilides by rat mammary tissue isolated from rats during late pregnancy, peak lactation and late lactation. The rate of hydrolysis depended upon the chemical nature of the aminoacyl-p-nitroanilide compound and the physiological state of the donor animals. Thus, mammary tissue isolated from rats during late pregnancy and peak lactation hydrolysed aminoacyl-p-nitroanilides in the order L-met-p-nitroanilide=L-leu-p-nitroanilide>L-lys-p-nitroanilide>gamma- glu-p-nitroanilide. The order of activity was the same for mammary tissue taken from rats during late lactation except that L-lys-p-nitroanilide was hydrolysed at the same rate as the neutral aminoacyl-p-nitroanilides. Mammary tissue from peak lactating rats also hydrolysed alpha-L-glu-p-nitroanilide and alpha-L-asp-p-nitroanilide but to a lesser extent than the other compounds tested. The anionic aminoacyl-p-nitroanilides were able to trans-stimulate D-aspartate efflux from mammary tissue explants and the perfused mammary gland via the high-affinity anionic amino acid carrier. The clearance of gly-L-phe by the perfused mammary gland was markedly inhibited by L-phe. The results suggest that mammary tissue expresses a variety of dipeptidases at the basolateral aspect of the mammary epithelium which are capable of hydrolysing peptides extracellularly. These enzymes may be important for providing amino acids for milk protein synthesis and/or inactivating signal peptides.

Vascular sources of phenylalanine, tyrosine, lysine, and methionine for casein synthesis in lactating goats.

The contribution to casein biosynthesis of peptides derived from blood was examined in late lactation goats (254 to 295 d in milk). Ratios of mammary uptake of free amino acids (AA) in blood to output of AA in milk protein and ratios of the enrichments of Phe, Tyr, Met, and Lys at isotopic plateau in secreted milk casein to the free AA in arterial and mammary vein blood were monitored during the last 5 h of a 30-h continuous i.v. infusion of [1-13C]Phe, [2H4]Tyr, [5-13CH3]Met, and [2-15N]Lys on two occasions: before (control) and on d 6 of an i.v. infusion of Phe (6 g/d). During the control, uptakes of free Phe and Met were less than their output in milk. This result was comparable with the labeling kinetic results, suggesting that vascular peptides contributed 5 to 11% of Phe and 8 to 18% of Met. Free Tyr and Lys uptakes during the control were sufficient for milk output; however, the labeling kinetics indicated that 13 to 25% of the Tyr and 4 to 13% of the Lys were derived from peptides. Infusion of Phe increased the uptake of free AA but reduced the contribution of peptides toward Phe (0 to 3%) and Tyr (8 to 14%) supply for casein synthesis. Whole body hydroxylation of Phe to Tyr increased from 10 to 18% with the infusion of Phe; within the mammary gland, this conversion was lower (3 to 5%). Results suggest that the mammary utilization of peptides containing Phe and Tyr appears to depend on the supply of free AA in blood.

Current concepts of amino acid and protein metabolism in the mammary gland of the lactating ruminant.

Milk protein responses to protein nutrition are typically poor and, in part, may be due to the low efficiency (approximately 25 to 30%) of converting dietary N into milk. Posthepatic availability of amino acids (AA) is not limited, yet only approximately 30% is converted into milk. The poor capture of AA by the mammary gland may relate to the imbalanced and uncoordinated timing of nutrient delivery to the gland. The infusion of essential AA improves the efficiency of utilization (0.31); however, further catabolism of AA within the mammary gland suggests that AA transport is not a major limitation. These losses may serve ancillary or functional roles, but mammary oxidation of some AA occurs only when AA extraction exceeds the stoichiometric requirements for milk protein synthesis. Intracellular substrate supply may be more limiting than is the appartus for protein synthesis. Studies utilizing isotope labeling and conducted in vitro and in vivo now suggest that circulating peptides and proteins can serve as sources of perhaps all AA for casein synthesis, but the source of these remains elusive. Constitutive protein and casein turnover contribute significantly (42 to 72%) to mammary protein synthesis. All AA are extensively channeled through an intermediary protein pool or pools that have rapid turnover rates. The AA are then incorporated into casein, which appears to be fixed in association with protein turnover. The mammary gland is a major controller of its metabolism, and the mechanisms of AA extraction and conversion into milk protein are linked to secretion events. Blood flow may be a key point of regulation whereby mechanisms sense and respond to nutrient supply and balance to the gland via alterations in hemodynamics.

