Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References

Discussion
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PKA Activities and Distribution in Crayfish

Figure 1 shows the effect of increasing lengths of anoxia exposure on the percentage of PKA present as the free catalytic subunit in crayfish tail muscle (A) and hepatopancreas (B). Total enzyme activity was nearly 10-fold higher in tail muscle compared with hepatopancreas but total activity did not change over the course of anoxia exposure in either tissue (data not shown). However, the percentage of the enzyme present as the free catalytic subunit, PKAc, did change over the course of anoxia exposure with a similar pattern in both tissues. Thus, the early response to anoxia exposure was a strong increase in PKAc content, rising from 72-76% in tissues of control animals to 94-96% PKAc within the first hour of submergence in nitrogen-bubbled water (Figure 1). PKAc remained high after 2 h anoxia exposure but had begun to decline to 3 h anoxia and by 4 h was significantly lower than the initial control values in both tissues. With prolonged anoxia exposure, PKAc remained at about 55 % in tail muscle but continued to decline in hepatopancreas to a final low of 30 % after 20 h anoxia.

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Fig. 1: Percentage of cAMP-dependent protein kinase present as the active, free catalytic subunit in crayfish tail muscle (A) and hepatopancreas (B) over the course of anoxia exposure.
PKA activities were assayed in the absence (free catalytic subunit)
versus presence (total PKA) of cAMP. Values are means +/- SEM; n = 4 animals.

Subcellular Fractionation

Table 1 depicts the subcellular fractionation of crayfish tail muscle and hepatopancreas taken from control animals and those exposed to 20 hours of anoxia, and thus the distribution of the marker enzymes lactate dehydrogenase (LDH, found in the cytosol or MC/CY), 5’nucleotidase (5NT, plasma membrane or PM), and citrate synthase (CS, mitochondria or MT). The values for activity clearly show subcellular fraction-specific activities in both tissues when compared with crude and other fractions. Tables 2 and 3 show the total PKA activity and the percent distribution of PKAc in crayfish tissues, respectively. In the tail muscle, the total activity drops significantly from 31.3 +/- 6.6 mU/mg to 11.7 +/- 2.7 in the PM fraction following 20 hours of anoxia. There is also a decrease in percent PKAc distribution from 31 % +/- 5 % to 15 % +/- 1 %. This corresponded with an increase in the distribution of PKAc in the MC/CY fraction, from 40 % +/- 0.2 % to 65 % +/- 4 %, with total activity rising from 88.5 +/- 2.4 mU/mg to 125.4 +/- 7.3 mU/mg in the same fraction. Similar results were found for the hepatopancreas, where the total activity of PKAc dropped to less than half in the PM fraction under anoxic conditions, representing a drop in percent distribution from 53 % +/- 2 % to 24 % +/- 4 %. The MC/CY fractions showed an increase in percent distribution of PKAc from 29 % +/- 4 % to 46 % +/- 3 %.

Table 1. Activities of marker enzymes in subcellular fractions of Orconectes virilis tail muscle and hepatopancreas.

                        CRUDE                  PM                  MT               MC/CY
                                             (mU/mg protein per fraction)
Tail Muscle
LDH                  125.2 +/- 25.5          6.7 +/- 0.6        89.9 +/- 0.1      711.8 +/- 37.5
5NT                    1.3 +/- 0.3           2.7 +/- 0.3         0.6 +/- 0.05      1.95 +/- 0.06
CS                    39.7 +/- 2.4           3.1 +/- 0.3       350.5 +/- 14.2       0.8 +/- 0.06
Hepatopancreas
LDH                    4.9 +/- 0.5           7.4 +/- 0.7         8.8 +/- 0.9       35.4 +/- 3.7
5NT                    1.6 +/- 0.2           3.1 +/- 0.02        1.4 +/- 0.1        0.5 +/- 0.1
CS                   117.1 +/- 0.6          77.1 +/- 6.7       482.7 +/- 19.6      16.3 +/- 1.6

Values are means +/- SEM, n = 6. Abbreviations are: LDH, lactate dehydrogenase; 5NT, 5' nucleotidase; CS, citrate synthase.

Table 2. Total activity for PKA in control and 20 hrs anoxic Orconectes virilis tail muscle and hepatopancreas following subcellular fractionation

                                 CRUDE                  PM               MT                     MC/CY
                                                                 mU/mg protein per fraction
Tail Muscle
Control                 40.9 +/- 1.3        31.3 +/- 6.6         49.7 +/- 8.1        88.5 +/- 2.4
20 hrs anoxic           44.5 +/- 1.6        11.7 +/- 2.7*        46.2 +/- 9.2       125.4 +/- 7.3**

Hepatopancreas
Control                 75.1 +/- 8.4       179.9 +/- 25.4        74.5 +/- 10.0      116.1 +/- 8.9
20 hrs anoxic          119.2 +/- 1.9**      99.6 +/- 10.0*       61.7 +/- 7.3        79.7 +/- 2.3**

Values are means +/- SEM, n = 6. *Significantly different from corresponding control values, P<0.05. **Significantly different from corresponding control values, P<0.005.

