Search

Coral holobionts are multi-species assemblages, which adds significant complexity to genotype-phenotype connections underlying ecologically important traits like coral bleaching. Small scale heterogeneity in bleaching is ubiquitous in the absence of strong environmental gradients, which provides adaptive variance needed for the long-term persistence of coral reefs. We used RAD-seq, qPCR and LC-MS/MS metabolomics to characterize host genomic variation, symbiont community and biochemical correlates in two bleaching phenotypes of the vertically transmitting coral Montipora capitata. Phenotype was driven by symbiosis state and host genetic variance. We documented 5 gene ontologies that were significantly associated with both the binary bleaching phenotype and symbiont composition, representing functions that confer a phenotype via host-symbiont interactions. We bred these corals and show that symbiont communities were broadly conserved in bulk-crosses, resulting in significantly higher survivorship under temperature stress in juveniles, but not larvae, from tolerant parents. Using a select and re-sequence approach, we document numerous gene ontologies selected by heat stress, some of which (cell signaling, antioxidant activity, pH regulation) have unique selection dynamics in larvae from thermally tolerant parents. These data show that vertically transmitting corals may have an adaptive advantage under climate change if host and symbiont variance interact to influence bleaching phenotype., (© 2022. The Author(s).)

This study used a digesta transfer protocol to determine the site of absorption of lysine synthesized by the gastrointestinal microflora of pigs. Eight pigs were used, four with reentrant cannulas in the terminal ileum, two with simple T cannulas in the terminal ileum, and two intact. All pigs were given, for 5 days, the same low-protein diet that included fermentable carbohydrates. The diet of two pigs with reentrant cannulas (donor) and of the two intact (control) pigs was supplemented with (15)NH(4)Cl. The two other pigs with reentrant cannulas (acceptor pigs) and those with simple cannulas (used to supply unlabeled digesta) were given the same diet but unlabeled NH(4)Cl. Ileal digesta were collected continuously from all of the reentrant cannulas and kept on ice. All digesta from each donor pig were reheated and returned to the distal cannula of its companion acceptor, whose ileal digesta were discarded. Unlabeled ileal digesta from the pigs with simple cannulas were instilled into the distal cannulas of the donor pigs. At the end of the experiment, the average (15)N enrichment in the plasma free lysine of control pigs was 0.0407 atom % excess (APE); that of donor pigs was 0.0322 APE (79% of controls), whereas that of acceptor pigs was only 0.0096 APE (24% of controls). Due to nitrogen recycling, acceptor pigs had labeled lysine in the digesta of the stomach and small intestine, and donor pigs had labeled lysine in the digesta of the large intestine. If account is taken of the higher (15)N enrichment of microbial lysine in the large compared with the small intestine, it can be estimated that >90% of the absorption of microbial lysine took place in the small intestine.

The synthesis of essential amino acids by the gut microflora of pigs, and their absorption, were assessed from the incorporation of (15)N from dietary (15)NH(4)Cl and of (14)C from dietary (14)C-polyglucose into amino acids in the body tissues of four pigs. Both (15)N and (14)C were incorporated into essential amino acids in body protein. Because pig tissues cannot incorporate (15)N into lysine or (14)C into essential amino acids, the labeling of these amino acids in body protein indicated their microbial origin. The absorption of microbial amino acids was estimated by multiplying the total content of each amino acid in the body by the ratio of the isotopic enrichment of the amino acid in the body to that in microbial protein. Because the ratio of (14)C:(15)N in body lysine was closer to that in the microflora of the ileum than to that of the cecum, absorption was assumed to take place exclusively in the ileum. The estimates of microbial amino acid absorption from (14)C-labeling were as follows (g/d): valine 1.8, isoleucine 0.8, leucine 2.0, phenylalanine 0.3 and lysine 0.9, whereas for lysine, the estimate from (15)N-labeling was 1.3 g/d. We conclude that the gastrointestinal microflora contribute significantly to the amino acid requirements of pigs.

