TCA-soluble Fraction Research Articles

Influence of dietary grape pomace supplementation on chemical and sensory properties of ewes' cheese

The nutritional and sensorial properties of Pecorino cheese made from milk of ewes fed a diet formulated with grape pomace (GP), a winemaking by-product, were evaluated. Dietary inclusion of GP (GP+) did not influence cheese moisture or total lipid content; however, a significant reduction of C14:1 cis-9 was noted, assumed to be a dietary effect on stearoyl-CoA desaturase function. The amount of β-casein was increased in GP+ samples, but total and TCA soluble fraction nitrogen did not indicate any marked effect on the proteolytic processes. Analysis of volatile compounds showed lower relative percentages of aldehydes, ketones, esters and lactones in GP+ cheeses, indicating improved oxidative stability during ripening. There was a marked decrease of lightness and an increase of hardness, gumminess and resilience (Young's modulus) in the GP+ samples. Sensory analysis of 120-day ripened cheeses confirmed these findings, and highlighted an increase in odour intensity and saltiness.

Characteristics of Miniature Cheddar‐Type Cheese Made by Microbial Rennet from Bacillus amyloliquefaciens: A Comparison with Commercial Calf Rennet

Miniature Cheddar-type cheeses were produced using microbial rennet from Bacillus amyloliquefaciens (milk-clotting enzyme [MCE]) or calf rennet (CAR). With the exception of pH, there were no significant differences in gross composition between MCE-cheese (MCE-C) and CAR-cheese (CAR-C). The pH value of CAR-C was significantly higher than that of MCE-C at 40 and 60 d of ripening. The total nitrogen content of the pH 4.6-soluble fraction obtained from MCE-C was higher than that obtained from CAR-C. However, nitrogen content of the 12% TCA-soluble fraction was similar between CAR-C and MCE-C. The extent of α(s1)-casein and β-casein hydrolysis, measured by urea-PAGE, was similar in both cheese samples. The hydrolysis of β-casein was lower than that of α(s1)-casein. Different reverse phase-high-performance liquid chromatography peptide profiles of ethanol-soluble and ethanol-insoluble fractions were obtained from CAR-C and MCE-C. The peptide content in the 2 cheese samples increased throughout ripening; the ratio of hydrophobic to hydrophilic peptides was lower in MCE-C than in CAR-C. Compared with CAR-C, MCE-C was softer as a result of higher protein hydrolysis. Microbial rennet from B. amyloliquefaciens contributed to higher proteolytic rates, which reduced ripening time.

Proteolysis of Cacioricotta cheese made from goat milk coagulated with caprifig (Ficus carica sylvestris) or calf rennet

A study was undertaken on Cacioricotta, a traditional Italian goat’s cheese obtained from overheated milk (90 °C) without use of starter. The profile of proteolysis in the artisanal type, made with vegetable coagulant (latex released from caprifig branches) as milk clotting agent, was compared to that of the “industrial” one, manufactured with calf rennet. Particular aim of the investigation was to study the differences and, possibly, establish a useful tool for distinguishing the two types of cheese. The study was based on the quantification of the water soluble, 15% TCA soluble and amino acid nitrogen fractions, RP-HPLC separation of low molecular weight peptides and their identification by mass spectrometry (MALDI-ToF MS). The use of fig latex was associated to higher amounts of the nitrogen fractions and to RP-HPLC chromatograms very rich in peptides, in contrast to an almost complete lack of peptides in the industrial counterpart. These results confirm the strong proteolyitic activity exerted by the caprifig clotting enzymes in spite of the intense overheating of the milk, which is considered to cause reduction of the rate of casein degradation in cheese. The MS-based identification of several peptides provided a support at the molecular level for the characterization of Cacioricotta made with this vegetable coagulant and could be useful for “tracing back” purposes. In conclusion, the peptide pattern determined by the use of caprifig for milk coagulation can be considered a particular feature of the artisanal Cacioricotta, giving confirmation of its vocation to EU protection as typical product.

Effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1

Glycosylation is generally applicable as a strategy for increasing the activity of bioactive proteins. In this study, we examined the effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1 (GLP-1) as a bioactive peptide for type 2 diabetes. Noninvasive imaging studies were performed using a gamma camera after the intravenous administration of (123)I-GLP-1 or (123)I-α2, 6-sialyl N-acetyllactosamine (glycosylated) GLP-1 in rats. In ex vivo biodistribution studies using (125)I-GLP-1 or (125)I-glycosylated GLP-1, organ samples were measured for radioactivity. Plasma samples were added to 15% trichloroacetic acid (TCA) to obtain TCA-insoluble and TCA-soluble fractions. The radioactivity in the TCA-insoluble and TCA-soluble fractions was measured. In the noninvasive imaging studies, a relatively high accumulation level of (123)I-GLP-1 was found in the liver, which is the major organ to eliminate exogenous GLP-1. The area under the time-activity curve (AUC) of (123)I-glycosylated GLP-1 in the liver was significantly lower (89%) than that of (123)I-GLP-1. These results were consistent with those of ex vivo biodistribution studies using (125)I-labeled peptides. The AUC of (125)I-glycosylated GLP-1 in the TCA-insoluble fraction was significantly higher (1.7-fold) than that of GLP-1. This study demonstrated that glycosylation significantly decreased the distribution of radiolabeled GLP-1 into the liver and increased the concentration of radiolabeled GLP-1 in plasma. These results suggested that glycosylation is a useful strategy for decreasing the distribution into the liver of bioactive peptides as desirable pharmaceuticals.

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.

A method for radiolabeling Artemia with applications in studies of food intake, digestibility, protein and amino acid metabolism in larval fish

The present paper describes a method for radiolabeling Artemia that has potential applicability in the study of food intake, Artemia digestibility, protein and amino acid (AA) metabolism in early stages of marine fish larvae. Artemia nauplii were labeled by adding a uniformly labeled 14C protein hydrolysate to its hatching or enrichment media. Both newly hatched and enriched nauplii assimilated rapidly the radiolabel and a very high proportion of the uptaken label was incorporated into the TCA precipitate (mostly protein) fraction after a short period of time—84–91%, at the end of the hatching period and 81–87%, after 12 h of enrichment. In addition, it was observed that the amount of radiolabel per Artemia, as well as its distribution between the TCA precipitate and soluble (free AA) fractions, was relatively stable (up to 8 h) in conditions simulating transfer to larval feeding tanks. During the radiolabeling trials a substantial amount of the total initial radioactivity in the media was not recovered at the end of the hatching or enrichment period (38% and 29%, respectively). An experiment was designed to study the release of the radiotracer into the atmosphere, as 14CO 2. It was concluded that although the Artemia catabolism may be responsible for some release of the radiolabel into the atmosphere, bacterial activity in the enrichment media has a major impact, being responsible for the loss of 19.2% of the total initial radiolabel from the system. The digestibility and transfer rates of the labeled nutrients from Artemia to fish larvae was analysed “in vivo” by feeding radiolabeled newly hatched nauplii to herring ( Clupea harengus) larvae and following the metabolic fate of the label. The results revealed a high digestibility (around 60%) of the Artemia, with 20% being retained in the body and 39% catabolised by the larvae, 24 h after feeding. The digestion process and subsequent absorption of the digested radiolabeled nutrients were quite rapid, given that 40% was recovered in the body only 2 h after feeding and 6% was found in the CO 2 trap at this time.

Effect of Dietary Fiber on Endogenous Nitrogen Flows in Lactating Dairy Cows

The effect of dietary fiber on endogenous N secretion was studied using a 15N isotope dilution technique in four fistulated Holstein cows. Two isonitrogenous diets differing only in fiber (NDF and ADF) content were used in a crossover design. One diet (HF) contained 37.4% NDF, while the other (LF) contained 23.3%. A new model was developed to estimate endogenous N secretions and losses for the preintestinal, intestinal, and the total sections of the gastrointestinal tract. Three precursor pools: TCA-soluble fraction of plasma, intestinal mucosa, and milk were compared. Although endogenous losses estimated with the model were numerically different for each precursor pool selected (TCA-soluble fraction>mucosa>milk), treatment effects were similar. As intestinal mucosa is probably closest to the precursor pool, these data are discussed. Non-urea N endogenous secretions contributed 13% of the duodenal N flow but were not affected by the fiber content of the diet. The nonurea N endogenous flow at the duodenum was comprised of approximately equal inputs from endogenous N direct, and that incorporated into the microbial biomass. Total endogenous N flows at the duodenum exceeded, by nearly twofold, estimated inputs of urea-N to microbial biomass. Metabolic fecal output averaged 17% of fecal N and was not affected by level of dietary fiber, but net losses from secretions occurring in the small intestine were higher with the low fiber diet. Overall, endogenous N secretions represented 30% of total digestive tract protein synthesis.

