Liberated Amino Acids Research Articles

Changes in protein-bound and free amino acids in the muscle of the tiger prawn Penaeus esculentus during starvation

Using the starvation technique, changes in protein and free amino acids were examined in Penaeus esculentus Haswell collected from Moreton Bay, Australia, by trawling in 1985. Prawns of 17.7±0.26 g wet weight were held at 25°C until 2 d after moulting. Groups of seven or eight were then starved fro 5, 10, or 15 d, with appropriate control groups. At the end of each period, ecreted amino acids were collected for 24 h and whole-muscle amino acids and free amino acids (FAA) g-1 in each prawn were analysed. Concentrations of whole-muscle amino acids showed only minor changes with starvation, but concentrations of many of the FAA changed significantly. Total FAA averaged 1 182±45 μmol g-1 dry weight. Individual FAA, in order of abundance, were glycine, arginine, proline, taurine, threonine, hydroxyproline, alanine, glutamic acid, valine, aspartic acid and lysine; the remaining FAA each contributed <0.2% of the total. Only taurine and alanine did not show significant changes with starvation. Concentrations of glycine, arginine, hydroxyproline, glutamic and aspartic acid increased, while those of proline, threonine, valine and lysine decreased with starvation, that of proline approaching zero after 15 d starvation. Excreted amino acid-nitrogen represented <2% of excreted ammonianitrogen ornithine being the most abundant (35%), followed by leucine (22%) and lysine (17%). The relative abundance of excreted amino acids did not correspond with those of the FAA. It is suggested that, as starvation progresses, the muscle protein is progressively hydrolysed, but with the remaining muscle maintaining its amino acid composition. The liberated amino acids enter the FAA pool and become available for energy production. Proline may have an important role as an energy source, but the ability to synthesise proline may be limited, and thus the artificial food of penaeid prawns may be improved by its addition.

Photoproduction of amino acids by mutant strains of N2-fixing cyanobacteria

Mutant strains of the N2-fixing cyanobacterium bacterium Anabaena variabilis resistant to 6-fluorotryptophan or to ethionine were isolated. Many of these strains liberated amino acids into their media in the absence of 6-fluorotryptophan and ethionine. Nitrogenase activity was higher in mutant strains than in the parent strain. Mutant strains were immobilised in calcium alginate and sustained photoproduction of amino acids has been demonstrated.

Extracellular hydrolysis of formyl peptides and subsequent uptake of liberated amino acids by alveolar macrophages

The mechanism of accumulation of radioactive label from fNle-Leu-[ 3H]Phe by guinea pig alveolar macrophages was investigated. The binding of fNle-Leu-[ 3H]Phe to macrophages reached equilibrium within 5 min at 4°C, but equilibrium could not be achieved at temperatures where fNle-Leu-Phe stimulated superoxide anion production is observed (e.g., 21–23°C). At this temperature a rapid phase of initial binding of fNle-Leu-[ 3H]Phe to its receptor was followed by continued accumulation of cell-associated radioactivity which was linear and was dependent on the extracellular pH, i.e., the rate increased as the pH was lowered from pH 8 to pH 6. Examination for possible intracellular hydrolysis of fNle-Leu-[ 3H]Phe revealed the presence of extensive amounts of [ 3H]phenylalanine, both cell-associated and in the medium. The increases in cell-associated [ 3H]phenylalanine correlated in time and pH with cell-associated radioactivity that was accumulated after stimulation with fNle-Leu-[ 3H]Phe. The addition of 1 mM unlabelled phenylalanine blocked the long term accumulation of label from fNle-Leu-[ 3H]Phe by macrophages. 1 mM phenylalanine had no measureable effect on fNle-Leu-Phe stimulated O − 2 production, fNle-Leu-[ 3H]Phe hydrolysis or on fNle-Leu-[ 3H]Phe binding to its receptor. These results indicated that the long term accumulation of radioactivity by alveolar macrophages was due to extracellular hydrolysis of fNle-Leu-[ 3H]Phe followed by transport of liberated [ 3H]phenylalanine into the cells. A high affinity ( K m = 3.56·10 −8 M) transport system for phenylalanine was measured in alveolar macrophages, which was not stimulated by the addition of fNle-Leu-Phe. The extracellular hydrolysis of fNle-Leu-[ 3H]Phe could not be attributed to release of macrophage enzymes into the medium. The responsible proteinase appears to be membrane bound and has a K m for the hydrolysis of fNle-Leu-[ 3H]Phe of 2.6·10 −7 M which is similar to the K d (1.5·10 −7 M) for fNle-Leu-Phe binding. Taken together, these data suggest that for the alveolar macrophage: (1) formyl peptides are not internalized by a receptor-mediated process; (2) a surface proteinase can catalyze the hydrolysis of formyl peptides; and (3) [ 3H]phenylalanine formed by fNle-Leu-[ 3H]Phe hydrolysis is transported into the interior of the macrophage.