Peptide aminonitrogen transport by the lactating rat mammary gland.

Recent studies have shown that the lactating mammary gland is able to utilize plasma-derived dipeptides for milk protein synthesis. However, it was not clear whether the peptides were hydrolysed followed by uptake of the constituent amino acids or were taken up intact. In view of this, we have designed experiments to investigate (a) whether the lactating rat mammary gland is capable of transporting hydrolysis-resistant dipeptides and (b) whether or not mammary cells are able to hydrolyse peptides, including glutathione, extracellularly. The uptake of the hydrolysis-resistant dipeptides D-[3H]Phe-L-Gln and D-[3H]Phe-L-Glu by the perfused rat mammary gland was low. Concomitant addition of L-Leu-L-Ala (50 mM) had no effect on the clearance of either labelled dipeptide suggesting that the small, albeit significant, uptake of the dipeptides is not via a high affinity peptide transporter (PepT1/PepT2). All anionic dipeptides tested (L-Glu-L-Ala, L-Asp-L-Ala, L-Ala-L-Asp, L-Asp-Gly, Gly-L-Asp and Gly-L-Glu) with the exception of D-Phe-L-Glu were able to trans-accelerate the efflux of labelled D-aspartate from preloaded rat mammary tissue (explants and perfused mammary gland). It appears that these peptides were being hydrolysed extracellularly followed by the uptake of free anionic amino acids via the mammary tissue high affinity, Na+-dependent anionic amino acid carrier operating in the exchange mode. Glutathione was able to trans-accelerate D-aspartate efflux from lactating rat mammary tissue in a fashion which was sensitive to the peptidase inhibitor acivicin. This suggests that gamma-glutamyltranspeptidase hydrolyses glutathione to produce L-glutamate which is subsequently transported via the high-affinity anionic amino acid carrier. Hydrolysis of peptides followed by uptake of the constituent amino acids may provide an important source of amino acids for milk protein synthesis.

Application of a U-13C-labeled amino acid tracer in lactating dairy goats for simultaneous measurements of the flux of amino acids in plasma and the partition of amino acids to the mammary gland.

A preliminary study was conducted using lactating British Saanen goats (n = 5) at 109 to 213 d in milk that yielded 1.67 to 3.68 kg of milk/d to examine the application of a U-13C-labeled amino acid (AA) mixture obtained from hydrolyzed algal proteins as a tracer for measuring plasma flux (n = 5) and partition to the mammary gland (n = 3; arteriovenous difference) of 13 AA simultaneously. Except for Ile and Ser, there was incomplete (6 to 54%) equilibration of the tracer with AA from packed blood cells (> 90% erythrocytes) during the 6-h infusions. This result agreed with the large ratio of packed cells to gradients for plasma AA concentration that was also observed. However, net mass and isotope removals by the mammary gland were predominantly from plasma, indicating that the erythrocytes did not participate in kinetic exchanges. Plasma AA fluxes (millimoles per kilogram of metabolizable protein intake per kilogram of body weight 0.75) differed among goats that consumed different protein sources; however, overall rates were lowest for Met (5 to 14) and His (8 to 17) and highest for Leu (48 to 70) and Ala (53 to 88). On average, 25% of plasma flux was partitioned to the mammary gland. Less than 20% of His, Ser, Phe, and Ala were directed to the mammary gland; 20 to 30% of Arg, Thr, Tyr, and Leu were directed to the mammary gland; and 30 to 40% of Pro, Ile, Lys, and Val were directed to the mammary gland. The unidirectional AA flux in the mammary gland (AA apparently available for protein syntheses, oxidation, and metabolite formation) did not match the pattern that is required for casein synthesis, suggesting differences in the metabolic requirements of AA for nonmilk protein synthesis.

Quantification of circulating peptides and assessment of peptide uptake across the gastrointestinal tract of sheep.