Nitrogen and in vitro Fermentation of Nitrogenous Substrates in Caecal Contents of the Pig

Marounek M., T. Adamec, V. Skfiivanova, N.I. Latsik: Fractions of Nitrogen and in vitro Fermentation of Nitrogenous Substrates in Caecal Contents of the Pig. Acta Vet. Brno 2002, 71: 429-433. Caecal contents of four 28-week-old pigs were analysed and used as inocula of cultures supplied with plant proteins (gluten, gliadin, zein), mucin, ribonucleic acid (RNA) and glucose. Pigs were fed ad libitum a 14.1% crude protein diet containing barley, wheat, wheat bran, extracted soybean meal and a feed supplement. The caecal digesta contained dry matter, total N, protein, trichloroacetic acid (TCA)-soluble N and NH3-N at 120, 3.1, 14.9, 0.64 and 0.27 mg/g (on average), respectively. One g of the caecal content contained 70 µmol of glycine equivalents of amino acids in TCA-soluble fraction. Peptides rather than amino acids were the primary component of the TCA-soluble ninhydrin-reactive material in the caecum. Fermentation of mucin yielded more volatile fatty acids (VFA) than fermentation of other nitrogenous substrates. Zein was the least fermentable substrate tested. Acetate was the principal fermentation end-product, followed by propionate and butyrate. Fermentation pattern differed in cultures grown on glucose and proteins: propionate molar proportions were higher and those of branched-chain VFA lower in former cultures than in cultures supplied with gluten, gliadin and zein. Low proportions of branched-chain VFA were also in VFA produced from mucin and RNA. It can be concluded, therefore, that caecal microorganisms convert nitrogenous substrates, which would be lost in faeces, into valuable products (VFA), utilizable by the host.

In vitro caecal fermentation of nitrogenous substrates in rabbits

Caecal contents of 3-month-old rabbits were analysed and used as inoculi of cultures supplied with plant proteins (gluten, gliadin, zein), mucin, casein, ribonucleic acid (RNA) and glucose. The caecal digesta contained dry matter, total N, protein, trichloroacetic acid (TCA)-soluble N and NH3-N at 234, 10·2, 42·6, 3·2 and 0·27 mg/g (on average), respectively. One g of the caecal digesta contained 180 μmol of glycine equivalents of amino acids in TCA-soluble fraction. Because ninhydrin reaction was almost three times greater after HCl hydrolysis, peptides rather than amino acids were the primary component in this fraction. Urea concentration was only 2·3 μg N/g.Net production of volatile fatty acids (VFA) in cultures with casein and mucin was higher than in cultures with gluten, gliadin, zein and RNA. Zein was the least fermentable substrate tested. Acetate was the principal fermentation end-product, followed mostly by butyrate and propionate. Branched-chain VFA proportions in VFA produced in cultures with casein, gluten, gliadin and zein were higher than in those supplied with glucose, mucin and RNA. In the former cultures, branched-chain VFA accounted for 10·2–17·7 molar % in the total VFA. In cultures supplied with proteins, there was a significant correlation between methane and VFA production, and between VFA and ammonia. The highest concentration of ammonia was found in cultures with casein. Production of methane was increased on glucose and nitrogenous substrates containing a carbohydrate moiety (mucin, RNA). It can be concluded that the caecal contents of rabbits are rich in nitrogenous compounds. The caecal fermentation of nitrogenous substrates supplies nutrients both for caecal microorganisms (branched-chain VFA, NH3) and for the host (VFA).

Disposition of radioactivity after injection of liver-targeted proteins labeled with 111In or 125I. Effect of labeling on distribution and excretion of radioactivity in rats

The effect of radiolabeling liver-specific proteins on the in vivo disposition of radioactivity was investigated. The suitability of 111In and 125I as radiolabels for protein disposition studies in vivo was examined. Galactosylated and cationized bovine serum albumin were labeled with either 125I by the chloramine-T method or 111In, using 1-(4-isothiocyanatobenzyl)ethylenediaminetetraacetic acid (SCN-BZ-EDTA) or diethylenetriaminepentaacetic acid (DTPA) as bifunctional chelating agents (BCAs) and administered intravenously to rats. 125I radioactivity disappeared rapidly from the liver with subsequent excretion in the urine and bile, mainly in the TCA soluble fraction. 111In-associated radioactivity, on the other hand, remained in the hepatic tissue in considerably higher amounts during the experiment and was excreted in the bile and urine to a lower extent when compared with 125I. When the effect of BCA on excretion of 111In radioactivity was compared, no significant differences were observed in the urinary clearances. However, biliary excretion was significantly higher for 111In-SCN-BZ-EDTA-bound radioactivity. In conclusion, when compared with 125I, 111In labeling seems to more accurately characterize the in vivo distribution of liver-targeted proteins after their iv administration in rats and allows a more accurate pharmacokinetic evaluation to be performed.