In vitro degradation of angiotensin II (A-II) by human placental subcellular fractions, pregnancy sera and purified placental aminopeptidases.

The degradation of angiotensin II (Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8: A-II) by human placental particulate and soluble fractions, pregnant and non-pregnant sera, and highly purified placental enzymes such as placental leucine aminopeptidase P-LAP (microsomal), retroplacental serum P-LAP (soluble), aminopeptidase A and post-proline endopeptidase, was studied by measuring liberated amino acids by high performance liquid chromatography. Placental particulate and soluble fractions degraded A-II almost completely into single amino acids. The purified P-LAP (microsomal) actively liberated five amino acids from the N-terminal. The placental particulate fraction containing P-LAP (microsomal) also actively liberated these amino acids. The purified aminopeptidase A liberated Asp1 very actively as expected. When the ratio of the velocity of liberation of each amino acid to P-LAP activity measured with leu-p-nitroanilide as a substrate was calculated, placental soluble fraction liberated Asp1 very actively, but the liberation rate of Asp1 with the purified P-LAP (soluble) was very low. Therefore it seems that the enzyme in the placental soluble fraction and pregnancy serum responsible for the Asp1 liberation is not P-LAP (soluble), but aminopeptidase A. The mixture of purified P-LAP (soluble) and aminopeptidase A showed higher liberation rate of Arg2 and Val3 than that with purified aminopeptidase A alone, demonstrating that once the N-terminal Asp1 was liberated, the P-LAP (soluble) attacks the shorter peptide (angiotension III) very actively. It was concluded that P-LAP (microsomal) together with aminopeptidase A seem to contribute greatly to the degradation of A-II in pregnant women.

On the purification and characterization of three anionic, serine-type peptide hydrolases from antarctic krill, Euphausia Superba

Abstract 1. 1. Three enzymes effecting hydrolysis of TAME have been purified from E. superba using gel filtration, affinity chromatography and FPLC-anion exchange chromatography. 2. 2. The enzymes had molecular weights of 30,000 (Enzyme I), and 31,000 (Enzymes II and III), respectively, as estimated from SDS-PAGE. 3. 3. pH-optima of about 8.2 were observed for all three enzymes. 4. 4. The enzymes were inhibited by phenyl methyl sulphonyl fluoride (PMSF), tosyl lysyl chloromethyl ketone (TLCK) and soybean trypsin inhibitor (SBTI), whereas tosyl phenyl chloromethyl ketone (TPCK) had no effect on the activity. 5. 5. The enzymes were stable at neutral pH, and only slowly inactivated at highly alkaline pH. Low pH rapidly inactivated all three enzymes. 6. 6. Enzyme I liberated amino acids from casein, whereas Enzymes II and III did not; the latter two being true endopeptidases which effected enhanced amino acid production from casein when mixed with Enzyme I.

The enzyme stability of dehydro-enkephalins

Dehydro-enkephalins [ΔAla 2]-, [ΔAla 3]-, [ΔPhe 4]-, and [ΔLeu 5]enkephalins, were examined for their stability to enzymatic hydrolysis by carboxypeptidase Y [EC 3.4.16.1]. The successively liberated amino acids were determined quantitatively by amino acid analyses. The saturated leucine-enkephalin was rapidly hydrolyzed from the COOH-terminus. However, peptide linkages with α,β-dehydroamino acid residues placed in the enkephalin molecule were strongly resistant to the enzyme at the carboxyl side and completely resistant at the amino side of the dehydro residue.

Fluorimetric assay for prolinase and partial characterisation in cultured skin fibroblasts

A sensitive assay for the estimation of prolinase has been developed, using o-phthaldialdehyde to detect the liberated amino acid. Substrates of the form proline-X (X = amino acid with one amino group) can be used. Prolinase can be estimated in cells used for diagnostic purposes with as little as 1 μg cell protein. Partial characterisation of the enzyme indicates the presence of at least two forms of the enzyme in skin fibroblasts which needs consideration when testing for a prolinase deficiency.