Four rats were housed in cages with mesh floors; another four rats were housed in tubular anticoprophagy cages, in which they could not turn round to reach their own faeces. Both groups were fed for 6 d on a low-protein diet containing fermentable carbohydrates and 15NH4Cl. At the end of the experiment the rats were killed and their carcasses were homogenized, lysine was isolated by ion-exchange chromatography and its 15N enrichment measured by isotope-ratio mass spectrometry. The 15N enrichment in the lysine of the microbial fraction of faeces and the total amount of lysine in the body were also determined in order to estimate the amount of microbial lysine absorbed. The 15N enrichment in body lysine of non-coprophagic rats was not different from that previously measured in rats given unlabelled NH4Cl, but in coprophagic rats it was significantly higher. The daily absorption of microbial lysine by the coprophagic rats accounted for 20.7 (SE 2.55) mg/kg body weight0-75, but was only 0.5 (SE 1.04) mg/kg body weight0-75 for the non-coprophagic rats. This value was not significantly different from zero. The utilization of microbial amino acids via coprophagy resulted in a higher weight gain (adjusted for intake) in the coprophagic group (15.5 g/6 d) than in the non-coprophagic rats (3.1 g/6 d). It was concluded that, in rats, the utilization of microbial lysine occurred exclusively via coprophagy.

The absorption of lysine synthesised by the gastrointestinal microflora was estimated by comparing the 15N incorporated into body lysine in four germ-free (15N-GF) and four conventional (15N-CV) rats. They were fed for 10 d on a protein-free diet containing fermentable carbohydrates and 15NH4Cl; another four conventional rats (control), fed on the same diet but with unlabelled NH4Cl, were used to estimate the natural abundance of 15N. The eviscerated carcass of each rat was homogenized and a sample hydrolysed. Lysine was isolated by ion-exchange chromatography and its 15N enrichment was measured by isotoperatio mass spectrometry. The 15N-CV rats significantly incorporated 15N into their body lysine. The 15N-GF rats had a statistically significant, although small, incorporation of 15N into their body lysine, probably arising from a measurement artifact. It was concluded, therefore, that all [15N]lysine was of microbial origin. The total lysine content in the body and the 15N enrichment of lysine in the microbial fraction of the faeces of the 15N-CV rats were also determined. The amount of microbial lysine absorbed by the 15N-CV rats was estimated by dividing the total amount of [15N]lysine in the body by the enrichment of microbial lysine. It was estimated that the daily absorption of microbial lysine by the conventional rats was 21.3 (SE 2.04) mg/kg body weight0.75.

The rates of [3H]N(tau)-methylhistidine (3-MH) accumulation in the medium, following pulse labelling of cells for 48 h with [3H]methionine, were used to measure myofibrillar protein degradation. In fused C2C12 myotubes, incubation for 24 or 48 h after the labelling period gave rates of myofibrillar degradation of 38 and 42%/day. In a leucine free medium, these rates were similar; 40 and 47%/day, respectively. Using identical conditions +/- leucine, but in the absence of [3H]-methionine, rates of protein accretion and synthesis over 24-48 h were measured. From these data, rates of total protein degradation were calculated by difference and were similar to myofibrillar degradation rates. We have used the same pulse labelling protocol to assess whether the method is applicable to non-muscle cell lines based on the knowledge that 3T3 fibroblasts contain actin in the cytoskeleton. 3-MH was detected both in protein and upon its release into the medium. Actin degradation measured over a 48 h period gave a value half that obtained for total degradation, but the results suggest that the release of 3-MH by fibroblasts in vivo could be appreciable. The development of this methodology should provide a useful tool to investigate signalling mechanisms regulating actin degradation in a variety of cell types.

Nitrogen retention and lysine oxidation were measured in growing pigs given diets which supplied 0, 0.2 or 0.8 of the lysine requirement, with other amino acids in relative excess. Eight groups of three female littermate pigs were used: one of each group was given each of the three diets. In half the pigs (four groups) N retention was measured at body weights (W) of approximately 25, 35 and 45 kg. The other four littermate groups of three pigs were given the same three diets; when they reached 35 kg W they were given a continuous (6 h) primed infusion of L-[6-3H]lysine. Lysine oxidation was estimated from the production of tritiated water. Rates of both N retention and lysine oxidation increased significantly with lysine intake; mean values (g/kg (W)0.75 per d) for the three diets respectively were for N retention, 0.00, 0.32 and 1.22, and for lysine oxidation 0.051, 0.058 and 0.078. From the N balance results (assuming a constant lysine concentration in body protein) the efficiency of utilization of absorbed lysine was estimated to be 0.85; from the oxidation results (assuming lysine absorbed but not retained is oxidized) the estimate was 0.95.

