Inhibition by Peptides of Amino Acid Uptake by Bacterial Populations in Natural Waters: Implications for the Regulation of Amino Acid Transport and Incorporation

To investigate the regulatory interactions of amino acid transport and incorporation, we determined the effects of dipeptides on amino acid uptake by bacteria in an estuary and a freshwater lake. Dipeptides noncompetitively inhibited net transport and incorporation of amino acids into macromolecules but had no effect on the ratio of respiration to incorporation. Nearly maximum inhibition occurred at peptide concentrations of <10 nM. In contrast, the initial uptake rate of glycyl-[¹⁴C]phenylalanine was not affected by glycine or phenylalanine. Net amino acid transport appeared to be inhibited by the increased flux into the intracellular pools, whereas the incorporation of labeled monomers into macromolecules was isotopically diluted by the unlabeled amino acids resulting from intracellular hydrolysis of the dipeptide. Chloramphenicol, sodium azide, and dinitrophenol all inhibited the initial uptake rate of leucine and phenylalanine. These results suggest that in aquatic environments amino acids are taken up by active transport which is coupled closely to protein synthesis.     

Ammonia Production by Ruminal Microorganisms and Enumeration, Isolation, and Characterization of Bacteria Capable of Growth on Peptides and Amino Acids from the Sheep Rumen

Excessive NH₃ production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH₃ production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH₃ production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH₃ production from Trypticase varied from 1.8 to 19.7 nmol mg of protein⁻¹ min⁻¹ depending on the substrate, its concentration, and the method used. Monensin (5 μM) inhibited NH₃ production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 μM monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH₃ at >250 nmol min⁻¹ mg of protein⁻¹, depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH₃ production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

The aerobic mineralization of organic compounds in the saline Lake Grevelingen (The Netherlands) (VI)

Summary During a one year period the uptake of aspartic acid and of a mixture of amino acids was determined using¹⁴C-labeled substrates as described by WRIGHT and HOBBIE (1966). By this technique the activity is analyzed of that part of the bacterial population which is able to utilize the added substrate. For comparison purposes the activity of the total heterotrophic bacterial population was determined by measurement of the oxygen consumption rate. From the oxygen consumption rate (mg O₂.l^–1.h^–1) the carbon mineralization rate (mg C.l^–1.h^–1) was calculated by applying a conversion factor of 0.29.Aspartic acid was respired for 80% and the amino acid mixture for 43%. From the maximum uptake rates, the potential yearly uptake of the substrate in question can be calculated. These data indicate the relative importance of the several subpopulations in the carbon mineralization process as a whole. The highest value of the potential yearly uptake was obtained for the amino acid mixture; the comparable value for the uptake of aspartic acid was slightly lower.The carbon mineralization rate as calculated from the oxygen uptake experiments was about 150–200 g C.m^–2.year^–1. The potential yearly uptake as determined with the¹⁴C-labeled amino acid mixture was only 2.8% of the amount of mineralized carbon, as calculated from the oxygen uptake experiments. This percentage is very low in view of the fact that 35–55% of the organic carbon of living phytoplankton and zooplankton consists of protein (HAGMEIER, 1961) and that the aerobic mineralization of amino acids is a very common property among the heterotrophic bacterial population (SEPERS, 1979). The value of the applied activity measurements was investigated in order to obtain information about the relation between the uptake process as measured with¹⁴C-labeled substrates and the activity of the bacterial population in situ. The results of this study have been published bij SEPERS and VAN ES (1979).     

Characteristics of the active transport of peptides and amino acids by germinating barley embryos

Use of two different assays involving either radioactively labelled substrates or a fluorescent-labelling procedure, gave good agreement for the rates of transport of peptides and amino acids into the scutellum of germinating grains of barley (Hordeum vulgare cv. Maris Otter, Winter). However, evidence was obtained for the enzymic decarboxylation of transpored substrate, which can cause underestimates of transport rates when using radioactively labelled substrates. The peptide Gly-Phe, was shown to be rapidly hydrolysed after uptake, and autoradiography of transported Gly-[U-¹⁴C]Phe indicated a rapid distribution of tracer, i.e. [U-¹⁴C] phenylalanine into the epithelium and sub-epithelial layers of the scutellum. The developmental patterns of transport activity indicate that peptide transport is more important nutritionally during the early stages of germination (1–3 d) whereas amino acids become relatively more important later (4–6 d). A range of amino acids is shown to be actively transported and several compete for uptake. At physiological concentrations, e.g. 2mM, transport of peptides and amino acids is inhibited about 80% by protonophore uncouplers, but at higher concentrations (10–100 mM) passive uptake predominates.Abbreviations Gly glycine - Leu leucine - Phe phenylalanine - Pro proline     

