Proteolytic activity of ruminal digesta during the feeding cycle in sheep receiving grass hay/concentrate or maize silage/concentrate diets

Proteolytic activity was measured by the digestion of 14C-labelled casein in digesta removed from the rumen of four sheep receiving a grass hay/concentrate diet and four sheep receiving a maize silage/concentrate diet. Samples were removed immediately before feeding and at 2-h intervals after feeding up to 12 h. Animals on both diets produced similar proteolytic activities (1.83 (S.D. 0.41) and 2.14 (S.D. 0.61) mg 14C-casein hydrolysed (ml ruminal fluid)-1 h-1 with the maize silage- and grass hay-based diets, respectively). Time after feeding had no effect on proteolytic activity, but between-animal variation was consistent and highly significant, with the highest-activity animals having activities 64 and 74% higher than the lowest-activity animals on the two diets, respectively.     

Natural variability and diurnal fluctuations within the bacteriophage population of the rumen.

To investigate the impact of nutritional and environmental factors on bacteriophage activity in the rumen, it is first valuable to determine the extent of natural variations and fluctuations in phage populations from different animal species, and from animals located together and separately, and variation in animals over time. Differences in phage populations between sheep on different diets, between sheep and goats, and within the rumen over time were investigated by using pulsed-field gel electrophoresis and comparing total phage DNA in ruminal fluid. It was found that no two individuals had similar DNA banding patterns, even when similarly fed and penned together, indicating there is considerable individual diversity in phage populations between animals. Despite these individual differences, the quantities, but not the banding patterns, of phage DNA were similar for animals within groups but varied between groups, suggesting that nutritional factors may influence overall phage activity in the rumen. In sheep fed once daily, a distinct diurnal variation in the phage population was observed. Two hours postfeeding, total phage DNA dropped to its lowest level. The phage population then increased, reaching a maximal level 8 to 10 h postfeeding before declining over the next 4 h to reach a stable concentration for the rest of the cycle. The general trend in phage DNA concentration appeared similar to previously recorded diurnal fluctuations in ruminal bacterial populations in cattle fed once daily.     

Specificity of Rumen Ciliate Protozoa in Cattle and Sheep*

SYNOPSIS. Six protozoa-free sheep, 3 fed alfalfa hay and 3 fed a concentrate diet, were inoculated with rumen contents from a steer fed the same alfalfa hay. All 24 species of protozoa in the inoculum became established in the sheep fed alfalfa hay, while only 9 species established in the sheep fed concentrate. Percentage species composition in the alfalfa-fed sheep was fairly similar to that of the inoculum. Rumen volumes of the alfalfa hay-fed sheep were significantly higher than those of the concentrate-fed sheep; however, fluid turnover rates were similar. Total protozoan numbers per ml of rumen contents were significantly higher in the concentrate-fed sheep, but after adjustment for rumen volume, there was no significant difference in the total number of protozoa in the rumen.     

Sulphate recycling and metabolism in sheep and cattle.

Merino wethers and Brahman x Shorthorn steers, offered lucerne or spear grass hay, were used to study the movements of sulphate through pools in plasma and ruminal liquor. The irreversible loss of sulphate from ruminal liquor was 60 and 76% of sulphur ingested for both species fed lucerne and spear grass respectively. The irreversible loss of sulphate from the plasma averaged 67 and 56% of sulphur ingested for animals fed lucerne and spear grass respectively. Daily recycling of sulphate to the rumen of sheep was 98 mg sulphur on the lucerne diet and 3.9 mg sulphur on the spear grass diet. Sulphate recycling in cattle fed lucerne was 533 mg sulphur; in cattle fed spear grass the value was 234 mg sulphur. Over 6 days following an intravenous injection of [35S]sulphate into sheep and cattle fed lucerne, 5-10% of the dose was excreted in the faeces and c. 10% was retained. Corresponding values for animals fed spear grass were 23-31% in faeces and 40-51% of the dose retained. After intraruminal injections of [35S]sulphate, animals fed lucerne excreted 15-18% of the dose in the faeces and retained 25-30% of the dose over 6 days. Values for animals fed spear grass were 22-26% in faeces and 62-70% retained. It was concluded that sulphate recycling to the rumen is a limiting factor in microbial synthesis for sheep fed low-quality roughage, and that secretion of endogenous sulphur into the postruminal tract of ruminants is of importance in the metabolism of sulphate.     