Homogenates of semitendinosus muscle from malignant hyperthermia (MH)-susceptible pigs produced threefold more pentane than those from MH-resistant pigs, indicating enhanced free radical-mediated peroxidation of n-6 fatty acids. This did not reflect a deficiency in tissue antioxidants or antioxidant-enzymes but glutathione concentrations and glutathione peroxidase activities were increased in the tissue from MH-susceptible swine, consistent with an adaptive response to a sustained oxidant stress. A lower proportion of linoleic acid (18:2 n-6) in phospholipids and neutral lipids in muscle from MHS pigs indicated increased peroxidation or metabolism (desaturation and elongation). The increased oleic acid (18:1 n-9) in the MHS muscle indicated that desaturase activity was elevated in all lipid classes. The results are consistent with the hypothesis that enhanced free radical activity and lipid peroxidation contributes to the abnormalities in Ca2+ homeostasis and polyunsaturated fatty acid metabolism in MH.

L-Arginine concentrations have been measured in benign and malignant breast and colonic neoplasms and compared with the macrophage content and arginase activity within these tumors. Our study confirmed previous findings of elevated plasma arginine concentrations in malignancy and demonstrated that tissue free-arginine concentrations are substantially higher in malignant (mean 9.8 mumol/g protein) than benign (2.8 mumol/g protein) breast disease. Similarly, malignant colonic neoplasms had a higher free-arginine concentration than benign colonic polyps (14.0 vs. 7.0 mumol/g protein). The macrophage content of the malignant tumors was also significantly higher than in the benign conditions (278 vs 29/high power field in breast disease), but despite this, there was no detectable difference in the arginase activity. These findings suggest that tumor-infiltrating macrophages are not able to produce this enzyme, and/or its activity is inhibited within the tumor cell milieu. The differences observed in the arginine concentrations within these lesions has potentially important implications for the pathway of arginine metabolism and local host antitumor responses.

1. The validity of the urinary excretion of N tau-methyl histidine (N tau-MH) by pigs as an index of muscle protein breakdown in vivo was tested using the criterion of the rate of recovery of radioactivity in urine following an intravenous dose of N tau-[14CH3]methyl histidine. 2. Urinary recoveries of radioactivity from five animals were less than 21% of dose in 7 d after which the daily recovery was less than 0 . 3% per day. 3. The incomplete recoveries of radioactivity were associated with the presence in muscle of a large pool of non-protein-bound N tau-MH, the concentration of which increased with age. 4. The N tau-MH in this pool was present as free N tau-MH and in a dipeptide which constituted more than 90% of the total non-protein-bound N tau-MH. The contribution of the peptide increased with age, reaching 99. 8% in older animals. 5. The pool of non-protein-bound N tau-MH was maintained and increased in both established and newly accreted tissue by retention of some of the N tau-MH released by muscle protein breakdown, only a proportion of which was therefore available for excretion. Hence, the urinary excretion of N tau-MH is not a valid index of muscle protein breakdown in pigs.

1. The recoveries of radioactivity in cattle urine following the intravenous administration of N tau-[14CH3]methyl histidine were essentially quantitative in 5--7 d in non-lactating cows, bulls and steers and did not change with age. 2. The N tau-methyl histidine was excreted unchanged in urine. 3. N tau-methyl histidine occurred in muscle extracts both in the free form and as a perchloric acid-soluble, acid-labile form which accounted for approximately 85% of the total non-bound N tau-methyl histidine in muscle and appeared identical to a similar component identified in muscle extracts of sheep and pigs. 4. There was probably an age-related decrease in the concentration of the acid-labile component in muscle but which did not produce a measurable change in recovery of radioactivity in urine. 5. The daily excretion of N tau-methyl histidine (E, mumol) by male cattle was highly correlated with live weight (W, kg) by the equation: E = 50 . 4 + 3 . 536 (+/- 0 . 044)W (r 0. 997). The excretions progressively decreased from 4 . 04 mumol/d per kg at 100 kg weight to 3 . 62 mumol/d per kg at 600 kg. 6. By the criterion of the rate of clearance of labelled N tau-methyl histidine from the body, the excretion of N tau-methyl histidine in urine appears to be a valid index of muscle protein breakdown in cattle.