Acidic amino acid clearance from CSF in the neonatal versus adult rat using ventriculo-cisternal perfusion

The acidic amino acids aspartate and glutamate are excitatory neurotransmitters in the CNS. The clearance of this group of amino acids from CSF of adult and neonatal (7-day-old) rats was investigated. Ventriculo-cisternal perfusions with 14C-amino acids and 3H-dextran were carried out for up to 90 min. Uptake of the amino acids by the whole brain was measured, and the loss to blood was calculated. 3H-Dextran was included in the perfusate for measurement of CSF secretion rate. After 90-min perfusion, both aspartate and glutamate showed a similar uptake into the whole brain, and this did not change with age (p>0.05). However, clearance from CSF was greater in the adult, as was entry into blood from CSF. Addition of 5 mM excess unlabelled amino acid resulted in reduction in the brain uptake of both 14C-amino acids in the adult rat. In the neonate, addition of aspartate also reduced brain aspartate uptake, whereas addition of glutamate increased brain neonatal [14C]glutamate uptake. The rate of CSF secretion was significantly greater in the adult, 1.26+/-0.18 microl x min(-1) x g(-1), than in the neonate, 0.62+/-0.08 microl x min(-1) x g(-1), and the turnover of CSF was greater in adults (p<0.01). In summary, both aspartate and glutamate showed greater clearances from CSF in the adult than the neonate. This clearance was found to be by carrier-mediated mechanisms.     

Ammonia production by ruminal microorganisms and enumeration,isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen

Excessive NH(3) production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH(3) production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH(3) production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH(3) production from Trypticase varied from 1.8 to 19.7 nmol mg of protein(-1) min(-1) depending on the substrate, its concentration, and the method used. Monensin (5 micro M) inhibited NH(3) production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 micro M monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH(3) at >250 nmol min(-1) mg of protein(-1), depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH(3) production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.     

Preparation of 14C-labeled tuberculin purified protein derivative

(14)C-labeled tuberculin purified protein derivative ((14)C-tuberculin PPD) has been prepared from culture filtrates of Mycobacterium tuberculosis var. hominis grown in a culture medium containing uniformly labeled (14)C-amino acids. With a mixture of (14)C-amino acids (an acid hydrolysate of (14)C-Chlorella protein) in the medium, the recoveries of (14)C in the final product were higher than with (14)C-labeled-l-glutamic acid. (14)C-tuberculin PPD was separated into tuberculoprotein and nucleic acid by paper electrophoresis. The specific radioactivity of tuberculoprotein was substantially greater than that of the nucleic acid. (14)C-tuberculin PPD is advocated as a means to measure the adsorption of tuberculin to glass or other surfaces. It could also prove useful as a means to study the structure and mode of action of tuberculin.     

Uptake and utilization of lipids by Paramecium tetraurelia

Uptake rates of a variety of 14C-labeled fatty acids and complex lipids by Paramecium tetraurelia during 48 h of log-phase growth varied. Fatty acid uptake was maximal during lag phase of growth when phagosome (food vacuole) formation was minimal. Food vacuole formation was shown to be suppressed by the presence of exogenous lipids and by starvation. The rates of uptake of lipids were significantly greater than those of small organic compounds such as amino acids, cyclitols, fatty acid precursors and metabolic intermediates. Significant amounts of radioactivity from 14C-labeled fatty acids were metabolized to 14CO2. The uptake rates of different saturated, straight-chain fatty acids of even carbon numbers were different and were not correlated with chain length, results suggesting that the primary mechanism for uptake of these compounds was neither by bulk transport nor simple diffusion and that carrier-mediated processes could possibly be involved.     

