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The phosphorylated or phosphate-accepting site of pyruvate kinase from pig and rat liver was removed without inactivation by incubation with subtilisin. At different time intervals the subtilisin was inactivated with phenylmethylsulfonyl fluoride and the amount of remaining phosphorylatable or phosphorylated sites of pyruvate kinase estimated by incubation with an excess of [32P]-ATP and protein kinase. It was found that to get the same rate of modification the subtilisin concentration required to modify unphosphorylated pyruvate kinase was approximately ten times higher than that used for removal of the phosphorylated site of phosphorylated site of phosphorylated enzyme. It was shown that the proteolytically-modified pyruvate kinase had an increased apparent Km for phosphoenolpyruvate without a change in V, when compared to unmodified unphosphorylated and phosphorylated pyruvate kinase. The removal of the phosphorylated site was not associated with loss of the allosteric sites for ATP and Fru-1,6-P2. The possibility that phosphorylation of the pyruvate kinase increases its degradation rate in vivo is briefly discussed.

Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.

Liver cell sap from normally fed rats, rats fed with a high-carbohydrate diet and fasted rats was chromatographed on DEAE-cellulose (pH 7.0). The chromatogram from each diet group was analyzed for pyruvate kinase activity and endogenous substrates of cyclic AMP-stimulated protein kinase. The materials were pooled into five phosphorylatable fractions, in each of which phosphate incorporation at 0.1 mM and 1.0 mM [32P]ATP in the presence of cyclic AMP and protein kinase was determined. For characterization of the phosphorylatable components, thin-layer gel chromatography on Sephadex G-200 and polyacrylamide gel electrophoresis in detergent were used for determination of native and minimal molecular weights, respectively. Except for pyruvate kinase, eight components which incorporated at least 0.05 nmol of [32P]phosphate/g of liver were detected. The phosphorylation of four of them was stimulated by cyclic AMP. Their minimal molecular weights were 42000, 21000, 52000 and 49000. The component with a minimal molecular weight of 42000 seemed to have a native molecular weight of 160000. Both the 21000 and the 52000 component had a native molecular weight of about 110000-120000. The protein with a minimal molecular weight of 49000 could not be correlated with certainty to a native molecular weight. The proteins whose phosphorylation was not stimulated by cyclic AMP had minimal molecular weights of 54000, 39000, 34000 and 22000.

A protein kinase active on fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) was demonstrated in rat liver cell sap. The protein kinase activity was stimulated by cyclic AMP and coincided with the activity of cyclic AMP-dependent protein kinase type I. In addition, three different peaks of phosphoprotein phosphatase active on [32P] phosphofructose-bisphosphatase were found on chromatography of rat liver cell sap on a DEAE-cellulose column. These phosphatases needed divalent cations for full activity. 5'-AMP, a negative modulator of fructose-bisphosphatase, had no effect on the phosphorylation-de-phosphorylation reactions of the enzyme. ATP and Ca2+ did not influence the dephosphorylation reaction of fructose-bisphosphatase.

One dominating peptic phosphopeptide, Asx-Thr-Lys-Gly-Pro-Glx-Ile-Glx-Thr-Gly-Val-Leu-Arg-Arg-Ala-(³²P)SerP-Val-Ala-Glx-Leu, was obtained from rat liver pyruvate kinase (type L) phosphorylated by cyclic 3′,5′-AMP-stimulated protein kinase from the same tissue. The sequence around the phosphorylated serine residue is similar to that of a corresponding but smaller peptic phosphopeptide previously isolated from pig liver (type L) pyruvate kinase, Leu-Arg-Arg-Ala-(³²P)SerP-Leu.

Phosphorylation of fructose-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) by the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle decreased the K0.5 for fructose-bisphosphate from 21 to 11 microM. When the phosphorylated fructose-bisphosphatase was treated with trypsin the K0.5 increased to 22 microM. The K0.5 also increased when the phosphoenzyme was treated with a partially purified phosphatase from rat liver. There was no difference between the unphosphorylated and phosphorylated enzyme with respect to pH dependence, the pH optimum being about 7.0 for both. Limited treatment of fructose-bis-phosphatase with subtilisin, which cleaves the enzyme at its unphosphorylatable N-terminal part, increased the pH optimum more than limited treatment with trypsin, which releases the phosphorylated peptide at the C-terminal part of fructose-bisphosphatase. The phosphorylated site on the phosphorylated fructose-bisphosphatase was more easily split off by trypsin treatment than the corresponding unphosphorylated site. The results suggest in addition to the glucagon-induced phosphorylation of fructose-bisphosphatase described by Claus et al. [1] that the phosphorylation-dephosphorylation of fructose-bisphosphatase could be of importance for the hormonal regulation of the enzyme in vivo.