Excreta were collected for four consecutive days from 4- to 18-week-old cockerels following subcutaneous injection of N tau-[14CH3]methylhistidine. The recoveries of radioactivity in excreta were incomplete and progressively decreased with increasing age. Most of the radioactivity not recovered in excreta after 4 d was found in skeletal muscle where greater than 55% of the radioactivity present was in the N tau-methylhistidine-containing dipeptide, balenine. This peptide appeared to be relatively stable so that most of the labelled N tau-methylhistidine incorporated was not released during the period of the recovery measurements. The total pool of non-protein bound N tau-methylhistidine (free (free N tau-methylhistidine + balenine) in pectoral and mixed thigh muscles increased with age and relative to the daily excretion of N tau-methylhistidine. At 18 weeks the pool was 3.3 times the daily excretion of N tau-methylhistidine. These observations account for the decreasing recoveries of radioactivity in excreta described previously, due to progressive dilution of labelled N tau-methylhistidine in an expanding pool of non-protein-bound N tau-methylhistidine, part of which was relatively stable. It is concluded that excretion of N tau-methylhistidine by 4- to 18-week-old cockerels cannot be used as a reliable index of muscle protein breakdown in vivo.

The claim of Millward, Bates, Grimble, Brown, Nathan & Rennie [(1980) Biochem. J. 190. 225--228] that muscle actomyosin contributes as little as 25% of urinary N tau-methylhistidine is not consistent with other published data from that group [Bates, DeCoster, Grimble, Holloszy, Millward & Rennie (1980) J. Physiol. (London) 303, 41 P] or with literature values. It appears likely that the turnover rate of muscle actomyosin has been considerably underestimated and that when realistic rates of protein turnover are used, muscle tissue remains the major contributor of N tau-methylhistidine in urine.

The metabolism of N tau-[Me-14C]methylhistidine by mice was influenced by both the sex and the phenotype of the animal. After subcutaneous injection of labelled N tau-methylhistidine, recoveries of radioactivity in excreta were incomplete, and the distributions of radioactivity showed that different mouse phenotypes degraded N tau-methylhistidine in vivo to different extents. Hence the excretion of N tau-methylhistidine by mice cannot be used as an index of protein breakdown in vivo.

The digest of penicillopepsin (EC 3.4.23.7) with protease II from Myxobacter AL-1 gave five fragments which were separated on a Biogel P-100 column in 70% formic acid. The fragments were from 16 to 125 amino acids long. Two fragments were also isolated from a digest with a protease from Staphylococcus aureus. The analysis of these fragments by automatic sequencer gave a number of overlaps of the chymotryptic and thermolytic peptides. The available amino acid sequence data for penicillopepsin described in this paper and the accompanying papers (Kurosky, A. and Hofmann, T.:Can. J. Biochem. 54, 872 (1976);Rao, L and Hofmann, T.:Can. J. Biochem.54,885 (1976); Harris, C.I.,Rao, L., Shutsa, P., Kurosky, A. and Hofmann, T.: Can. J. Biochem. 54,895 (1976) have been combined and yield 15 fragments which range in lengths from 3 to 112 amino acid residues. These unique fragments account for virtually all the amino acids of the fungal protease. Four of the fragments with a total of 194 residues (about 60% of the molecule) have been aligned with corresponding sections of pig pepsin (EC 3.4.23.1) and with part of the N-terminal sequence available for calf chymosin (EC 3.4.23.4). In the alignments about 37% of the residues in the fungal enzyme are identical with at least one of the mammalian enzymes. An additional 20% are chemically similar. These results, together with previously reported active site directed modifications, show conclusively that penicillopepsin is an evolutionary homologue of the mammalian acid proteases.

1. The validity of the urinary excretion of N tau-methyl histidine (N tau-MH) in sheep as a measure of the breakdown of muscle protein in vivo was assessed from the urinary recovery of radioactivity following the intravenous administration of N tau-[14CH3]methyl histidine. 2. Recoveries of radioactivity in urine from animals of 4 weeks to 7 years of age were incomplete in 7 d but progressively increased with the age of the animal, becoming almost quantitative (90%) in older animals after recovery for 3 weeks. 3. The incomplete urinary recoveries were not due to partial excretion of N tau-MH in faeces or its oxidation and elimination in expired gases but were related to the presence in muscle of a pool of non-protein-bound N tau-MH which was several times larger than the expected daily urinary excretion. 4. This pool in newly accreted muscle tissue was maintained by retention of some of the N tau-MH released by breakdown of muscle protein. Hence, only a proportion of the N tau-MH released from protein breakdown was available for excretion. This proportion increased with the age of the animal and was probably the main determinant of the improved recoveries of radioactivity obtained in urine from older animals. 5. The non-protein-bound N tau-MH in muscle consisted of free N tau-MH and a dipeptide containing N tau-MH, the latter comprising on average approximately 82% of the total non-protein-bound N tau-MH in muscle. This proportion did not change appreciably with the age of the animal. 6. The dipeptide appeared to be synthesized in muscle from free N tau-MH and was not a terminal product of protein breakdown. 7. The results show that urinary excretion of N tau-MH is not a reliable index of muscle protein breakdown in sheep.

