Dephosporylation with Alkaline Phosphatase of Histone and Fibrinogen Phosphorylated with Protein Kinase C in vitvo

Alkaline phosphatase from calf intestinal mucosa dephosphorylated histone H1 and fibrinogen that had been phosphorylated with protein kinase C. The reaction velocity was dependent on the ionic strength of the buffer; decreasing with increasing concentration. The pH optimum was around 7, which is lower than pH-optima described for other kinds of substrates. ( P)phosphorylated fibrinogen was dephosphorylated about 20 times faster than (32P)phosphohistone on a weight basis and the reaction continued linearily with time f o r the longest time tested (3 hs) even at 37 C. As alkaline phosphatase is present in the blood the possible physiological significance of the dephosphorylation of phosphofibrinogen is discussed.

Alkaline phosphatase belongs to a group of phosphatases known to show a broad substrate specificity and the physiological function of which still is unknown. Synthetic substances like dinitrophenyl phosphate, nucleotides and proteins phosphorylated with protein kinase A or tyrosine kinase are some of the known substrates in vitro (8,121. Both phosphoester and phosphoanhydrid bonds can be cleaved by the enzyme. As fibrinogen phosphorylated with protein kinase C was found to be dephosphorylated ( 5 ) it was of interest to investigate the optimal conditions f o r this dephosphorylation together with that of another substrate of protein kinFse C, histone H1, as alkaline phosphatase could be a usefuI tool in the investigation of the physiological role of protein kinase C phosphorylation. Also, increased information on thr possible role of the phosphatase in biological systems could be obtained by such an investigation.

MATERIALS AND METHODS
Histone H 1 , l,Z-diolein, L-a-phosphatidylserine were from Sigma (USA).
(32P)hTP was a product of NEN (England) and Lphadex G-50 w a s bought from Pharmacia (Sweden). Alkaline phosphatase was from Boehringer-Mannheim (Germany). All other chemicals were of highest grade commercially available.
Preparation of (32P)pho~phohi~tone. Histone Hl (1.0 mg/ml) was phosphorylated with 1500 U of protein kinase C and 0. m M NaC1. The radioactivity was measured as Cerenkov radiation ( 9 ) . The amount of { Plhistone was determined according to Bradford (2) with histone as reference protein and the material was stored at -2OOC until used. Dephosphorylation of ( P)phosphohistone and ( P)phosphofibrinogen with alkaline phosphatase. The dephosphorylation was performed as follows unless otherwise stated. In a total volume of 100 1-11 the incubations contained 20 mM Tris-HC1 buffer, pH 7.5, 0.9% (w/v) NaC1, alkaline phosphatase, and 1-3 pg of ( P)phosphohistone and 20-40 pg of ( P)phosphofibrinogen, respectively.  found that the activity decreased around 10% when the concentration of this substance was increased 10 times ( Fig. 2 ) with fibrinogen as substrate.
Therefore, the phosphatase activity seen in Fig. i with different bufier substances was, if any, little influenced by the buffer substance itself but was a result of pH and ionic strenght.
The dependence of the phosphatase activity on the pH and divalent cations The pH-optimum for dephosphorylation w a s found to be around 7 with a slight decrease in activity towards the alkaline pH-region (Fig. 3).Magnesium (1.0 mM) is often used in the reaction mixture when alkaline phosphatase is used, so I tried that cation and also zinc (0.1 mM).

2+ 2+
This resulted in no effect on the dephosphorylation by these ions when histone was used as substrate. The dephosphorylation of fibrinogen was not effected by magnesium but zinc inhibited the activity by 60%. The phosphatase activity as a function of the amount of enzyme. The amount of phosphatase was varied at a constant concentration of histone and fibrinogen. The result is given in Figure 4. A 30% dephosphorylation is seen at 3900 U of phosphatase/mg histone after 30 min at 3OoC (Fig. 4). A simil:!iactivity with fibrinogen was obtained wi$h 21 U of phosphatase/mg ( substrates showed that alkaline phosphatase dephosphorylated fibrinogen about 20 times faster than histone. The same difference can also be deduced from the results given in Figure 2.  Fig. 6 The dependence of dephosphorylation on amount of ( P)phosphohistonc The amount of enzyme was 47 U, the total volume 80 p1 and the ixubation time was 10 min. Otherwise the reaction was performed as described under Materials and Methods. Inserted in the Fir.1r-e is the corresponding Lineweaver-Burke plot.

DISCUSSION
It was found that alkaline phosphatase can be used as a tool for dephosphorylation of proteins phosphorylated with protein kinase C. The following parameters are then of value to consider: The substance buffering the system did not have any pronounced effect ( Fig. 1) but with fibrinogen as substrate the activity of the phosphatase decreased with increasing ionic strenght from 100% (at 167mM) to 75% (at 77'2 mM) (Fig.2).
The optimum pH was 7. The decrease in alkaline phosphatase activity at higher pH values was not more than 5% so the reaction could be performed at higher pH if necessary (Fig. 3). The optimal pH is dependent on the protonised form of the substrate and therefore shifts towards a more acidic: pH-optimum at low concentrations of substrates (11).
Ca that but that It ions did not interfcre with the reactior., with one exceptio:i, zinc2+ inhibited the dephosphorylation of fibrinogen. Zinc is a known inhibitor s also often used as an activating cation ( 4 , 6 ) . It therefore seems, if possible, no divalent cations should be added.
was possible to dephosphorylate the substrate linearly for a long time.
even at 37'C. This is known and used when alkaline phosphatase is coupled to the secondary antibody in Western techniques. In the case of protein kinase C phosphorylated substrates it was important that they did not contain any contaminating proteases if the reaction was prolonged.
Phosphorylated fibrinogen was a better substrate of alkaline phosphatase than histone. Even so, the K of 7.5 pM for phosphohistone indicated a high affinity of the enzyme for this substrate. However, the Vmax and the turnover number that can be calculated ( O .~X~O -~ mol/mol enzyme x s ) are low. In summary, the alkaline phosphatase can be used to dephosphorylate substrates phosphorylated with protein kinase C.

ACKNOWLEDGEMENT
project was supported by The Swedish Medical Research Council 13x50.
The excellent work of Urban Smangs is hereby greatly acknowledged. The