The chemical composition of Phyllanthus discoideus and its effect on the ruminal ammonia and volatile fatty acid concentration when fed to west african dwarf sheep

The feeding value of Phyllanthus discoideus (also called Margaritaria discoidea) leaves was evaluated using eight two‐year‐old West African Dwarf sheep fed natural grass hay. Four of the animals were fistulated ruminally and used for ammonia and volatile fatty acid determination in the fluid. Dried leaves of Phyllanthus discoideus were offered at two levels (25% and 50% of DMI, diets D25% and D50%, respectively) as supplements to the basal hay diet. The CP content of the control, D25% and D50% diets were 11.5, 12.6 and 13.6%, respectively, and their digestible energy amounted to 58.2, 61.1 and 56.9%, respectively. Rumen liquor was sampled one hour before and one, three and five hours after the morning feeding.

Sheep fed the control diet had a higher ruminai ammonia concentration than those fed diet D25%. Similarly, ruminai ammonia concentration was higher in sheep fed the control diet than those fed the diet D50%. Five hours after feeding the ruminai ammonia concentration was significantly lower than one hour after feeding.

The VFA concentrations in rumen fluid of sheep fed the control diet was inferior to those fed diets D25% and D50%. Sheep fed diet D50% showed significantly higher VFA concentrations than those fed diet D25%. Digestibility of organic matter and digestible energy did not show any significant difference. However, a marginal increase in organic matter digestibility of 3.5% was observed in diet D25% compared with the control diet. There was no significant difference in the N‐digestibility in sheep fed the control, D25% and D50% diets. Nevertheless, a marginal improvement in N‐digestibility (1.5%) and N‐retention (2.7%) was observed with the highest level of Phyllanthus discoideus (D50%).

In conclusion, Phyllanthus discoideus appears as a particularly valuable feedstuff because it contains low levels of condensed tannins (12.8 g/kg), high CP content (156 g/kg) and a relatively high GE content (19.3 kJ/gDM). Although the improvement in N‐digestibility and N‐retention were only marginal the feeding of Phyllanthus discoideus could be justified under extreme shortage of feed resources during dry season. It should also be mentioned that a much more pronounced effect by supplementation with Phyllanthus discoideus could be expected when poor quality grass hay prevalent in West Africa during the dry season is fed. Phyllanthus discoideus could serve as a supplement to poor quality grass at 25% to 50% of supplementation.     

Involvement of an acrosinlike proteinase in the sulfhydryl-induced degradation of rabbit sperm nuclear protamine

Previous studies demonstrated that proteolytic activity is associated with isolated rabbit sperm nuclei and is responsible for the degradation of nuclear protamine that occurs during thiol-induced in vitro decondensation of the nuclei (Zirkin and Chang, 1977; Chang and Zirkin, 1978). In this study, we present the results of experiments designed to characterize this proteolytic activity. Basic protein isolated from rabbit sperm nuclei incubated with 5 mM dithiothreitol (DTT) and 1 percent Triton X-100 for increasing periods of time exhibited progressively faster migrating bands on acid-urea polyacrylamide gels, reflection the progressive degradation of protamine. Ultimately, a specific and characteristic peptide banding pattern resulted. When sperm nuclei were treated with the esterase inhibitor nitrophenyl-p-guanidino benzoate (NPGB) to inhibit the nuclear-associated proteolytic activity and then incubated with one of several exogenous proteinases in addition to DTT and Triton X-100, characteristic peptide banding patterns were seen for each exogenous proteinase employed. For trypsin, chymotrypsin, pronase, and papain, the peptide banding patterns differed from one another and from the pattern characteristic of protamine degradation by the nuclear-associated proteinase. By contrast, when rabbit acrosin served as the exogenous proteinase, the peptide banding pattern seen was identical to the pattern characteristic of the nuclear-associated proteinase. These results demonstrate directly that the proteinase associated with rabbit sperm nuclei and involved in sperm nuclear decondensation in vitro is acrosinlike.     