Characteristics of the prolactin stimulation of iodide uptake into mouse mammary gland explants.

We have recently reported that prolactin (PRL) stimulates iodide uptake into cultured mouse mammary tissues. This effect occurs in both TCA soluble and insoluble tissue fractions. The effect of PRL apparently involves an RNA-DNA-dependent mechanism, since actinomycin D and cyclohexamide abolish the PRL stimulation of iodide uptake and its incorporation into protein. Perchlorate and thiocyanate, inhibitors of the iodide transporter, also abolish the PRL effects on iodide uptake and incorporation. Similarly, propylthiouracil and aminotriazole, inhibitors of peroxidase, abolish both effects of PRL. Finally, the extent of iodide uptake in mammary cells is suppressed by about 50% in sodium-free medium. These studies thus suggest the existence of a sodium-iodide symporter in the mammary gland which has characteristics similar to the iodide transporter in the thyroid gland-that is, it is sodium dependent and is inhibited by perchlorate and thiocyanate. The fact that both iodide transporter inhibitors and peroxidase inhibitors abolish PRL-stimulated iodide uptake and incorporation suggests that there may be a coupled mechanism involving the iodide transporter and the peroxidase enzyme.

An Index of Proteolysis Degree in Ewes' Milk and Ewes' Milk Yoghurt, by Single Strains and Combinations of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, Isolated from Traditional Greek Yoghurt.

The proteolysis degree in ewes' milk by 5 Streptococcus thermophilus and 4 Lactobacillus delbrueckii subsp. bulgaricus strains, as well as by their 20 combinations in ewes' milk yoghurt, was determined. Samples for analysis were taken after 24 h and 5 days' storage at 4°C. Determination of proteolysis degree in the 2% and 12% TCA soluble fractions was performed by the photometric ninhydrin method. The proteolysis degree varied among the single strains and their combinations. The proteolytic activity of Lb. bulgaricus strains was greater than or at the same level as the activity determined for Str. thermophilus strains. In yoghurt, the proteolysis determined was higher than that of the single strains. Changes in the proteolysis degree were also observed between the 24-h and 5-day samples. The highest total amount of free amino acids/peptides soluble in the 2% and 12% TCA fraction, in the 24-h as well as in the 5-day samples, was produced by Str. thermophilus strain CNRZ 404, Lb. bulgaricus strain CNRZ 1159 and the combinations CNRZ 1198/CNRZ 1159, ACA-DC 116/CNRZ 1159.

Evaluation of the 15N-isotope dilution technique for determining the recovery of endogenous protein in ileal digestion of pigs: effect of dilution in the precursor pool for endogenous nitrogen secretion.

Three barrows, average initial BW of 35 kg, were fitted with a re-entrant cannula at the distal ileum and catheters in each of the external jugular veins. The barrows were fed twice daily a diet containing barley as the sole source of protein. A 1-d intravenous infusion of saline was followed by an 8-d continuous infusion of [15N]leucine at a rate of 30 mg/(kg BW x d). Blood samples, taken hourly for 9 d, were pooled over the 12-h period between feeding. Ileal digesta were collected hourly on d 3 and 8 of the [15N]leucine infusion and pooled over 12 and 24 h. Enrichments (atom percentage excess 15N) in nitrogen (N), leucine, isoleucine, valine and alanine were measured in the trichloroacetic acid (TCA)-soluble and -insoluble fractions of plasma and in ileal digesta. Although the enrichments in N were similar in all pools, enrichments in amino acids in the TCA-soluble fraction of plasma were at least twice that in the TCA-insoluble fraction and in ileal digesta. The estimated contributions of endogenous N and amino acids to total N and amino acids in ileal digesta were 82.3 +/- 9.38, 39.0 +/- 3.83, 43.8 +/- 6.50, 51.9 +/- 3.24, and 51.9 +/- 9.80 for N, leucine, isoleucine, valine and alanine, respectively. The results from this study indicate that enrichments in total N in the TCA-soluble fraction of plasma may not accurately reflect enrichments in endogenous N secreted into the intestinal lumen. This factor should be considered when using the 15N-isotope dilution technique to estimate the recovery of endogenous protein in ileal digesta of pigs fed protein-containing diets.