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. (32P) phosphorylated fibrinogen was dephosphorylated about 20 times faster than (32P)phosphohistone on a weight basis and the reaction continued linearily with time for the longest time tested (3 hs) even at 37 degrees C. As alkaline phosphatase is present in the blood the possible physiological significance of the dephosphorylation of phosphofibrinogen is discussed.

Tyrosine protein kinase activities have been demonstrated in transformed and normal cell systems. So far, few data on the quantity of protein-bound phosphotyrosine in intact cells have been published. A knowledge of the stoichiometric increase in phosphotyrosine in cells after hormonal induction could be of interest when evaluating the importance of the tyrosine protein kinase activities found. By the addition of a known amount of unlabeled phosphotyrosine to the precipitated protein of 32P-phosphate-labeled cells it was possible after alkaline hydrolysis to spectrophotometrically follow the phosphotyrosine during consecutive chromatographies of the material. From the specific radioactivity of the purified phosphotyrosine the initial concentration of [32P]phosphotyrosine could be calculated. The method proved to be useful for the determination of [32P]phosphotyrosine is small amounts of cells. The minimum detectable amount of [32P]phosphotyrosine was about 1 pmol, and as an example, only 2.5 X 10(6) fibroblasts were needed. By this method it was shown that platelet-derived growth factor increased protein-bound [32P]phosphotyrosine from 600 to 3,200 pmol/g of fibroblasts, while insulin only increased the [32P]phosphotyrosine from 110 to 120 pmol/g of hepatocytes.

Using H1 as substrate the protein kinase C activity of rat liver cell sap was increased about fourfold by treatment with DEAE-cellulose at pH 7.5 at an intermediate ionic strength due to removal of protein inhibitors. The activity of cell sap from rat spleen, brain or muscle was about doubled by the same treatment. In contrast, when a specific synthetic peptide substrate was used the corresponding increase of enzyme activity was not obtained when the inhibitors were removed. This shows that this type of substrates should be preferred for reliable assays of protein kinase C in crude extracts. The possible role of the protein inhibitors for the substrate specificity of protein kinase C is briefly discussed.

A Ca/+-activated protease from rat liver cell sap was prepared. It was shown to act on rat liver pyruvate kinase that had been phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, the activity being optimum at neutral pH. The modified pyruvate kinase had the same Vmax as the phosphoenzyme but showed a lower affinity for the substrate phosphoenolpyruvate. The possibility that this proteolytic attack is the step that initiates further degradation in the cell is discussed.

An assay for protein-bound phosphate with a capacity to determine 100 pmol of phosphate is described. It is based on the combination of two well known methods: the alkaline hydrolysis of phosphate from seryl and threonyl residues in phosphoproteins and the quantification of the released phosphate by the use of malachite green and phosphomolybdate.

Glucokinase, purified from rat liver, was phosphorylated to an extent of 1 mol [32P]-phosphate/mol of enzyme when incubated with [32P]ATP and protein kinase A from pig or rabbit muscle. The phosphate was bound to serine residues. K0.5 increased and Vmax decreased upon phosphorylation. The phosphate group was removed during incubation of the phosphorylated glucokinase with alkaline phosphatase. Enzymatically inactive glucokinase was not phosphorylated by the protein kinase.

A set of stereoisomeric nonapeptides KRPSQRAKY with one, two, or all L-amino acid residues replaced by the corresponding D-amino acids, and two analogs with L- and D-threonine instead of serine, were synthesized and tested as substrates for protein kinase C. All of the peptides were phosphorylated by the enzyme. The maximal rate of the reaction with the all-D peptide was more than one order of magnitude lower than that for all-L peptide with serine. The same applied to the peptides with D-Ser or with D-Arg in position +2 with respect to Ser. The Km values for the peptides containing one D-amino acid were close to that for the prototype peptide (53 microM). On the other hand, when two or more D-amino acids were present, the Km value increased considerably. Replacement of serine by threonine also reduced the phosphorylation rate and increased the Km values. One can conclude that the stereospecificity of protein kinase C is much less pronounced than that of protein kinase A, which is in agreement with the less clearly pronounced substrate specificity of the former enzyme.

Rat liver endothelial cells cultivated in the absence of serum and activated with thrombin released up to 10% of the total protein kinase activity into the cell medium using casein or fibrinogen as the phosphate acceptor protein. The activity was partly inhibited by heparin, indicating that it was of the casein kinase II type. The release of kinase started directly after the addition of thrombin (2 NIH U/ml) to the media with two maxima; one after about 10 min and the second after around 30 min. The phosphorylating activity of media from cells incubated for longer times was less dependent on thrombin-induction which probably indicated the start of destruction of the cells. The results reported suggest that phosphorylation of fibrinogen could occur in the blood under acute phase conditions.