Activity patterns and resource use by sheep and red deer grazing across a grass/heather boundary

Diet selection and activities of sheep and red deer grazing alone and together across a grass (primarily Lolium perenne/Agrostis capillaris ) and heather ( Calluna vulgaris ) boundary, between July and October, are described.
Throughout the experiment, both animal species had a much higher proportion of grass than heather in their diet. Time spent grazing on each type of vegetation was similarly greater on grass, and the bite rates of the animals were also more rapid on the grass than on the heather.
Red deer spent more time grazing per 24 hours than did sheep, with sheep grazing less during darkness hours than red deer. Total grazing time of both species did not decrease with decreasing daylength from July to October.
There were no consistent differences in the diurnal patterns of activity of sheep and red deer, with both species showing 2-3 large grazing peaks per day.
There were no consistent effects of mixing sheep and deer on their grazing behaviour and diet composition.     

Breakdown of different peptides byPrevotella (Bacteroides) ruminicola and mixed microorganisms from the sheep rumen

Several di-, tri-, and oligopeptides were incubated individually in vitro with rumen fluid from two sheep receiving a mixed grass hay/concentrate diet and with washed cells ofPrevotella (formerlyBacteroides)ruminicola M384 andP. ruminicola B₁4. The rates of breakdown of most peptides were similar in the rumen fluid from the two sheep. Acidic and proline-containing peptides tended to be more slowly degraded than neutral or basic peptides. The dipeptide at the N-terminus of higher peptides was observed as an early product of hydrolysis, confirming that a dipeptidyl aminopeptidase type of activity was present. The relative rates of breakdown of dipeptides byP. ruminicola were different from that of rumen fluid, but the hydrolysis of higher peptides followed a similar pattern, and dipeptides from the N-terminus were detected as early products.     

The Microflora and Concentrations of Volatile Fatty Acids in The Rumen of Cattle Fed on Single Component Rations

The composition of the rumen microflora and the volatile fatty acids were examined in cattle free-grazing on grass or stall-fed on hay, grass pellets, oats or dried beet pulp with molasses. Total and viable counts of anaerobic bacteria were highest on the grass feeding, but viable counts as a percentage of total counts were highest when oats or beet pulp with molasses were fed. Counts of cellulolytic bacteria were lowest on these latter 2 diets, and highest on grass or grass pellet diets. Studies of the anaerobic flora showed that the composition in animals fed on grass pellets resembled more that found in animals free-grazing on grass than in those fed on hay. Counts of aerotolerant bacteria were only a small percentage of the total count, but were highest on the hay diet. On this latter diet and on grass-feeding the streptococci (identified as Streptococcus bovis) were predominant, but contrary to expectation, streptococci were found only in small numbers on the oats diet, where coryneform rods were the major type present. Although a period of 4–6 weeks was allowed for the animals to adapt to the feeds, the 2 periods of feeding on oats and dried beet pulp with molasses markedly affected the composition of the rumen flora in the subsequent periods of feeding grass pellets and hay. Ruinen volatile fatty acid analysis showed a propionogenic effect of oats and the highest percentage of butyric acid when beet pulp with molasses was fed. The expected propionogenic effect of grass pellets was not observed.     

Rumen function in red deer, hill sheep and reindeer in the scottish highlands.

Red deer, sheep and reindeer grazing on their normal hill ranges were examined at intervals over a period of four years. Samples from the digestive tract were taken at different seasons and processed in the field. The Red deer and reindeer were killed before samples were taken; rumen samples from the sheep were taken by stomach tube, but a number of animals were also killed at different seasons to correlate stomach tube and whole rumen samples. The animals sampled were representative of the general condition of the herds. Examinations were made for parasites and any pathological conditions. In most instances parasitic infections were slight. Apparent seasonal changes were found in the compositions of the diets. The Red deer and sheep ate principally heather and grass, the proportion of heather increasing in the winter. The reindeer ate mainly grass in the summer, with lichens and grass forming the winter diet, and these animals seemed to have a higher nutritional status in the winter than did the other two species. The weights of the animals and of their rumen contents, the concentrations of rumen ammonia and volatile fatty acid, and the rates at which different dietary components were fermented are recorded. Rumen fermentation was low in winter and the diets were generally inadequate for the animals. A lack of nitrogen seemed to be a major factor. Some data on caecal contents are also given.     