Gastrointestinal absorption of peptides was examined in sheep fed a forage-based diet. Peptide concentrations were determined in arterial, portal, and mesenteric blood and plasma by quantification of amino acid concentrations before and after acid hydrolysis of samples that had been first deproteinized then subjected to Sephadex G-15 gel-filtration to remove residual protein. In contrast to other studies of ruminants, peptide concentrations for individual amino acids were lower than for the corresponding free amino acids with peptide (expressed as a proportion of total nonprotein amino acid) representing not more than .25 to .3 of total amino acid. Peptide concentrations in arterial, mesenteric, and portal blood and plasma were similar, indicating that on this diet there was no net uptake of peptides from the small intestine (mesenteric-drained viscera, MDV) or the whole tract (portal-drained viscera, PDV). Increasing the intake of alfalfa pellets from 800 to 1,200 g/d, while increasing the absorption and net flux across the MDV and PDV of free amino acids, had no effect on peptide absorption. Preparation of blood and plasma samples for peptide analysis with methods used in studies in which substantial peptide absorption has been reported indicated no net MDV or PDV flux of peptide. Such conflicting data on the extent of gastrointestinal peptide flux are discussed in the context of methodological differences and the importance of diet and physiological state of the animal.

Hepatic response to increased exogenous supply of plasma amino acids by infusion into the mesenteric vein of Holstein-Friesian cows in late gestation.

The hepatic responses of late gestation, dry dairy cows to acute (6 h) infusions of an amino acid (AA) mixture (Synthamin; 0.0, 1.1, 2.2, 4.4, 8.8 and 17.6 mumol/min) into the mesenteric vein were determined. Neither blood flow nor O2 consumption across the portal-drained viscera (PDV) and liver was significantly altered by infusion. Similarly, there were no effects on net absorption, or hepatic removal, of acetate, propionate, butyrate or NH3. Glucose PDV appearance was unchanged but hepatic glucose production increased (P = 0.032) by 0.2 mumol/min per mumol/min of AA infused. Additional extraction of alanine, glycine (both infused) and glutamine (not infused) by the liver was sufficient to account for most of the extra C required for glucose synthesis. The N that would be liberated from these glucogenic AA would also account for a large proportion of the increase in urea-N produced in response to the AA infusion. This supports the concept of a correlation between gluconeogenesis and ureagenesis. Furthermore, the amide-N liberated from the extracted glutamine would contribute up to 0.17 of hepatic NH3 flux and assist in balancing N inputs into the carbamoyl phosphate and arginosuccinate entry points of the ornithine cycle. Rates of fractional extraction of the various AA by the liver were best fitted by linear equations, indicating that even at the highest rates of administration (approximately twice maximal physiological absorption) the transport systems were not saturated. Hepatic fractional extractions of infused essential AA were highest for methionine (0.83) and phenylalanine (0.87) with the lowest proportion removed observed for valine (0.25), leucine (0.30), lysine (0.31) and isoleucine (0.49). For the non-essential AA, the highest apparent fractional extractions were for glycine (0.73), arginine (0.79) and tyrosine (0.63) followed by alanine (0.54), proline (0.47) and serine (0.37). Hepatic removal of AA-N exceeded the increase in urea-N formation such that, at the highest rate of infusion, approximately 10 mmol/min of the extracted AA was apparently available for hepatic anabolism, more than is required to account for assumed increases in liver mass and export protein synthesis. Similarly, the amount of AA available for peripheral tissue protein gain, when assessed against phenylalanine supply as the limitation, would be the equivalent of a maximum of 0.5 g protein retained/min (6 mmol AA-N/min). This would provide sufficient AA for replenishment of peripheral (muscle) protein stores plus support of the placenta and fetus.

Effect of intravenous amino acid infusion on leucine oxidation across the mammary gland of the lactating goat.

Changes in the kinetics of leucine in the mammary gland were examined in four lactating goats (25, 38, 45, and 135 DIM) that were given an i.v. infusion of a mixture of 18 AA, not including leucine, to alter the availability of leucine to the gland relative to other AA. Arteriovenous monitoring of [1-13C]leucine kinetics across one-half of the mammary gland was conducted on the last day (d 6 or 7) of the saline (control) and the AA infusion periods. Although blood flow to the mammary gland and the arterial concentration of most AA other than leucine were increased by the AA infusion, milk and protein yields did not change. For goats in early lactation (n = 3), arterial leucine concentrations fell considerably during AA infusion; however, the arteriovenous difference of leucine was maintained, resulting in uncommonly low leucine concentrations in venous plasma (8 microM). Whole body leucine flux (protein synthesis plus oxidation) was unaffected by AA infusion, but, because whole body leucine oxidation was reduced, whole body utilization of leucine for protein synthesis increased. The AA infusion reduced mammary oxidation of leucine to approximately one-third of control values. These results suggest that leucine oxidation can be reduced considerably without affecting milk protein output; thus, leucine oxidation may not be an irrevocable consequence of mammary metabolism. If catabolism of other AA either by the gland or in the whole body can be reduced, then the efficiency of milk yield can be improved.

