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Interest in flaxseed and products derived from flaxseed has increased considerably in the past decade. In the development of nutraceutical foods, several flaxseed components including lignans and alpha-linolenic acid, have been recognized to have health benefits. There are many patents and health claims to these components; however, relatively little research and information is found on flaxseed proteins. The objective of the present study was to isolate and characterize flaxseed proteins from defatted flaxseed meal. / Proteins were extracted from defatted flaxseed meal with NaOH. NaCl and NaCl/Papain. Protein solubility with these extractants ranged from 18 to 25%. Proteins were precipitated from the extracts using one or more of the following precipitation techniques; isoelectric precipitation (IP) gave yields and protein contents ranging between 22--25% and 67--73% respectively. Co-precipitation with soy and whey proteins gave yields of 26.95 and 35.78% respectively for a NaOH extract. Chemical hydrolysis of flaxseed meal with NaOH, NaCl and NaCl/Papain extraction and IP increased protein solubilization (35--43%) and protein yields (19--37%). / Proteins in the extracts and isolates were characterized by polyacrylamide gel electrophoresis (PAGE). Proteins extracted with NaOH and NaCl gave two bands by native-PAGE with molecular weights (MW) of 320 and 514KDa; proteins extracted with NaCl/Papain gave a single band at 188KDa. SDS-PAGE of isolates extracted with NaOH, NaCl and NaCl/Papain gave subunits with MW ranging from 6.5 to 56KDa. The major fractions isolated from NaOH-IP by RP-HPLC showed subunits with MW ranging from 6.5 to 40.1KDa by SDS-PAGE and 5.9 to 42.5KDa by ESI-MS. Subunits characterized by ESI-MS have not been reported previously in the literature.

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Electrophoresis is a widely used analytical technique in the medical and biotechnology industries. It can provide for the determination of thousands of individual compounds on this small scale. The operating conditions are quite conducive to use with the delicate products of genetic engineering. Due to other complexities associated with scale-up, the process is not widely used on a large scale. A novel recycling electrophoretic instrument was investigated as a preparative protein separation device. The process occurs in a thin film of liquid between two flat plates, in a direction perpendicular to the flow. This device was unique in its use of a relatively high flowrate, and recycling of the process fluid as a method for increasing residence time. The apparatus was operated in three modes, isoelectric focusing (IEF), zone electrophoresis (ZE) and isotachophoresis (ITP). For use in the ITP mode, a computer was used for data acquisition and control functions. Model systems included monoclonal antibodies and lentil lectins.

An attempt to develop a short and reliable method of caldesmon purification led to the development of three procedures of caldesmon purification. The first method was seldom used because of its low yield and the lack of caldesmon endogenous kinase activity. However, it allowed us to purify MLCK (myosin light chain kinase). The second and third methods gave respectively, a caldesmon sample with and without kinase activity. We were able to localize the endogenous kinase in the 0-30% ammonium sulfate precipitated DEAE pellet but we were unsuccessful at purifying the kinase to homogeneity. We found that caldesmon can also be phosphorylated by rat brain Ca²⁺-calmodulin-dependent kinase II at sites identical to those of caldesmon endogenous kinase but different to those of kinase C. In addition, caldesmon and its endogenous kinase are two different proteins. Furthermore, our study of caldesmon inhibition of actomyosin ATPase activity showed that further research needs to be done to refute F-actin bundling process as a possible cause of caldesmon inhibition of actomyosin ATPase activity. In addition, our studies of caldesmon inhibition of HMM and S-1 ATPase activity suggest that S-2 might be partially involved in the inhibition mechanism. Finally, caldesmon did not affect the 6S-10S transition of myosin conformation and since caldesmon cannot compete against higher affinity calmodulin-binding protein such as MLCK thus, the flip-flop theory is untenable.

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