Evaluation of dietary influences on Escherichia coli O157:H7 shedding by sheep.

The effect of diet, an abrupt diet change, and fasting on the shedding of Escherichia coli O157:H7 was investigated with experimentally inoculated sheep as a ruminant model. Sheep were fed a grass hay diet (G), which was low in protein and digestible energy and high in fiber, or a mixture of corn and pelleted alfalfa (C), which was high in protein and digestible energy and low in fiber. After a single oral inoculation of E. coli O157:H7, all the animals shed fecal E. coli O157:H7. However, sheep that were fed G shed the bacterium almost twice as long as, and in larger numbers than, did sheep that were fed C. The number of culture-positive animals increased after the diet was abruptly changed from C to G and decreased with the opposite change (G to C). A 24-h fast did not influence E. coli O157:H7 shedding. Horizontal transmission of infection between animals occurred. Recent shedding of E. coli O157:H7 did not affect recolonization with E. coli O157:H7. The findings presented in this study indicate that preharvest control of diet may reduce the risk of E. coli O157:H7-positive animals entering the food chain.     

Assessment of ciliates in the sheep rumen by DGGE

AIMS: This work was carried out to develop a rapid molecular profiling technique to screen ciliate populations in the rumen of sheep. METHODS AND RESULTS: DGGE was used to study the ciliate diversity in the rumen of sheep. There was considerable variation between sheep which were co-housed, and fed the same diet. However, no difference in the major banding patterns was detected, when samples were collected from a single sheep sampled at different points. Following dietary changes, use of a pair-wise comparison of lanes, demonstrated that although there was still diversity between the ciliate population of sheep, the effects as a result of dietary changes were greater. CONCLUSIONS: The technique generated molecular profiles which are sufficiently different to allow comparison between samples, and to permit molecular ecological studies on the rumen ciliate population. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of this study means that ciliate diversity in the rumen may now be studied by those unfamiliar with morphological identification of these organisms.     

Differences in mean retention time of sheep and goats under controlled feeding practices

The objective of this study was to compare the mean retention time (MRT): (a) between sheep and goats when they fed in groups with the same diet, the same amount of food and forage to concentrate (F/C) ratio and (b) between group and individual feeding in each animal specie (the same diet, the same amount of food and F/C ratio). Twelve, 3-4 years old, Friesian crossed dairy ewes and twelve, 3-5 years old, Alpine crossed dairy goats were used for the experiment. The animals were fed with a diet, consisted of alfalfa hay, wheat straw and concentrate, in two treatments. In Treatment A the animals fed on a group basis as it is traditionally used in practise, while in Treatment B the diet was offered individually toin each animal, in order to have fully comparable feeding level among animals and between treatments. Each Treatment (A and B) lasted 3 weeks. The results of this study have shown that: sheep had significantly longer rumen retention time (RRT) (30.03 vs. 14.43) and mean retention time (MRT) (40.80 vs. 27.81), and shorter transit time (TT) (8.86 vs. 11.49) than goats, while the caecum retention time (CRT) did not differ between the two species in Treatment A (group feeding). The comparison between group (Treatment A) and individual feeding (Treatment B) in goats or in sheep has shown that there were no significant differences as RRT, CRT, TT and MRT concerns. In conclusion, animal species (sheep vs. goat) was the main factor which affected the MRT, RRT and TT when the animals were fed in groups with the same diet, amount of food and F/C ratio, while the feeding technique (group vs. individual) had no effect on those parameters in both animal species.     

