Thermodynamics of Pb(ii) and Zn(ii) binding to MT-3, a neurologically important metallothionein.

Isothermal titration calorimetry (ITC) was used to quantify the thermodynamics of Pb(2+) and Zn(2+) binding to metallothionein-3 (MT-3). Pb(2+) binds to zinc-replete Zn7MT-3 displacing each zinc ion with a similar change in free energy (ΔG) and enthalpy (ΔH). EDTA chelation measurements of Zn7MT-3 and Pb7MT-3 reveal that both metal ions are extracted in a tri-phasic process, indicating that they bind to the protein in three populations with different binding thermodynamics. Metal binding is entropically favoured, with an enthalpic penalty that reflects the enthalpic cost of cysteine deprotonation accompanying thiolate ligation of the metal ions. These data indicate that Pb(2+) binding to both apo MT-3 and Zn7MT-3 is thermodynamically favourable, and implicate MT-3 in neuronal lead biochemistry.

Evaluation of rumen-protected methionine sources and period length on performance of lactating dairy cows within Latin squares.

Multiparous Holstein cows (n = 16) were used in a replicated (n = 4) Latin square design with 2-wk periods to determine whether length of the experimental period chosen within Latin square designs would influence experimental outcomes for performance-related variables. Cows were fed a basal TMR formulated to supply Lys in excess of the predicted requirements and either no rumen-protected Met (RP-Met; control), or 6 (M6) or 12 (M12) g/d of RP-Met (Mepron), or 12 (S12) g/d of RP-Met (Smartamine M). Performance outcomes were evaluated separately using data collected at the end of each week of each period, and as overall means for each period. Milk yield was not affected by treatment in any period. Supplementation of RP-Met from all sources tended to increase milk fat percentage when evaluated using wk-1 data, but responses evaluated using wk-2 data only were not significant. Supplementation of M12 and S12 tended to increase milk fat percentage over the entire experimental period. Supplementation of M6 and M12 tended to increase milk true protein percentage when evaluated using wk-1 data only; however, responses to treatments during wk 2 were not significant. All supplemental RP-Met treatments either tended to increase (M6 and S12) or increased (M12) milk true protein content over the entire experimental period relative to the control treatment. Yields of fat and true protein were not affected by treatment. Carryover effects of treatment on production variables were largely not significant. Predictions of nutrient supply with 3 models used in dairy ration formulation and evaluation (CPM-Dairy, AminoCow, and 2001 NRC Dairy) indicated that energy, protein, and Met all were supplied in excess of requirements for all treatments. Overall, results suggest that interpretation of performance outcomes can vary depending on selection of the experimental period in Latin squares, and responses to increased Met supply based on ratio-based formulation may not be independent of the grams of Met supplied.

Effect of dietary protein concentration and degradability on response to rumen-protected methionine in lactating dairy cows.

An incomplete 8 x 8 Latin square trial (4-wk periods; 12 wk total) using 32 multiparous and 16 primiparous Holstein cows was conducted to assess the production response to crude protein (CP), digestible rumen-undegraded protein (RUP), and rumen-protected Met (RPM; fed as Mepron; Degussa Corp., Kennesaw, GA). Diets contained [dry matter (DM) basis] 21% alfalfa silage, 34% corn silage, 22 to 26% high-moisture corn, 10 to 14% soybean meal, 4% soyhulls, 2% added fat, 1.3% minerals and vitamins, and 27 to 28% neutral detergent fiber. Treatments were a 2 x 2 x 2 factorial arrangement of the following main effects: 15.8 or 17.1% dietary CP, with or without supplemental rumen-undegraded protein (RUP) from expeller soybean meal, and 0 or 9 g of RPM/d. None of the 2- or 3-way interactions was significant. Higher dietary CP increased DM intake 1.1 kg/d and yield of milk 1.7 kg/d, 3.5% fat-corrected milk (FCM) 2.2 kg/d, fat 0.10 kg/d, and true protein 0.05 kg/d, and improved apparent N balance and DM and fiber digestibility. However, milk urea N and estimated urinary excretion of urea-N and total-N also increased, and apparent N efficiency (milk-N/N-intake) fell from 33 to 30% when cows consumed higher dietary CP. Positive effects of feeding more RUP were increased feed efficiency and milk fat content plus 1.8 kg/d greater FCM and 0.08 kg/d greater fat, but milk protein content was lower and milk urea N and urinary urea excretion were elevated. Supplementation with RPM increased DM intake 0.7 kg/d and FCM and fat yield by 1.4 and 0.06 kg/d, and tended to increase milk fat content and yield of milk and protein.

Effect of supplementing rumen-protected methionine on production and nitrogen excretion in lactating dairy cows.

Two 4 x 4 Latin square trials (4-wk periods; 16 wk total) were conducted to see whether supplementing rumen-protected Met (RPM; fed as Mepron) would allow feeding less crude protein (CP), thereby reducing urinary N excretion, but without losing production. In trial 1, 24 Holsteins were fed 4 diets as total mixed rations containing [dry matter (DM) basis]: 18.6% CP and 0 g of RPM/d; 17.3% CP and 5 g of RPM/d; 16.1% CP and 10 g of RPM/d; or 14.8% CP and 15 g of RPM/d. Dietary CP was reduced by replacing soybean meal with high-moisture shelled corn. All diets contained 21% alfalfa silage, 28% corn silage, 4.5% roasted soybeans, 5.8% soyhulls, 0.6% sodium bicarbonate, 0.5% vitamins and minerals, and 27% neutral detergent fiber. There was no effect of diet on intake, weight gain, or yields of protein, lactose, and solids-not-fat. However, production was greater at 17.3% CP plus RPM and 16.1% CP plus RPM than on the other 2 diets. Apparent N efficiency (milk N:N intake) was greatest on the lowest CP diet containing the most RPM. Linear reductions in milk urea N and urinary N excretion were observed with lower dietary CP. In trial 2, 32 Holsteins were fed 4 diets as total mixed rations, formulated from ingredients used in trial 1 and containing 16.1 or 17.3% CP with 0 or 10 g of RPM/d. On average, cows were calculated to be in negative N balance on all diets because of lower than expected DM intake. There was no effect of RPM supplementation on any production trait. However, higher CP gave small increases in yields of milk, protein, and solids-not-fat and tended to increase DM intake and lactose yield. Apparent N efficiency was greater, and milk urea nitrogen was lower, on 16.1% CP. In trial 1, feeding lower CP diets supplemented with RPM resulted in improved N efficiency and reduced urinary N excretion. However, in trial 2, reducing dietary CP from 17.3 to 16.1% reduced milk secretion, an effect that was not reversed by RPM supplementation at low DM intakes when cows were apparently mobilizing body protein.