Glycogen as Primordial Carbon Reserve andα-Glucosidase in the GeneraLynghya-Phormidium-Plectonema, Thermophilic Cyanobacteria

This work was done to characterize the structure of a photosynthetic polysaccharide and its metabolizing enzymes in cyanobacteria, which represent a link between bacteria and green plants in evolutionary terms. Filamentous cyanobacteria, occurring in an alkaline hot spring (45–50oC, pH 8.5–9.0) in Kagoshima Prefecture, were morphologically classified in the genera Lyngbya-Phormidium-Plectonema (LPP). We found a thermostable neutral (α-glucosidase with optimum pH 6.5 in the LPP. A polysaccharide isolated from the TCA-soluble fraction of the LPP was characterized as glycogen that resembled animal glycogen in structure. We also recognized the presence of the TCA-insoluble glycogen at 32–38% of the total amount of glycogen, most of which was bound non-covalently to protein and had a similar iodine absorption spectrum to that of the TCA-soluble glycogen.

Rapid plasma clearance of albumin-acrolein adduct in rats

Protein adducts are used as markers of chemical exposure. Determination of the clearance rate of these adducts from the blood circulation will provide the time frame for their measurement. Radioactive albumin was prepared biosynthetically by repeated intraperitoneal injections of l-[4,5- 3H]lysine to a rat. After an affinity purification, an aliquot of this native [ 3H-lysine]albumin was adducted with 5 mM acrolein. Both the native albumin (A-treated group) and the albumin-acrolein adduct (AAA-treated group) were intravenously injected to separate groups of rats, and the clearance of radioactivity from the plasma was measured as a function of time. At the end of the experiment (33 h after the injection), radioactivity in the whole plasma, and in homogenates of liver, kidney and spleen and their trichloroacetic acid(TCA)-soluble and -insoluble fractions in both A- and AAA-treated groups, was measured. The results, at the initial 11 h after the injection, showed that the radioactivity was cleared from the circulating plasma more rapidly in the AAA-treated group (32% of the injected radioactivity remained) than the A-treated group (52%). At 33 h after the injection, 22% of the injected radioactivity remained in the plasma in the AAA-treated group as compared to 32% in the A-treated group. The whole homogenates of liver and kidney and their corresponding TCA-soluble fractions showed higher radioactivity in the AAA-treated group as compared to the A-treated group. However, the TCA-insoluble fractions from livers and kidneys of the AAA-treated group showed lower radioactivity as compared to the A-treated group. These results indicated that the albumin-acrolein adduct was removed more rapidly from the circulation than the native albumin, and degraded more rapidly by the liver and kidney. There was no preferential removal or degradation of the adducted albumin by the spleen.

Identification of Proteolytic Activities of Gel-Degrading Factors in Three Lizardfish Species.

The inhibitory effects of several selected proteinase inhibitors, soybean trypsin inhibitor, leupeptin, antipain, E-64, NEM and EDTA, on myosin heavy chain (MHC) degradation of unwashed and washed meat gels from three lizardfish species, maeso or true lizardfish Saurida undosquamis, wanieso Saurida wanieso, tokageeso or shortfin lizardfish Saurida elongata, were investigated in order to identify the proteolytic activities of gel-degrading factors (GDFs) existing in mat. It was apparent that the proteolytic activities of GDFs responsible for MHC degradation of meat gels from all species at 40°C resulted from two myofibril-associated (Mf) type proteinases, mainly of serine proteinase and slightly of cysteine proteinase. In addition, as indicated by the amino acid patterns obtained from TCA-soluble fraction of meat gels with the presence of various inhibitors, these proposed proteinases were not distinct in these three species. With regard to the MHC degradation at 60°C, the proteolytic activity present in GDFs of maeso, proposed as myofibril-associated gel degrading factors (Mf-GDFs), belonged to cysteine proteinase while that of wanieso and tokageeso were sarcoplasmic-type serine proteinases. The Mf-GDF corresponding to the MHC degradation at 60°C, which occasionally occurs in wanieso meat gel, showed the proteolytic activities of serine proteinase.