Protein degradation in the mouse visual system I. Degradation of axonally transported and retinal proteins

The analysis of proteolysis in the nervous system is complicated by the heterogeneity of cell types, extensive reutilization of liberated amino acids, and artifacts that may arise when the integrity of the tissue is disrupted during experimentation. For these reasons, changes in proteolytic activity that are observed during brain development and in neuropathological states may often be difficult to interpret. To minimize these problems, we have developed a technique that permits protein degradation to be investigated specifically within axons of the mouse retinal ganglion cell (RGC). In the present study, the method has been used to examine the degradation of proteins conveyed in the slow phases of axoplasmic transport. When adult C57B1/6J mice were injected intravitreally with L-[ 3H]proline, labeled proteins within the primary optic pathway (optic nerve and tract) after 5 days were almost exclusively the slow phase axonal proteins. The rate of degradation of these proteins was then determined within the excised, but otherwise intact, optic pathway by measuring the release of acid soluble radioactivity at 37°C in vitro. At physiological pH, the amino acids released by proteolysis were extensively reutilized. Unless amino acid reutilization was prevented, protein degradative rates were artifactually lowered 3-fold. At least two proteolytic systems within RGC axons actively degraded the slowly transported axonal proteins. A ‘neutral’ system, stimulated by exogenous calcium ions, was optimally active within the physiological pH range (pH 7.0–7.8). The rate of protein degradation at pH 7.4 was uniform along the RGC axon. An ‘acidic’ system was optimally active when the incubation was carried out at pH 3.8. This proteolytic activity was calcium-independent and exhibited a proximodistal gradient within the RCG axon with higher activity proximally. Similar proteolytic activities were present in isolated intact retinas but in different proportions. The half-lives of axonal and retinal proteins were comparable to CNS protein half-lives estimated in vivo by methods that take amino acid reutilization into account. These and other recent findings demonstrate the utility of this neuron-specific approach in characterization proteolytic processes within one cell type that may otherwise be obscured by proteolytic events in other cells when brain tissue is analyzed by conventional methods.

Degradation of soluble and insoluble proteins by Bacteroides amylophilus protease and by rumen microorganisms.

Various soluble and insoluble proteins (6.25 mg) were incubated at 37 C with partially purified protease from Bacteroides amylophilus (156 micrograms) in 2.0 ml of .1 M potassium phosphate buffer, pH 7.6, for 2, 4, 6 and 18 hr, and the liberated amino acids were determined by the ninhydrin method. Results showed that (1) although soluble, serum albumin and ribonuclease A were resistant to hydrolysis; (2) soluble and insoluble proteins of soybean meal were hydrolyzed at almost identical rates; (3) soluble proteins from soybean meal, rapeseed meal and casein were hydrolyzed at different rates, and (4) treatment of resistant proteins (serum albumin, ribonuclease A and insoluble fish meal and rapeseed meal proteins) with mercaptoethanol in 8 M urea or oxidation with performic acid rendered these proteins susceptible to hydrolysis. It is concluded that (1) solubility or insolubility of a protein is not by itself an indication of the protein's resistance or susceptibility to hydrolysis by rumen bacterial protease; (2) structural characteristics of the properties which renders feed protein resistant to degradation is the presence of crosslinking disulfide bonds.

Automated colorimetric determination of acid proteinase activity in fermentation samples using a trinitrobenzenesulphonic acid reagent.

An automated method for the determination of acid proteinase activity has been developed. The enzyme is incubated with a haemoglobin substrate after which the reaction solution is dialysed. The concentration of the liberated amino acids and peptides is determined by reaction with a trinitrobenzenesulphonic acid reagent. The technical parameters of the process and the general analytical conditions have been optimised. The method offers great advantages in its flexibility of selecting substrate and buffer independently of the conditions for the colour reaction. The final result is an automated method with high sensitivity, precision and speed. The sensitivity of the colour reaction is 1 mg l–1 of nitrogen from leucine.

Alkaloid production by some local fungi

Summary The ability of five different local fungi to synthesize ergoline-alkaloids was studied. High alkaloid production was confined to a strain of Cunninghamella blakesleana where 0.13 % alkaloids were formed. TLC analysis of the alkaloids produced by the different organisms showed that Sepdonium sp., C. blakesleana, and Curvularia lunata could synthesize both peptidetype and clavine alkaloids. The identity of each individual peptide type alkaloid was confirmed by acid hydrolysis and by identification of the liberated amino acids.

Purification and some properties of a new metallo carboxypeptidase of Streptomyces griseus K-1.