Evidence for the utilization of peptides for milk protein synthesis in the lactating dairy goat in vivo.

Precursors for milk protein synthesis have been examined in lactating dairy goats using arteriovenous difference and isotope kinetic techniques. Certain amino acids, such as phenylalanine and histidine, are taken up by the mammary gland in quantities that are insufficient to account for their output in milk protein. Some amino acids have been shown to be present in significant quantities (10-30% of total non-protein-bound amino acids) as peptides (< 1,500 Da) in the arterial supply to the mammary gland, although methodological considerations make it difficult to accurately assess the extent of their uptake across the tissue bed. Indirect evidence for the utilization of peptides for milk protein synthesis in vivo has been obtained, however, by examination of the kinetics of milk casein labeling during long-term (24 h) systemic infusion of [1-13C]phenylalanine and [1-13C]leucine. Comparison of plateau enrichments for blood, plasma, and casein indicate that, although, for leucine, the plasma free pool seems to provide all the leucine for milk protein synthesis, sources other than the labeled plasma free amino acids contribute phenylalanine (10-20%) for casein biosynthesis. These findings raise questions relating to the type and source of amino acid precursors used by tissues for protein synthesis.

Responses in milk constituents to intravascular administration of two mixtures of amino acids to dairy cows.

Four Holstein-Friesian cows were used to investigate the effects of intravascular infusions of AA mixtures on milk constituents. Cows were in wk 11 to 28 of lactation and were fed a basal concentrate (142 g of CP/kg of DM) and grass silage (149 g of CP/kg of DM) in a 60:40 ratio (percentage of DM). Cows were fed hourly, and feed intake was fixed at 95% of ad libitum intake for each experimental period. Each cow received a 4-d jugular saline infusion, followed by a 5-d jugular infusion of a mixture of AA. Two mixtures of AA were used in a crossover design. The first mixture contained both the essential AA and non-essential AA found in milk protein (total AA); this mixture was infused at 400 g of AA/d. The other mixture represented the essential AA fraction only and was infused at 208 g/d. Infusion of total AA increased milk protein concentration from 32.4 to 35.0 g/kg, and essential AA increased milk protein concentration from 32.5 to 36.9 g/kg; milk protein yield increased by 87 g/d (total AA) and 143 g/d (essential AA). Intravascular administration of AA specifically stimulated milk protein concentration, and the efficiency with which the AA were used was higher than had been previously reported when AA supply was increased either by dietary supplementation or by abomasal infusion.

Leucine and protein metabolism in the lactating dairy cow mammary gland: responses to supplemental dietary crude protein intake.

Mammary gland protein metabolism, determined by an arteriovenous difference technique, was monitored in four Holstein-Friesian dairy cows in response to supplemental dietary protein (provided as rumen-protected soyabean meal) during late lactation (weeks 24-30). Each cow was offered two isoenergetic diets composed of grass silage (170 g crude protein/kg dry matter) plus either a low (108 g/kg) or medium (151 g/kg) crude protein concentrate in a single crossover design involving two 21 d periods. On day 21, arteriovenous measurements across the mammary gland were made during a 13 h continuous i.v. infusion of [1-(13)C]leucine and with frequent (2 hourly) milk sampling during the final 6 h. Although total milk yield was slightly increased (+1 kg/d) by protein supplementation, milk protein yield was not significantly affected. Whole body protein flux (protein synthesis plus oxidation) was not significantly affected by supplementation. Total mammary gland protein synthesis (milk plus non-milk protein) was also not affected by supplementation but on both diets gland synthesis was always greater (by 20-59 percent) than milk protein output. The fractional oxidation rate of leucine by the mammary gland was significantly increased by protein supplementation (0-047 v. 0-136). Although the enrichment of leucine in secreted milk protein continued to increase, the final value (at 13 h) was 0-94 of the arterial plasma free leucine plateau value (not significantly different), suggesting almost exclusive use of plasma free leucine for milk protein synthesis. Based on current feeding schemes for dairy cattle, a fixed proportion (0-65 0-75) of the additional protein intake (+490 g/d) should have been partitioned into milk protein. Instead, leucine oxidation by the mammary gland was increased. Whether oxidation of other amino acids was also enhanced is unknown but if amino acid oxidation and the 'additional' non-milk protein synthesis occurring in the gland are not crucial to milk synthesis, then by reducing such activities improvements in the efficiency of converting absorbed amino acid into milk protein can be achieved.