The role of holotrichs in the metabolism of dietary linoleic acid in the rumen

The uptake and metabolism of linoleic acid by rumen holotrichs (mainly Isotricha prostoma and I. intestinalis) has been examined in in vitro infusion experiments. Maximum absorption and metabolism of [1-14C]linoleate by 2 . 10(6) Isotricha suspended in 100 ml buffer was obtained using an infusion rate of 1.6 mg linoleate/h. After 90 min, 84% of the added substrate was recovered within the cells, mainly as free fatty acid or phospholipid. There was a rapid incorporation of radioactivity into phospholipid, mainly phosphatidylcholine, at the commencement of linoleate infusion but no further incorporation after about 40 min. The presence of bacteria during incubations, in approximately the same Isotricha/bacteria ratio as found in the rumen, reduced the uptake of linoleate and the accumulation of free fatty acid by holotrichs but the incorporation into phospholipid remained similar to that obtained in the absence of bacteria. Very little biohydrogenation of linoleic acid occurred in incubations with holotrichs alone. Bacterial suspensions converted linoleic acid to mainly trans monoene and a small amount of stearic acid, but in incubations containing both bacteria and holotrichs, both stearic acid and trans monoene were major products. Using the latter mixed culture, about 20% of the added [1-14C]linoleic acid was present in holotrich phospholipid of which 62% remained as octadecadienoic acid. The Isotricha population was 3 . 10(3)--2 . 10(4)/ml rumen fluid and it contributed about 23% of the linoleic acid in the rumen of a cow on a hay diet.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

Inhibition of protein synthesis in Krebs 2 ascites cells and cell-free systems by phenylalanine and its effect on leucine and lysine in the amino acid pool

1. The inhibition of incorporation of ¹⁴C-labelled amino acids into protein of whole cells by phenylalanine has been reproduced in a cell-free system. In both cases only the l-isomer was inhibitory. 2. The effect of phenylalanine on incorporation of [¹⁴C]leucine and [¹⁴C]lysine into protein was different in both whole cells and cell-free systems. 3. In whole cells inhibition of incorporation of leucine at 2·5μg./ml. was very rapid, but when the concentration was increased to 100μg./ml. the inhibition was not apparent for about 1hr. The kinetics of inhibition of lysine was the same at both these concentrations and was similar to that found with leucine at 100μg./ml. 4. Neither a lower specific radioactivity of the two amino acids in the pool nor a decrease in their pool size could be consistently related with inhibition of protein synthesis. 5. In the cell-free system l-phenylalanine inhibited the incorporation of leucine but not of lysine. 6. Charging of transfer RNA by leucine was markedly decreased in the presence of phenylalanine, whereas charging of transfer RNA by lysine was not.     

Effects of Essential Oils on Ruminal Microorganisms and Their Protein Metabolism

A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited (P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

Characterization and Molecular Mechanism of AroP as an Aromatic Amino Acid and Histidine Transporter in Corynebacterium glutamicum

Corynebacterium glutamicum is equipped with abundant membrane transporters to adapt to a changing environment. Many amino acid transporters have been identified in C. glutamicum, but histidine uptake has not been investigated in detail. Here, we identified the aromatic amino acid transporter encoded by aroP as a histidine transporter in C. glutamicum by a combination of the growth and histidine uptake features. Characterization of histidine uptake showed that AroP has a moderate affinity for histidine, with a K_m value of 11.40 ± 2.03 μM, and histidine uptake by AroP is competitively inhibited by the aromatic amino acids. Among the four substrates, AroP exhibits a stronger preference for tryptophan than for tyrosine, phenylalanine, and histidine. Homology structure modeling and molecular docking were performed to predict the substrate binding modes and conformational changes during substrate transport. These results suggested that tryptophan is best accommodated in the binding pocket due to shape compatibility, strong hydrophobic interactions, and the lowest binding energy, which is consistent with the observed substrate preference of AroP. Furthermore, the missense mutations of the putative substrate binding sites verified that Ser24, Ala28, and Gly29 play crucial roles in substrate binding and are highly conserved in the Gram-positive bacteria. Finally, the expression of aroP is not significantly affected by extracellular histidine or aromatic amino acids, indicating that the physiological role of AroP may be correlated with the increased fitness of C. glutamicum to assimilate extracellular amino acid for avoiding the high energy cost of amino acid biosynthesis.     