Correlated biochemical and radioautographic studies of protein turnover in developing rat incisor enamel following pulse-chase labeling with L-[35S]- and L-[methyl-3H]-methionine.

The movement of proteins into and out of enamel was followed over time using a highly sensitive microprecipitation technique to quantify the amount of TCA-insoluble radioactivity present within small pieces of freeze-dried enamel and cells (enamel organ) dissected from the mandibular incisors of rats injected with L-[35S]-methionine. Conventional image processing techniques were also used to estimate the number of silver grains over enamel and cells in radioautographs of mandibular incisors from rats similarly injected with L-[methyl-3H]-methionine. Data from both techniques indicated that the average half-life for labeled proteins secreted into enamel was about 8.9 days. Typically, radioactive proteins accumulated in increasing amounts for 8 hours after which they were lost slowly up to 4 days and more rapidly thereafter when enamel formed during the secretory stage underwent maturation. The half-life for radioactive proteins in cells was only about 20.7 hours. No significant accumulation of radioactivity could be detected in the TCA-soluble or TCA-insoluble fractions of cells as enamel development proceeded. Results from this study suggest that radioautographs provide an accurate estimate of changes occurring to proteins in enamel and cells except at early time intervals (less than 1 hour) when a high percentage of total radioactivity is present within the TCA-soluble fraction of cells.

A comparative study of uptake and release of glutamic acid and kainic acid by blue mussel (Mytilus edulis L.)

Glutamic acid (OA) and kainic acid (KA) are structurally related to domoic acid (DOM), a neurotoxic amino acid that caused human food poisoning in 1987, following consumption of toxic mussels from Prince Edward Island, Canada. To study the difference in response of mussels to a physiological amino acid (GA) and a non-physiological amino acid (KA), GA and KA were presented to mussels in dissolved form and as a food encapsulated in liposomes. Kainic acid was absorbed from solution to a lesser degree (0.4%), while GA was taken up more readily (1.1%). Both amino acids were absorbed across the gastrointestinal epithelium. Kainic acid was concentrated in the kidney, whereas glutamic acid showed equal distribution in all visceral tissues. Glutamic acid was steadily released from mussel tissues, whereas KA resisted depuration. Over 80% of intracellular KA was associated with the TCA soluble fraction of digestive gland tissue, while about 42% of GA was associated with the TCA insoluble fraction. Although KA is structurally related to GA, it was handled differently by mussels, but in the same way as DOM, suggesting that non-protein amino acids are recognized as undesirable compounds.

A method for determination of macropeptide by cation-exchange fast protein liquid chromatography and its use for following the action of chymosin in milk

SummaryCation-exchange chromatography on a Mono S column (Pharmacia) was used to separate macropeptide from whey proteins. Macropeptide was eluted by 0·1 M-NaCl in a 20 mM-KCl–HCl buffer, pH 2. This technique was suitable for quantitative determination of macropeptide in rennet whey and also for following the action of chymosin on κ-casein in skim milk. Precipitation at pH 4·6 was used to remove residual caseins and to keep macropeptide in solution. In comparison with other methods for determining macropeptide, the present one eliminates the need for pretreatment of samples with trichloroacetic acid (TCA) and allows the recovery of all the macropeptide. Quantitative determination of macropeptide in the 8% TCA-soluble fraction by cation-exchange chromatography showed that only 50–75% of the macropeptide was recovered. This chromatographic technique could also be applied for isolating and producing whole macropeptide on a preparative scale.

Distribution of dietary limiting amino acid injected into the prepyriform cortex

Diffusion or metabolism of the dietary limiting amino acid (DLAA) in the prepyriform cortex (PPC) may account for the time lag between injection of the DLAA into the PPC and the increase in intake of an amino acid-imbalanced diet. Results from the injection of [3H]Leu +/- [14C]Thr (DLAA) into the PPC indicated rapid (less than 15 min) and limited diffusion (85-90% of recovered label was less than or equal to 1 mm from the injection site). 3H and 14C decreased in the trichloroacetic acid (TCA)-soluble fraction and increased in the TCA-insoluble fraction during the first 1.5 h and remained constant in the TCA-insoluble fraction 1.5-6 h after injection. An increase (approximately 50%) in 3H in the TCA-insoluble fraction was found less than or equal to 30 min after injection of the DLAA. There was no affect of the DLAA on 3H in the TCA-soluble fraction. These results indicated that a change in metabolism within the PPC may be responsible for the delay in onset of the feeding response after injection of the DLAA into the PPC.