Calmodulin Purification Calmodulin (heated) was isolated from bovine testes purchased from a local slaughter house. A 20% (w/v) bovine testes homogenate was centrifuged at 25,000g for 1 hr. The resulting supernatant was brought to a rapid boil and centrifuged at 25,000g for 30 min. The supernatant was made 70% saturated with ammonium sulfate and centrifuged at 25,000g for 30 min. The resulting pellet was dissolved in 15 mL of Tris buffer, pH 7.5 and applied onto a phenothiazine-affinity column. The purified sample was lyophilized and stored at -20^oC. The heat treatment was omitted to obtain unheated calmodulin. The same samples were used in subsequent enzymatic, chromatographic and electrophoretic studies.

Gel Electrophoresis

A NOVEX mini-gel system with pre-cast gels were used:

Denaturing: NOVEX 4-12% Bis-Tris Gel with MES-SDS running buffer.

Non-denaturing: NOVEX 8-16% Tris-Glycine Gel with Tris-Glycine running buffer.

High Performance Liquid Chromatography

All HPLC experiments were performed on commercially available HPLC instruments: Spectra-Physics 8800 HPLC equipped with a 10 uL sample loop and a ternary pump with a spectra-Physics 8480XR scanning detector set at 280 and a Spectra-Physics 4270 integrator. 10uL of 20 ug/uL calmodulin samples were injected onto a Supelco TSK-Gel SW3000XL gel permeation column. Buffers ranging from pH 4.5 to 7.5 at a flow rate of 1 mL/min were used as the mobile phase.

Gel Electrophoresis

With only one band obtained from each calmodulin sample as can be seen in Figure 1a, SDS-PAGE supports the purity of both the heated and unheated calmodulin samples used. Figure 1b shows the separation of calmodulin samples in the non-denaturing gel, which indicates the presence of a high molecular weight aggregate in the unheated samples, although it was not present in a large concentration.

Fig. 1: Denaturing (a) and non-denaturing (b) gel electrophoresis results at various concentrations of calmodulin. A faint band in the non-denaturing gel indicates the presence of a 150 kD aggregate.,BR CLEAR="ALL">

HPLC Chromatograms

The retention times for the monomer and the aggregate of interest were determined to be ~12 sec and ~10 sec, respectively. The monomer to aggregate ratio (M/A) was calculated using the peak areas of the monomer and the aggregate.

Heated Calmodulin (HCaM)

Heated calmodulin samples were separated by HPLC at various pH values and a flow rate of 1 mL/min as can be seen in Figure 2. To aid in comparison, the peak areas and the monomer-to-aggregate ratios were plotted as a function of pH, which is shown in figure 3.

Fig. 2: Gel permeation HPLC chromatograms of 0.2 mg of heated calmodulin (HCaM) at various pH values and a flow rate of 1 mL/min.,BR CLEAR="ALL">

Fig. 3: Heated calmodulin peak areas obtained from gel permeation HPLC and monomer to aggregate ratios (M/A) are plotted against pH on a log scale.,BR CLEAR="ALL">

Unheated Calmodulin (HCaM)

Chromatographic results obtained from unheated calmodulin samples were at various pH values are summarized in Figure 4. Figure 5 shows the relationship between peak areas and monomer-to-aggregate ratios with pH.

Fig. 4: Gel permeation HPLC chromatograms of 0.2 mg of unheated calmodulin (UHCaM) at various pH values and a flow rate of 1 mL/min.,BR CLEAR="ALL">

Fig. 5: Unheated calmodulin peak areas obtained from gel permeation HPLC and monomer to aggregate ratios (M/A) are plotted against pH on a log scale.,BR CLEAR="ALL">

Electrophoretic data demonstrated evident aggregation in the unheated calmodulin. However, the HPLC results indicate that both heated and unheated calmodulin form an aggregate and aggregation is a function of pH. Furthermore, aggregation appeared to be more pronounced in the heated calmodulin (see Fig. 2). In addition to the aggregate of focus in this study (150 kD), the unheated calmodulin also forms aggregates of other molecular weights (see Fig. 4). This may result in a decrease in the net amount of "functional" calmodulin, leading to minimized enzymatic activation in the previous studies.

Aggregation may be involved in controlling calcium/calmodulin-modulated metabolism, i.e. calmodulin activity may be regulated by a disturbed equilibrium between monomer and aggregate as a function of subcellular local calcium concentrations and pH.

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