PROTEIN BIOSYNTHESIS IN SALT-SHOCKED CELLS OF STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

We have developed an in vivo¹⁴C-amino acid labelling procedure for monitoring protein synthesis in salt-shocked cells of Stichococcus bacillaris Naeg. This alga possesses an efficient transport system for the uptake of leucine, methionine, and phenylalanine and rapidly incorporates these amino acids into proteins. Of the three amino acids tested, ¹⁴C-phenylalanine is ideally suited for labelling proteins in S. bacillaris, as it establishes an early equilibrium between uptake and incorporation of the amino acid into proteins. The uptake of phenylalanine shows little inhibition following transfer of cells to higher salinities and is also not affected in short-term experiments by the presence of the protein inhibitors cycloheximide and chloramphenicol. While Stichococcus bacillaris grows slowly at salinities equal to, or higher than, 150% artificial seawater (ASW), it shows surprising rates of recovery of major physiological functions following considerable salt shocks. Cells transferred from 33 to 150% ASW show complete recovery of photosynthetic activity and protein synthesis within 10–15 min, and cell transferred from 33 to 300% ASW recover 50% of their capacity to synthesize proteins within. 1 h. Cytoplasmic and organellar protein synthesis appears to be equally sensitive to the effects of salt shocks according to studies with protein synthesis inhibitors.     

Oxidation of oleic and elaidic acids in rat and human heart homogenates

Parallel incubations with uniformly 14C-labeled oleic and elaidic acids were conducted to compare oxidation rates in tissue homogenates prepared from rat and human hearts. Radioactivity in 14CO2 and 14C-labeled chain-shortened acid-soluble products was used to measure the extent of oxidation. Oxidation rates (pmol/min per mg heart protein) determined on 14C-labeled acid-soluble products suggest that oleic acid was oxidized 35-40% faster than elaidic acid by both male and female rat heart homogenates, whereas human heart homogenates oxidized these fatty acids at equal rates. Rates for female heart homogenates were somewhat higher than those for males in rats and humans. Rates of formation of 14CO2 were the same for each acid in rat and human heart tissue. Comparative rates of formation of oxidation products expressed as oleic/elaidic ratios from parallel incubations confirm that preferential oxidation of oleic acid occurred with rat heart homogenates, but not with the human heart homogenates. These data suggest that the presence of the trans double bond in elaidic acid does not impair its utilization for energy by human heart muscle.     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Assessment of Reductive Acetogenesis with Indigenous Ruminal Bacterium Populations and Acetitomaculum ruminis

The objective of this study was to evaluate the role of reductive acetogenesis as an alternative H₂ disposal mechanism in the rumen. H₂/CO₂-supported acetogenic ruminal bacteria were enumerated by using a selective inhibitor of methanogenesis, 2-bromoethanesulfonic acid (BES). Acetogenic bacteria ranged in density from 2.5 × 10⁵ cells/ml in beef cows fed a high-forage diet to 75 cells/ml in finishing steers fed a high-grain diet. Negligible endogenous acetogenic activity was demonstrated in incubations containing ruminal contents, NaH¹³CO₃, and 100% H₂ gas phase since [U-¹³C]acetate, as measured by mass spectroscopy, did not accumulate. Enhancement of acetogenesis was observed in these incubations when methanogenesis was inhibited by BES and/or by the addition of an axenic culture of the rumen acetogen Acetitomaculum ruminis 190A4 (10⁷ CFU/ml). To assess the relative importance of population density and/or H₂ concentration for reductive acetogenesis in ruminal contents, incubations as described above were performed under a 100% N₂ gas phase. Both selective inhibition of methanogenesis and A. ruminis 190A4 fortification (>10⁵ CFU/ml) were necessary for the detection of reductive acetogenesis under H₂-limiting conditions. Under these conditions, H₂ accumulated to 4,800 ppm. In contrast, H₂ accumulated to 400 ppm in incubations with active methanogenesis (without BES). These H₂ concentrations correlated well with the pure culture H₂ threshold concentrations determined for A. ruminis 190A4 (3,830 ppm) and the ruminal methanogen 10-16B (126 ppm). The data demonstrate that ruminal methanogenic bacteria limited reductive acetogenesis by lowering the H₂ partial pressure below the level necessary for H₂ utilization by A. ruminis 190A4.     

Aminopeptidase C of Aspergillus niger Is a Novel Phenylalanine Aminopeptidase

A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from Aspergillus niger (CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine β-naphthylamide (F-βNA) and phenylalanine para-nitroanilide (F-pNA), but it also displayed activity towards other amino acids with aromatic side chains coupled to βNA; other amino acids with nonaromatic side chains coupled to either pNA or βNA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze N-acetylalanine-pNA, a substrate for acyl-peptide hydrolases.