The effect of dietary crude protein as protected soybean meal on mammary metabolism in the lactating dairy cow.

Metabolism in the mammary gland was related to changes in milk output in response to changes in dietary protein intake. Three diets of grass silage and concentrate were fed to four lactating dairy cows equipped with intravascular catheters across the mammary gland. Concentrates differed in the inclusion of protected soybean meal and provided 11.3, 15.4, and 20.1% CP, respectively. Blood samples were taken to assess the effect of protein percentage on the nutrient fluxes across the gland and their relationship to milk production. Milk production, milk protein yield, and milk protein concentration were all increased as CP intake increased, although these responses were not linear. Concentrations of urea in milk reflected those in plasma and increased as dietary protein intake increased. Uptake of glucose and BHBA by the mammary gland tended to increase as milk production increased. Arterial supply of essential AA increased as the dietary protein increased. Supply and uptake of nonessential AA were unchanged by dietary treatment, and uptake was insufficient to account for output of nonessential AA residues in milk protein. The supply of essential AA was not limiting for milk protein synthesis, and some alternative mechanism must have existed for the control of milk protein yield.

Evidence for a glycyl-proline transport system in ovine enterocyte brush-border membrane vesicles.

A peptide transport system in ovine enterocyte BBMV has been examined with glycyl-L-proline (Gly-Pro) as substrate. Transport is stimulated by an inwardly directed proton gradient (pH1 = 8.4; pH0 = 6.0). Gly-Pro uptake was saturable and conformed to Michaelis-Menten kinetics. Uptake was not affected by the presence of glycine but was inhibited by glycyl-L-phenylalanine (Gly-Phe). This is thought to be the first positive demonstration of the existence of a peptide transporter in the ruminant small intestine.

Evidence that protein kinase C and mitogen activated protein kinase are not involved in the mechanism by which insulin stimulates translation in L6 myoblasts.

Insulin stimulated a concentration-dependent increase in protein synthesis in L6 myoblasts which was significant at 1 nM. This response was not prevented by the transcription inhibitor, actinomycin D. The protein kinase C (PKC) inhibitor, Ro-31-8220, and downregulation of PKC by prolonged incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), had no effect on the ability of insulin to stimulate protein synthesis whilst completely blocking the response to TPA. In contrast, insulin failed to enhance protein synthesis significantly in the presence of either ibuprofen, a selective cyclooxygenase inhibitor or rapamycin, an inhibitor of the 70 kDa S6 kinase. When cell extracts were prepared and assayed for total myelin basic protein kinase activity, a stimulatory effect of insulin was not observed until the concentration approached 100-fold (i.e. 100 nM) that required to elicit increases in protein synthesis. Upon fractionation on a Mono-Q column, 100 nM insulin increased the activity of 3 peaks which phosphorylated myelin basic protein. Two of these peaks were identified as the 42 and 44 kDa forms of Mitogen Activated Protein (MAP) kinase by immunoblotting. In contrast, 1 nM insulin had no effect on the activity of these peaks. The data suggest that physiologically relevant concentrations of insulin do not stimulate translation in L6 cells through either PKC or the 42/44 kDa isoforms of MAP kinase and that this response is, at least in part, mediated through the activation of the 70 kDa S6 kinase by cyclooxygenase metabolites.

Kinetics of blood free and milk casein-amino acid labelling in the dairy goat at two stages of lactation.

The kinetics of blood free amino acids (AA) transfer into milk casein were compared in goats (n 4) at 61 (SE 5) d (Expt 1; post-peak, 4.51 (SE 0.26) kg milk/d) and at 180 (SE 6) d (Expt 2; late, 2.36 (SE 0.16) kg milk/d) of lactation during non-primed, continuous (Expt 1, 12 h; Expt 2, 16 h) intravenous infusions of mixtures of L-[1-13C]leucine and L-[1-13C]phenylalanine with either L-[1-13C]valine (Expt 1) or L-[5-13C]methionine (Expt 2). The 13C enrichments of blood free and casein-bound AA were fitted to a single exponential model to estimate isotopic plateaux and the fractional rate constant for milk casein labelling. Milk protein output and its contribution to whole-body flux was higher in Expt 1 (post-peak) than in Expt 2 (late lactation), but the kinetics of 13C labelling of the casein-bound AA were similar for all AA tracers in both experiments. At both stages of lactation the delay (6-8 h) between the attainment of isotopic plateau for the blood free AA and the corresponding attainment of plateau for the casein-bound AA indicated that the blood free pool was not the immediate precursor pool for milk casein biosynthesis. Plateau enrichments of casein-bound AA were generally higher than those for the corresponding blood free AA in both experiments. These results indicate that the relative contributions of different AA sources to the immediate precursor pool for milk casein biosynthesis are similar at different stages of lactation despite major changes in the partitioning of whole-body flux towards milk protein output. Non-milk protein fluxes were also similar in post-peak and late lactation.