A new carboxypeptidase was isolated from a mixture of proteolytic enzymes of St. griseus K-1 (pronase) and purified about 30-fold by a procedure including CM-cellulose chromatography, D-Arg-CH-Sepharose chromatography, Sephadex G-100 gel filtration, and ST-Sepharose chromatography with a yield of 55%. In these purification procedures, two kinds of substrates, Cbz-Gly-Leu and Bz-Gly-Arg, which are representative substrates of mammalian pancreatic carboxypeptidase A [EC 3.4.12.2] and B [EC 3.4.12.31], respectively, were used to detect the activity. The activities towards the two substrates were eluted at the same position, and the ratio of the activity with Cbz-Gly-Leu to that with Bz-Gly-Arg was always constant. On isoelectric focusing, the purified enzyme protein appeared as a single symmetrical peak at pH 5.2, and the two activities overlapped completely. Metal analysis of the enzyme showed 0.88 zinc atom per mol of the enzyme; its molecular weight determined by the sedimentation equilibrium method was 34,000. In studies on the enzymic properties, such as pH optimum, pH stability, stability at various temperatures, effect of buffer composition, and optimum activity temperature, no difference between the two activities could be observed. Reversible inhibitions of the activities towards Bz-Gly-Leu and Bz-Gly-Lys by reaction products were studied. Lys or Leu showed competitive-type inhibition and the K1 values for the two activities were parallel. The carboxypeptidase activity was tested with reduced and carboxymethylated pancreatic ribonuclease. The enzyme liberated amino acids sequentially, except for Pro. These observations led us to conclude that the carboxypeptidase of St. griseus K-1 has a new substrate specificity which includes the specificities of mammalian carboxypeptidases A and B.

Purification and properties of an aminopeptidase from rabbit skeletal muscle.

An aminopeptidase active on L-Val-L-Val-L-Val-L-Ala was purified from rabbit skeletal muscle by the method including ammonium sulfate precipitation, DEAE-cellulose chromatography, gel-filtration on Sephadex G-200, rechromatography on DEAE-cellulose, hydroxylapatite chromatography and rechromatography on Sephadex G-200. Polyacrylamide gel disc electrophoresis showed that the enzyme thus obtained was homogeneous. The specific activity of the purified enzyme was 1500 times that of the original muscle extract. The enzyme had an optimal pH in a range of 6.0_??_7.6 and was stable in pH 6.1_??_8.1. Molecular weight of the enzyme was estimated to be 160, 000 from the result of gel-filtration on Sephadex G-200. The enzyme showed specificity for tri-, tetra-, penta-, and hexapeptides. The analytical data of liberated amino acids showed that the enzyme was an aminopeptidase active on these oligopeptides. The enzyme was strongly inhibited by N-ethyl-maleimide and EDTA.

Absorption of two Tripeptides by the Human Small Intestine: A Study Using a Perfusion Technique

1. A double-lumen perfusion technique has been used in man to study intestinal absorption of two tripeptides, glycyl-glycyl-glycine and l-alanyl-glycyl-glycine. Intraluminal tripeptide hydrolysis has been investigated by incubating both tripeptides in vitro with fresh uncentrifuged saline aspirates. 2. Glycine was absorbed faster from a glycyl-glycyl-glycine solution (3·3 mmol/l) than from an equivalent (10 mmol/l) glycine solution, and both l-alanine and glycine were absorbed faster from 10 mmol/l l-alanyl-glycyl-glycine than from the equivalent amino acid mixture. Free amino acids and dipeptides were aspirated during the tripeptide perfusions. 3. l-Alanyl-glycyl-glycine was hydrolysed faster than glycyl-glycyl-glycine in vitro, and results indicated that hydrolysis of the tripeptides occurred from the N-terminal end of the molecule. Intraluminal tripeptide hydrolysis was not sufficient to account for the concentrations of free amino acids and dipeptides liberated during the tripeptide perfusion experiments in vivo. 4. It is suggested that the liberated amino acids and dipeptides either diffused back into the luminal contents after hydrolysis in the brush border had taken place, or effluxed from the mucosal cell after intracellular tripeptide hydrolysis.

Surface aminopeptidase in Moniliformis dubius and its relation to amino acid uptake.