Stability and stabilization of potential feed additive enzymes in rumen fluid*

Four commercial preparations of fibrolytic enzymes, from Irpex lacteus, Trichoderma viride, Aspergillus niger, and a mixture designed to be similar to the I. lacteus extract, were incubated in vitro with digesta taken from the rumen of sheep receiving a grass hay/concentrate diet, and the survival of major enzyme activities was measured. Some activities, including the beta-1,4-endoglucanase and xylanase from the extract derived from Aspergillus niger, were stable for at least 6 h in rumen fluid. The same activities in the other extracts also retained substantial activity for several hours. beta-Glucosidase and beta-xylosidase activities were much more labile, most being almost completely destroyed after 1 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most proteins in the extracts were digested extensively after up to 7 h of incubation. Adding bovine serum albumin (0.5 g/l) to the incubation increased the half-life of Trichoderma viride beta-glucosidase activity from less than 0.5 h to 3 h. Proteins extracted from plant materials, particularly the soybean 7S globulin fraction, also conferred protection from proteolytic breakdown, but none was as effective as bovine serum albumin. It was concluded that the stability of most fibrolytic enzymes in rumen fluid is not likely to be a limiting factor in the use of enzymes as feed additives for ruminants; but if the enzymes are not stable, means can be found for their stabilization.     

Age-dependent changes in extracellular proteins, aminopeptidase and proteinase activities in Frankia isolate BR

To investigate protein secretion by the nitrogen-fixing actinomycete Frankia isolate BR, we designed a rapid DEAE adsorption, salt elution and Biogel P6DG desalination method to concentrate protein from the growth medium. Secreted proteins reached a maximum concentration (5.6 gm l-1) in the medium at growth arrest. Analysis by SDS-PAGE detected up to 63 extracellular polypeptides when Frankia cells were grown under stirred conditions in BAP medium supplemented with phosphatidylcholine and MES buffer and 65 proteins in stirred BAP media alone. The pattern of extracellular polypeptides changed during growth. Several extracellular proteolytic activities were detected and compared with intracellular ones. The substrate specificity of the extracellular and intracellular aminopeptidase activities were the same. Also, the electrophoretic migration patterns of secreted and intracellular aminopeptidases could not be distinguished. Secretion of the proline-specific aminopeptidase FAP proteinase (PF) were secreted: 10 had the same electrophoretic mobility as their intracellular counterparts after SDS-gelatine-PAGE while five (PF - 39.5, PF - 38.5, PF - 36.5, PF - 25.5 and PF - 20.5 kDa) had a different electrophoretic mobility and, therefore, appeared to be exclusively extracellular. At least seven extracellular proteinases appeared to increase coordinately in activity shortly before growth arrest.     

Sulfur and methionine metabolism in sheep. I. First approximations of sulfur pools in and sulfur flows from the reticulo-rumen.

Sulfur pools in the rumen and sulfur flows from the rumen were investigated in two experiments with sheep on a diet containing equal parts of oaten and lucerne chaffs. The diet was fed at two levels, either chopped or pelleted, and with intraruminal DL-methionine supplements. Ruminal fluid volumes and fluid flows to the omasum were measured. None of the treatments influenced ruminal fluid volume. Fluid flow to the omasum, however, was increased by increasing dry matter intake (DMI), and was further enhanced by feeding chaffed hay rather than the same materials ground and pelleted; the DL-methionine supplement had no effect. First approximation of the ruminal sulfur pools and of sulfur flows to the omasum were derived from the concentration of sulfur in true digesta and the ruminal fluid volume or fluid flow. Increasing DMI from 500 to 1000 g/day resulted in larger ruminal pools of total (1096 v. 792 mg), neutral (1016 v. 731 mg) and protein (479 v. 419 mg) sulfur, but the reducible sulfur pools were not affected by the level of DMI. Infusions of DL-methionine increased the ruminal sulfide sulfur pool irrespective of level of DMI. The first approximation of total sulfur flow was increased by 1660 mg/day at the higher level of DMI, due mainly to increases of 710 mg S/day as protein sulfur and 859 mg S/day as non-protein neutral sulfur. Flows of inorganic sulfate and ester sulfate sulfur, although small in comparison with organic sulfur flows, increased with level of DMI. Sulfide sulfur flows were also increased at the higher level of DMI, and were almost doubled by intraruminal infusions of DL-methionine.     