Utilization of dipeptides by the caprine mammary gland for milk protein synthesis.

Specific use by the mammary gland in vivo of amino acids (AA) of peptide origin has been demonstrated in lactating dairy goats using a dual-labeled tracer technique involving close-arterial (external pudic artery, EPA) infusion of 13C-labeled dipeptides. The extent of utilization does not appear to differ for glycyl-L-[1-13C]phenylalanine and glycyl-L-[1-13C]leucine, perhaps indicative of a common mechanism by which AA are incorporated from peptide into milk protein. [1-13C]phenyl-alanine of peptide origin appears to be concentrated within the red blood cell, suggesting a role for the erythrocyte in peptide metabolism in vivo. In conclusion, it appears that the lactating mammary gland of goats has the ability to utilize AA of peptide origin for milk protein synthesis, and while the mechanism by which [1-13C]AA are incorporated into milk protein is not clear, it may involve peptide hydrolysis by either mammary cell surface or red blood cell hydrolases followed by uptake of liberated AA by the mammary gland.

The effect of supplementary protein on in vivo metabolism of the mammary gland in lactating dairy cows.

Four lactating cows equipped with rumen and duodenal cannulas were fed a diet of grass silage and concentrates containing either 12.4 or 17.2% CP (DM basis) in a change-over design. Additional protein was supplied as white fish meal. Fish meal did not affect molar proportions of VFA in the rumen, but duodenal NAN supply was increased .69 g/g of N in supplementary feed. In Experiment 2, three lactating dairy cows that had been prepared with catheters across the mammary gland were fed the same diets using a switchback design. Blood samples were taken to determine changes in metabolite flux to the mammary gland. In both experiments, milk production and protein yield were nonsignificantly increased by addition of fish meal. Milk urea output was increased from 3.18 to 4.74 g/d by fish meal supplementation, reflecting increased arterial concentrations of urea. Concentrations of glucose, VFA, and BHBA in blood showed no substantial changes because of dietary supplementation of fish meal. Supply of essential AA increased 26% with fish meal supplementation, mammary uptake increased 34%, but milk protein output only increased 5%. The low efficiency of conversion of supplementary protein to milk protein appears to be related to the inability of the gland to utilize the additional AA.

Inhibition and inactivation of monoamine oxidase by 3-amino-1-phenyl-prop-1-enes.

We have previously shown that E-3-amino-1-phenyl-prop-1-ene (E-cinnamylamine) is readily oxidised by monoamine oxidase (MAO) type B from either rat or bovine liver (Williams et al. (1988), Biochem. J. 256, 411-415) in each case producing a non-linear progress curve which was attributed to inhibition by the reaction product E-cinnamaldehyde. We have now found that although this aldehyde inhibits MAO B competitively (Ki 0.017 mM) this cannot account for the inhibitory process, since during a 60 min incubation with the substrate (0.5 mM; Km, 0.074 mM) more than 95% inhibition of MAO B was observed and the concentration of aldehyde had reached approx. 0.025 mM. Inhibition was relieved either by dialysis or dilution of inhibited samples. The activity of MAO A from rat liver was largely unaffected by E-cinnamylamine. Oxidation of N-methyl-E-cinnamylamine and its Z-isomer by MAO B produced progress curves similar to that obtained with the primary amine, but in these cases inhibition was not reversed either by dilution or dialysis. Partition ratios for the pair of N-methyl isomers with bovine MAO B were calculated to be 1640 (E-isomer) and 1430 (Z-isomer). The time-dependent inhibition process for all three amines obeyed pseudo-first-order kinetics. A tritiated form of N-methyl-E-cinnamylamine, incubated with MAO B from bovine liver, resulted in incorporation of radioactivity into the enzyme. This labelling was stable to dialysis and to SDS-PAGE.