Surface aminopeptidase (APase) in the acanthocephalan, Moniliformis dubius, was studied using leucylleucine, leucylglycylglycine, glycylglycine, alanylalanine, tri-alanine and tetra-alanine as substrates. In the presence of intact worms all peptides, except glycylglycine, were hydrolysed liberating amino acids and/or peptide subunits into the incubation medium. In 5 min incubations with 5mM leucylleucine, 93% of the liberated leucine was absorbed by the worms indicating a kinetic advantage for absorption of the leucine. In media with leucylleucine plus 10 mM methionine as an inhibitor of leucine uptake, the liberated leucine was not absorbed by the worms, but accumulated in the incubation medium. The inhibitory effects of leucylleucine and alanylalanine (or products of hydrolysis) on the uptake of 0·1 mM [14C]leucine in 2 min incubations were examined. Inhibition of [14C]leucine uptake by these peptides was non-linear up to 5 mM inhibitor concentration. Concentrations of either peptide greater than 5 mM did not produce further inhibition. A significant portion of [14C]leucine transport was not inhibited by these peptides. Inclusion of 0·5 mM Pb2+, an APase inhibitor, blocked the inhibition of [14C]leucine uptake by leucylleucine indicating that the inhibition is caused by the liberated leucine alone, and that interaction of the intact dipeptide and the leucine transport system is negligible. It is concluded that the surface membrane of adult worms has APase which can hydrolyse peptides in the ambient medium. The spatial arrangement of the APase and the leucine transport system is such that the hydrolysis of a peptide confers a kinetic advantage for absorption of the liberated amino acids. Encysted or mechanically excysted cystacanth larvae from the haemocoele of the cockroach showed no APase activity. Activation of the APase required a 30 min treatment in lipase, [Na+]taurocholate or other surface active agents. Since the APase in adult worms was unaffected by washing, the data strongly suggest that this enzyme is of parasitic origin.

Studies on the Utilization of Single Cell Proteins

To evaluate single cell proteins as food resources, both the baker's yeast (Saccharomyces) and the yeast of Candida family grown on n-paraffin were comparably subjected to the study. Extraction with heated alkali and successive precipitation at about pH 4.5 were tested to isolate protein from the cells of the above stated yeast. From the evidences experimentally obtained here, conditions in question in the present study, regarding the strength of applied alkali and its tempeature to obtain the hignest recovery of protein from the cell, were found to be as follows; extraction at 80° to 90°C for 30 to 60 minutes with 0.1 to 0.25N sodium hydroxide (approximately 100mg per g of the dried cell) was found to be the most favorable when the resulted liquid was examined in the soluble nitrogen, liberated amino acid potency, ammoniacal nitrogen level, and the total nitrogen of trichloracetic acid precipitates, of tungstate precipitates or of isoionic precipitates as the criteria. Conditions for extraction from Candida yeast were slightly milder than those conditions required for baker's yeast. Inferior extraction yield was observed in harder dehydrating conditions at 130°C of cell. Over 80% crude protein and crude ash less than 0.1% were recorded with the pH 4.5 isoionic precipitates from the supernatant liquid treated at 80°C for 60 minutes with 20 folds of 0.125N sodium hydroxide solution.

麹酵素による蛋白質の分解(第2報)

Oryzenin, the principal protein of milled rice, was fractionated into 3 fractions by Sepharose 4B gel filtration. The hydrolysis of oryzenin by the acid protease prepared from Taka-diastase was examined at several pH; 3.0, 4.0 and 6.0. (1) Optimum pH of the acid protease for the hydrolysis of oryzenin was found to be near 3.0. (2) Ratios of lower molecular peptides (phosphotungstic acid-soluble fraction) to higher molecular peptides (trichloroacetic acid-soluble fraction) liberated from oryzenin by the enzyme were determined occasionally during the long time reactions and it was found that the ratios were higher when reacted at pH 4.0 or 6.0 than at pH 3.0. (3) Elution patterns of the protein fractions of the reactants from Sepharose 4B column were obtained at times during the reactions. The gel filtration patterns were somewhat different depending on the reaction pH; Fraction I (eluted at void volume and considered to be the largest molecular fraction) disappeared rapidly and completely when reacted at pH 4.0 and the disappearance of Fraction II (eluted succesive to Fraction I) was comparatively rapid at pH 3.0. (4) Lower molecular fractions of the reactants were occasionally fractionated by Sephadex G-15 gel filtration during the reactions. It was also found that the gel filtration patterns were somewhat different depending on the reaction pH; When the protein was reacted at pH 3.0, there appeared several peaks in the gel filtration pattern between the high molecular fraction (void volume) and the small molecular fraction (eluting out volume)mainly consisted of free amino acids presumably, while these peaks were not appeared at all when the protein was reacted at pH 4.0 or 6.0. (3) Amino acid liberation patterns were again different depending on the reaction pH; Amounts of the liberated amino acids were larger when reacted at pH 4.0 or 6.0 than at pH 3.0.