Table 3. Percent distribution of PKAc in control and 20 hrs anoxic Orconectes virilis tail muscle and hepatopancreas following subcellular fractionation

                             PM                 MT                MC/CY

Tail Muscle
Control                  31% +/- 5%         32% +/- 5%         40% +/- 0.2%
20 hrs anoxic            15% +/- 1%*        21% +/- 2%         65% +/- 4%**

Hepatopancreas
Control                  53% +/- 2%         25% +/- 3%         29% +/- 4%
20 hrs anoxic            24% +/- 4%**       35% +/- 1%*        46% +/- 3%*

Values are means +/- SEM, n = 6. *Significantly different from corresponding control values, P<0.05. **Significantly different from corresponding control values, P<0.005.

The purification procedure developed for the cAMP-dependent protein kinase catalytic subunit from the tail muscle of O. virilis consisted of a rapid sequence of steps which could be completed in a matter of hours. The jump in specific activity following the Matrix Red step is accounted for by partial binding of the catalytic subunit to the column, which eluted off of the matrix column 2-3 fractions after the majority of the original crude had been collected. The purification of the catalytic subunit of Orconectes virilis tail muscle PKA is summarized in Table 4.

Table 4. Purification of the catalytic subunit of cAMP-dependent protein kinase from Orconectes virilis tail muscle

 Step                Total protein (mg) Total activity (U) Yield (%) Fold purification Specific activity (U/mg)

 Crude                    49.4               7.36           100%            1.000            0.15

 Matrix Red               0.844              7.18            98%            57                8.5					

 OH Apatite               0.587              6.0             81%            68.6             10.2

 Protamine Agarose        0.018              0.92            13%           352               52.4
                         

The catalytic subunit was purified 352-fold with a yield of 13 %, using a simple method, which manipulates the partial specificity of the catalytic subunit on Matrix Red and Hydroxylapatite. By the first two steps of this method nonspecific proteins are either immediately washed off of the column or trapped. The catalytic subunit finally elutes off of the protamine agarose column at a salt concentration of 250 mM. Elution of the catalytic subunit off of a protamine agarose column using a 0-825 mM KCl gradient clearly showed peak activity at around 250 mM KCl. The enzyme was judged to be homogeneous as assessed by the presence of a single band seen after SDS-polyacrylamide gel electrophoresis (Fig. 2). Molecular weight of crayfish PKA catalytic subunit was estimated to be 42.8 kDa. The results of enzyme elution from a Sephacryl S-300 gel filtration column indicated a molecular weight of 43.8 +/- 0.4 kD, a result that concurred with SDS-PAGE.

Fig. 2: Coomassie-stained SDS-Polyacrylamide gel showing the purification of PKA catalytic subunit from crayfish tail muscle.
Lane 1 shows the final preparation after elution from the protamine agarose column, lane 2 is the original crude extract, and lane 3 shows the subunit molecular weight standards ß-galactosidase (116 kDa), glycogen phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa).

Kinetic Characterization

The effects of different inhibitors on PKAc activity are shown in Table 5, along with Km values for substrates. The most effective inhibitor appeared to be H7 or 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, an known inhibitor of protein kinases A and C, with an I₅₀ of 7.9 +/- 0.6 pM. KCl had very little effect on PKAc activity, and its I₅₀ value was found to be 739 +/- 14.5 mM.

Table 5. Physical properties and kinetic constants of Orconectes virilis tail muscle PKA catalytic subunit.

KINETIC PARAMETER                   VALUE
Km KEMPTIDE (µM)                 31.6 +/- 5.2
Km Mg.ATP (µM)                    131 +/- 14.4
I50 KCl (mM)                      739 +/- 14.5
I50 PKA-I (nM)                    4.2 +/- 1.0
I50 H7 (pM)                       7.9 +/- 0.6
I50 H89 (nM)                      954 +/- 108
I50 KT-5823 (µM)                 1.8 +/- 0.2
Activation energy (kJ/mol)
    0-15°C                        42.3 +/- 2.8
    15-45°C                      17.0 +/- 1.5

Data are means +/- SEM, n = 3 separate preparations of the purified enzyme. Values determined at 22 °C.

The crayfish catalytic subunit had a Km value of 31.6 +/- 5.2 µM for kemptide and a Km of 131 +/- 14.4 µM for Mg.ATP. The effect of temperature on PKAc maximal activity is shown in Figure 3 as an Arrhenius plot. Two distinct linear segments were seen with a break in the plot at about 15°C. The calculated energy of activation for this protein kinase above 15°C over the range 15-45°°C was 17.0 +/- 1.5 kJ/mol. At lower temperatures, however, the activation energy increased by 2.5-fold to 42.3 +/- 2.8 kJ/mol for T < 15°C, from 0-15°C. The effect of pH on PKAc activity at 22°C is shown in Figure 4. The pH optimum was 6.8 and activity dropped off rapidly at both higher and lower pH values.

Fig. 3: Arrhenius plot showing the effect of temperature on the activity of purified crayfish tail muscle PKA catalytic subunit. Assay reagents were mixed and incubated at the designated temperatures for 10 min prior to the addition of PKA, which was measured under optimal assay conditions without cAMP added. Activity was assayed at intervals between 0-40°C. Data are the means of three separate determinations on separate preparations of purified enzyme with less than 10 % variation between runs.

Fig. 4: Effect of pH on the activity of purified crayfish tail muscle PKA catalytic subunit. Values representative of three separate analyses.

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