The mean molar ratio (0.0171) of 3-methylhistidine to creatinine in 24-h urine samples from carriers of Duchenne muscular dystrophy did not differ significantly from that of controls. The creatine/creatinine ratio was also normal.

The amino acid sequence of peptides isolated from a tryptic digest of penicillopepsin (EC 3.4.23.7), a subtilisin (EC 3.4.21.14) digest of maleylated penicillopepsin, and a chymotryptic digest of penicillopepsin modified with dinitrophenylsulfenyl (DNPS) chloride have been determined. The first two digests identified four of the five lysyl residues of the enzyme as well as the N-terminal peptide. The third digest provided overlaps at three of the tryptophanyl residues. The DNPS-tryptophan peptides were isolated on an affinity column prepared by coupling dinitrophenyl antibody raised in sheep to cyanogen bromide-activated Sepharose.

Isolated soleus and extensor digitorum longus muscles from small (40 or 70 g) rats developed a central and substantial (13-57%) loss of glycogen and alpha-glucan phosphorylase activity after incubation for up to 2 h in vitro. The central 'core' of the muscles showed a marked decrease in the rate of protein synthesis. It is suggested that during brief periods of incubation the central core of isolated rat muscles becomes hypoxic, and that consequently the viability of such muscles must be in question.

Twenty-two British Friesian bull calves were used in a comparative slaughter experiment to determine the effects of a beta-agonist (clenbuterol) on body composition and energy retention. Four calves were slaughtered at 18 d of age and constituted the initial slaughter group. Of the remaining calves, eight (group A, controls) were given milk replacer only, and ten calves (groups B and C, five calves per group) were given milk replacer plus clenbuterol (0.1 and 1.0 mg clenbuterol/kg milk replacer equivalent to approximately 2 and 20 micrograms/kg body-weight respectively over the 105 +/- 3 d of the experimental period). Calves were slaughtered over the weight range 146-177 kg. Clenbuterol had no significant effect on dry matter (DM) intake, daily live-weight gain or feed conversion ratio. DM digestibility of the milk replacer was not affected by treatment. Nitrogen balance was measured on three separate occasions starting when the calves weighed approximately 60, 110 and 130 kg. N retention was increased over the experimental period in clenbuterol-treated calves, although the effect only achieved significance in calves weighing approximately 110 kg live weight (P less than 0.05). Clenbuterol (20 micrograms/kg body-weight) increased estimated mean daily N retention in the carcass of the calves from 22 to 25 g whilst N retention in the non-carcass components decreased from to 8 g/d. Effects of clenbuterol on N retention occurred mainly in skeletal muscle. Fat in both carcass and non-carcass components was reduced by treatment with clenbuterol. The total energy content of live-weight gain was reduced from 1077 to 897 MJ in clenbuterol-treated calves and mean daily heat production was estimated to increase from 23.1 in controls to 25.9 MJ/d in calves in group C. In calves of mean live weight during balance of 120 and 136 kg, clenbuterol significantly increased daily urinary creatinine excretion and in 120 kg calves N tau-methylhistidine was significantly decreased (P less than 0.05). Based on estimates of muscle mass from urinary creatinine and protein degradation from N tau-methylhistidine excretion, the fractional breakdown rate of muscle protein in clenbuterol-treated calves was only 0.66 of that in the controls when the calves weighed 120 kg.

The occurrence of the dipeptide, balenine (beta-alanyl-N tau-methyl histidine), has been identified by comparative chromatography in extracts of muscles from 9 species of mammal, including man, and the chicken. The identity of the dipeptide from 6 mammalian species and chicken was confirmed by preparative isolation prior to determination of the amino acid composition and N-terminal residue. In all cases, the dipeptide contained equimolar amounts of beta-alanine and N tau-methyl histidine with beta-alanine as the N-terminus. The concentration of the dipeptide varied dramatically between species, being just detectable in the muscle of man and rat while present at concentrations of up to 14 mumol/g wet muscle in adult pigs. It is proposed that balenine, like carnosine and anserine, should be regarded as a normal constituent of muscle.

