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Nsph-CM and GM were fractionated into proteins greater than (Fraction A) and less than (Fraction B) 30 kDa using Amicon filters (Millipore).
We fractionated the nsph-CM into fraction A (>30 kDa) and fraction B (<30 kDa; Fig. 1B) and found that in low density cultures fraction A has nsph stimulatory activity approximating that of whole nsph-CM, and fraction B stimulated nsph formation by 1.5-fold (Fig. 1C).
Fraction B was further purified by silica gel CC eluting with a gradient of CHCl3 MeOH (50:1–10:1), to yield five fractions (B1 B5).
The insoluble but potentially degradable fraction ("b") of DM and NDF of the diets were linearly decreased and increased, respectively.
There was no effect of diet (P > 0.05) for the rapidly degraded (a) or degradable fraction (b) of dry matter.
For alfalfa hay, the disappearance of fraction A of DM, NDF, and ADF decreased and fraction B of DM and NDF increased with treatment.
The slowly degradable fraction (fraction B) of Fe increased (P<0.01) across both harvest dates, that of Zn and Cu decreased (P<0.01) on the 18 August harvest date, and fraction B of Zn increased (P=0.02) with N fertilization on the 30 May harvest date.
a Saccharide fraction; b amino acid fraction.
At the peak of induction, fraction B constituted 12% of the total protein synthesized.
A fraction eluting between 0.2 and 0.4 M NaCl (fraction B) did not contain this protein.
As compared to the initial total protein extract, Fraction B demonstrated increased Na-H+ exchange activity.
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