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The influence of phosphorylation on the properties of lactate dehydrogenase (LDH) has been studied. Data obtained using the immobilization approach support the assumption that the autophosphorylation of LDH discovered previously in the presence of ATP has no relation to protein kinase activity of the enzyme. Phosphorylation of native LDH by tyrosine kinases was shown to be inefficient. However, the efficiency of the phosphorylation considerably increased after the dissociation of LDH into non-native forms of the enzyme. Ca2+/calmodulin-dependent protein kinase catalyzes incorporation of 0.8-0.9 mole phosphate per mole of LDH tetramer. The phosphorylation results in an increase in activity by 25-30% and increases markedly the stability of the enzyme during cold inactivation. Phosphorylation of LDH by Ca2+/calmodulin-dependent protein kinase, unlike the phosphorylation on tyrosine residues, is supposed to be of importance for the control of cell metabolism.

Ca,phospholipid-dependent (PKC) and cAMP-dependent (PKA) protein kinases phosphorylate the alpha-subunit of the Na,K-ATPase from duck salt gland with the incorporation of 0.3 and 0.5 mol 32P/mol of alpha-subunit, respectively. PKA (in contrast to PKC) phosphorylates the alpha-subunit only in the presence of detergents. Limited tryptic digestion of the Na,K-ATPase phosphorylated by PKC demonstrates that 32P is incorporated into the N-terminal 41-kDa fragment of the alpha-subunit. Selective chymotrypsin cleavage of phosphorylated enzyme yields a 35-kDa radioactive fragment derived from the central region of the alpha-subunit molecule. These findings suggest that PKC phosphorylates the alpha-subunit of the Na,K-ATPase within the region restricted by C3 and T1 cleavage sites.

The regulatory subunit of type II cAMP-dependent proteinkinase was isolated from cytosol of the rabbit small intestinal mucosa by affinity chromatography. The preparation contained 3 proteolytic enzymes and occurred in two forms differing as regards cAMP affinity. The cAMP-binding capacity of the preparation was equal to 17 nmol cAMP/mg protein. To study the topography of the cAMP-binding center, use was made of cAMP analogs. It was demonstrated that introduction of the substituents into the 8th position of the purine ring and substitution with respect to the N6-exoaminogroup affected insignificantly the analog affinity for the cAMP-binding center. At the same time the substituents introduced into the first position of the adenine base, into the area of the 2'-hydroxyl group of ribose and into the cyclophosphate part of the cAMP molecule considerably decreased the analog affinity for the regulatory center of type II cAMP-dependent proteinkinase.

The cAMP-dependent protein kinase from the soluble fraction of pigeon breast muscle is represented by two forms, PK I and PK II. The ratio of the phosphotransferase activity of the two forms is 35-40% and 60-65% for PK I and PK II, respectively. The regulatory subunit of PK I was isolated in a homogeneous state by affinity chromatography on 8-(2-oxoethylthio)-cAMP immobilized on epoxy-activated Sepharose 4B. The molecular weight of the regulatory subunit of PK I as determined by SDS polyacrylamide gel electrophoresis is 45 000. The specific cAMP-binding activity is equal to 16 nmol of [3H]cAMP per mg of protein. The apparent dissociation constant (Kd') for cAMP equals to 380 nM. The preparation of the regulatory subunit of PK II obtained by affinity chromatography on the same adsorbent is made up of polypeptides with Mr 56 000, 39 000, 29 000, 17 000 and 11 000. The preparation possesses a cAMP-binding activity of 22 nmol of [3H]cAMP per mg of protein. The interaction of several analogs of cAMP containing substituents at different positions of the nucleotide molecule with the regulatory subunit of PK I was studied. Practically all the analogs with substituents at positions 8 and 6 of the adenine ring in the cAMP molecule had the affinity which was 2-9 times less than that of cAMP. The only exceptions were 8-carboxymethylamino- and 8-(2-oxyethyl)-amino-cAMP whose binding to the regulatory subunit was 100 and 53 times lower than that of cAMP. The substitutions in position N-1 of the cAMP molecule leads to a 30-50-fold decrease of the analogs affinity. beta-Bromoethyl ester of cAMP does not reveal the ability to bind to the regulatory subunit. The carboxymethyl ester of cAMP possesses the affinity for the cAMP-binding site that is 35 times less than that of cAMP. Modification of the 2'-hydroxyl of ribose (as in the case of 2'-amino-2'-deoxy-8-hydroxy-cAMP, 2'-deoxy-cAMP and 2'-O-acrylyl-cAMP) decreases the affinity of these compounds 125-, 313- and 126-fold as compared with cAMP. It was assumed that the cAMP molecule is bound to the regulatory subunit in the syn-conformation. A structural model of the cAMP-binding site in the regulatory subunit of cAMP-dependent protein kinase I from pigeon breast muscle is proposed.