Digestion of gluten peptides by normal human jejunal mucosa and by mucosa from patients with adult coeliac disease.

1. The ability of homogenates of jejunal mucosa to liberate amino acids from a peptic-tryptic digest of gluten was assessed. Mucosal specimens were obtained from thirty-two control subjects without malabsorptive disease, from twenty-six patients with untreated adult coeliac disease and from nine patients with adult coeliac disease in whom the intestinal mucosa was histologically normal as a consequence of treatment with a gluten-free diet. In addition nine of the untreated patients were restudied after institution of a gluten-free diet. 2. The ability of the jejunal mucosa from the patients with untreated adult coeliac disease to liberate amino acids from the gluten peptides was significantly less than that of the mucosa from control subjects. 3. No significant difference from normal was found when jejunal mucosa from patients with treated adult coeliac disease was studied irrespective of whether or not the mucosa was histologically normal. 4. These results indicate that the impairment of jejunal mucosal digestion of gluten in untreated adult coeliac disease is a secondary phenomenon and do not support the hypothesis that coeliac disease is due to the absence from the intestinal mucosa of an enzyme normally concerned in the digestion of gluten.

Alteration of Electrophoretic Pattern of Plasma Aminopeptidase in Avian Erythroblastosis

AMINOPEPTIDASE activity in starch gel electropherograms has been demonstrated indirectly and directly using a chromogenic substrate. In the indirect method the enzyme is first eluted from sections of the gel after completing the electrophoreticrun (Kowlessar et al., 1960). The enzyme activity is then measured in the eluates. In an established direct method there is visualization of enzyme activity using the chromogenic substrate, l-leucine β napthylamide (Lawrence et al., 1960). Another direct method for the demonstration of peptidase activity is based on the peroxide-forming activity of l-amino acid oxidase with the liberated amino acid (Lewis and Harris, 1967).Modifications of these two direct visualization techniques have been used in this laboratory to demonstrate aminopeptidase activity in the plasma of normal birds and birds with erythroblastosis (induced with Strain R virus). In the first method, after the runs with starch gel electrophoresis were completed, the gels were incubated in 0.1 M tris…

Hydrolysis of fat and protein in small cheeses made under aseptic conditions

SummaryAseptically drawn milk was made into small cheeses (100 g) under aseptic conditions. The influence of the cow's feeding régime on the composition of the cheese fat, and the hydrolysis of the cheese fat and protein by bacterial and native milk enzymes, was studied.In the absence of a strongly lipolytic bacterial cheese flora, the free fatty acid (FFA) content of cheese appeared to depend on several variables:1. The initial FFA content of the milk varied according to the mode of milking (the lowest was obtained by cannulation of the udder) and the feeding régime of the cow. When one of a pair of monozygotic twin cows was maintained on pasture and the other on winter feed (hay and concentrates), the cheese made from the milk of the latter contained less FFA than did that from the former.2. The major changes in the composition of the milk fat of the cows on winter regime were an increase of myristic and palmitic acids from 8 to 11% and 21 to 28%, respectively, and a decrease in stearic and oleic acid from 11 to 7·5% and 38 to 28%; minor changes in the other fatty acids were also observed.3. The milk lipase in raw milk cheese liberated substantially more FFA than did the weakly lipolytic lactic acid streptococci. Milk lipase was active at the low pH value of cheese, but was inactivated when the milk was pasteurized at 63°C for 30 min.4. Although rennet did not produce any amino acids in the mature cheeses it hydrolysed some of the cheese protein to nitrogen soluble in water (WS), trichloroacetic acid (TCA-S) or phosphotungstic acid (PTA-S). Some of the WS-N appeared to be utilized by the lactic acid streptococci. The native milk protease resisted the heat treatment used and liberated amino acids at the low pH value of cheese, but its contribution to the proteolysis was relatively less important than the contribution of the milk lipase to the fat breakdown in cheese.5. The free amino acids of the milk, which appeared in the whey, were not recovered in the cheese. The cheese made from unheated milk with δ-gluconic acid lactone contained low amounts of free amino acids apparently due to the action of milk protease. The proteolytic activity of the lactic acid bacteria increased the amino acid content appreciably.