Molecular diversity of rumen methanogens from sheep in Western Australia

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobrevibacter: Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.     

Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques for analysing the influence of diet on ruminal bacterial diversity

The objective of this study was to compare the automated ribosomal intergenic spacer analysis (ARISA) and the denaturing gradient gel electrophoresis (DGGE) techniques for analysing the effects of diet on diversity in bacterial pellets isolated from the liquid (liquid-associated bacteria (LAB)) and solid (solid-associated bacteria (SAB)) phase of the rumen. The four experimental diets contained forage to concentrate ratios of 70:30 or 30:70 and had either alfalfa hay or grass hay as forage. Four rumen-fistulated animals (two sheep and two goats) received the diets in a Latin square design. Bacterial pellets (LAB and SAB) were isolated at 2 h post-feeding for DNA extraction and analysed by ARISA and DGGE. The number of peaks in individual samples ranged from 48 to 99 for LAB and from 41 to 95 for SAB with ARISA, and values of DGGE-bands ranged from 27 to 50 for LAB and from 18 to 45 for SAB. The LAB samples from high concentrate-fed animals tended (p < 0.10) to show greater peak numbers and Shannon index values than those isolated from high forage-fed animals with ARISA, but no differences were identified with DGGE. The SAB samples from high concentrate-fed animals had lower (p < 0.05) peak numbers and Shannon index values than those from animals fed high-forage diets with ARISA, but only a trend was noticed for these parameters with DGGE (p < 0.10). The ARISA detected that animals fed alfalfa hay diets showed lower (p < 0.05) SAB diversity than those fed grass hay diets, but no differences were observed with DGGE. No effect of forage type on LAB diversity was detected by any technique. In this study, ARISA detected some changes in ruminal bacterial communities that were not detected by DGGE, and therefore ARISA was considered more appropriate for assessing bacterial diversity of ruminal bacterial pellets. The results highlight the impact of the fingerprinting technique used to draw conclusions on dietary factors affecting bacterial diversity in ruminal bacterial pellets.     

Listeriosis in Sheep

A comparison was made between a hay fed group, consisting of 23 ewes, and a grass silage fed group of 22 ewes, all pregnant. Excretion of Listeria monocytogenes (Lm) in the faeces and milk, antibody titres in sera and whey and delayed hypersensitivity against Lm, and several blood components were determined. The animals had previously been exposed to Lm, and Lm was isolated from the faeces from several animals when the experiment started. No significant difference in number of excretors between the 2 groups was found during the experimental period. The haemagglutination titres in both sera and whey were low and on the same level in both groups. The titres were higher in animals with 1 foetus than in animals with more than 1 foetus. In the first part of the experimental period the silage group had a reduced number of lymphocytes, lower total serum protein values and higher serum iron values, compared with the hay group. The silage group also had a stronger delayed hypersensitivity reaction against Lm than the hay group, and in the silage group the reaction was significantly stronger in ewes with 3 or more foetuses than in ewes with 1 foetus. In conclusion, the combined effect of some of the changes found in animals fed grass silage may leave them more susceptible to infections.     

Multiple forms of acetylcholinesterase from rat erythrocytes. Effect of fat-free diet.

Electrophoretic patterns of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from rat erythrocyte were studied. The enzyme was solubilized by the following treatments: a) Triton X-100, b) sodium deoxycholate, or c) ultrasonic irradiation. When the erythrocyte membrane was solubilized by Triton X-100 at concentrations higher than 0.3%, by 10 mM sodium deoxycholate, or by ultrasonic irradiation for more than 5 min, a single band of acetylcholinesterase activity appeared in the gel. Two bands of activity were stained in the gel when the membrane was solubilized by Triton X-100 at concentrations between 0.1--0.2%, or by ultrasound for 5 min. Electrophoretic patterns of acetylcholinesterase from rats fed a fat-sufficient diet were similar to those for the enzyme from animals fed a fat-free diet. The recombination of lipids with the enzyme eluted from the gels confirmed the "phenotypic allosteric desensitization phenomenon".