1. To study the potential of the constant-infusion technique for measuring rates of protein synthesis in New Zealand White rabbits, animals were infused for up to 6 h with radioactively-labelled tyrosine. 2. Labelled tyrosine from plasma and tissues was isolate from labelled metabolites by ion-exchange chromatography. 3. Analysis of serial blood and muscle biopsy samples removed under anaesthesia showed that the specific radioactivity (SR) of the free tyrosine pools reached an approximately constant value within 2 h. 4. Certain commercial preparations of L-[side-chain 2,3-3H]tyrosine were contaminated with 300 mg radioactive D-tyrosine/g. The D-isomer appeared to enter the muscle intracellular pool. 5. In constant-infusion experiments L-[3H]tyrosine could replace the uniformly-14C-labelled L-isomer for the determination of rates of protein synthesis in muscle. L-[side-chain 2,3-3H]tyrosine may not be suitable for use as a precursor for measuring rates of liver protein synthesis. 6. Evidence is presented that the precursor of liver protein synthesis may not be well defined by the SR for free tyrosine of the homogenate. 7. The technique was used to measure the rates of protein synthesis in adult rabbits. The rates of protein synthesis in liver and muscle were measured and from measurements of tyrosine flux the mean rate of whole-body protein synthesis was calculated as 13-8 g/kg per d.

1. The effects on growth performance, energy and nitrogen retention, and leucine metabolism of a subcutaneous combined implant of 140 mg trenbolone acetate (TBA)+ 20 mg oestradiol-17 beta (OE) have been examined in Hereford x Friesian beef steers (280-520 kg). Comparisons were made both with the same animals before implantation and with untreated control animals maintained under similar physiological and nutritional conditions. 2. Over a 10 week period the implanted steers showed an improvement in rate of live-weight gain (LWG) of 0.5-0.6 with an even greater proportional increase in N retention compared with control animals. Total energy retention was unaffected and thus the ratio, protein energy: total energy gain was 0.43 for implanted steers compared with 0.26 for untreated animals. 3. Estimates of protein synthesis and protein oxidation were obtained from the specific radioactivities of blood free-leucine and exhaled carbon dioxide during continuous infusions of [1-14C]leucine. Whole-body protein synthesis, based on metabolic size, and amino acid fractional oxidation remained similar for control steers throughout the experiment. Steroid-treated steers showed a slight decline in synthesis which was significant (P less than 0.05) at week +5 post-implant while amino acid oxidation was significantly lower at weeks +2 (P less than 0.01) and +5 (P less than 0.05) compared with control animals. The ratio, protein deposition: protein synthesis was 0.05 for control animals but 0.08-0.10 for steroid-treated animals after implantation. 4. There was a slight decrease in urinary N tau-methylhistidine elimination after implantation which suggested that muscle protein degradation may be reduced although the estimated decrease was insufficient to account for the total improvement in growth rate and N retention. 5. The results suggest that for both control and treated steers, less than 0.5 of total urine N elimination was derived directly from tissue catabolism of protein and amino acids. 6. The combined action of the exogenous steroids in the promotion of protein gain, primarily through a decrease in total protein degradation with little alteration of total energy retention, is compared with present understanding of the role of the endogenous sex hormones.

1. Adult rabbit skeletal-muscle myosin has been shown to contain 1.0 residue of mono-N(in)-methyl-lysine and 3.3 residues of tri-N(in)-methyl-lysine per molecule of molecular weight 500000. 2. The methyl-lysines appear to be located in the subfragment 1 portion of the myosin molecule. 3. Methyl-lysines are not present in actin, tropomyosin, inhibitory factor and calcium-sensitizing factor. 4. Enzymic methylation of histidine and lysine residues of myosin has been demonstrated in vitro. 5. The methylation of histidine and lysine of the total myofibrillar protein occurs after peptide-bond synthesis. 6. Although methylated lysines and 3-methyl-histidine could not be detected by analysis of hydrolysates, radiochemical evidence is provided for the presence of these residues in the soluble-protein fraction of rabbit skeletal muscle.

1. By the use of the extended elution system for basic amino acid analysis, 3-methylhistidine has been detected in hydrolysates of actin isolated from mammalian, fish and bird skeletal muscle. 2. Evidence is presented to indicate that 3-methylhistidine forms part of the primary structure and that in rabbit actin this residue is restricted to one peptide fraction obtained from the tryptic digest. 3. Rabbit skeletal-muscle actin has a 3-methylhistidine:histidine ratio 1:7.6, indicating a minimum molecular weight of 47600. 4. Adult rabbit myosin contains approximately 2 3-methylhistidine residues/mol. These residues are localized in the heavy meromyosin part of the molecule, and are restricted to the major component obtained after succinylation.