cAMP-dependent and casein proteinkinase were found in cytosol of the rabbit small intestine mucosa. cAMP-dependent proteinkinase of cytosol is represented by two forms of types I and II. The activity of enzymes of types I and II constitutes 10 and 90%, respectively. Casein proteinkinase is represented by a single form. The catalytic subunit of cAMP-dependent proteinkinase of type II was isolated in a homogenous state. The catalytic subunit phosphorylates histones H1, H2a, H2b and protamine and to a far less degree histones H3, H4 and casein (H2b greater than H1 greater than H2a greater than protamine much greater than H3 greater than casein). The Km value for histone H1 is equal to 65 mkM, and that for Mg-ATP 12 mkM. Chloromethylpyrophosphonate and adenosine p-fluorosulfobenzoate were studied as affine modifiers of the active center of the catalytic subunit from the small intestine mucosa. It was shown that only adenosine p-fluorosulfonate is an irreversible inhibitor of the catalytic subunit.

The catalytic subunits of cAMP-dependent protein kinases I and II were isolated from rabbit skeletal muscles in a homogeneous state. The specific phosphotransferase activities of homogeneous preparations of catalytic subunits were 8 mumol/mg X min (type I) and 6 mumol/mg X min (type II). In order to elucidate the mechanisms of the phosphotransferase reaction, the steady-state kinetics method and an inhibitory analysis involving the phosphotransferase reaction products, ADP and phosphohistone H1, were used. It was shown that phosphorylation of histone H1 catalyzed both by protein kinases I and II occurs via a random "bi-bi" mechanism. The values of constants for kinetic equation of the phosphotransferase reaction coincide with those for the catalytic subunits of both protein kinase types and are equal to 11 microM (KmATP), 60 microM (KmH1), 5.0 microM (KSATP) and 27 microM (KSH1). The value of the competitive inhibition constant for Mg-ADP (KiADP) is also identical for the catalytic subunits of types I and II and is equal to 30 microM. In both cases, the phosphorylated histone H1 inhibits the phosphotransferase reaction; this inhibition is partly competitive with respect to histone H1.

The phosphorylating enzymes of human skin were studied in bioptic samples of normal tissue (18 samples) and tumors (21 cases), melanomas and basaliomas, which developed in different regions of head and face. The activity of cAMP-dependent and cAMP-independent protein kinases was tested in skin extracts using histone HI and casein as substrates of phosphorylation, respectively. In most tumors the casein kinase activity was found to be significantly elevated (about 10-fold) as compared with normal counterparts. The histone kinase activity was only slightly elevated in tumors (by 30%). For each bioptic sample the ratio of histone kinase activity versus casein kinase activity was calculated. In normal skin this ratio constituted from 1 to 3.7, while in 90% of samples it was higher than 1.5. In all tumors except one the ratio was decreased down to 0.2-0.9. The effect did not depend upon the type of malignancy and tumor location. The change in the protein kinase ratio is considered to be a specific feature of transformed tissue.