Expression and regulation of casein kinase 2 during heat shock in Verticillium dahliae.

A homogeneous preparation of casein kinase 2 has been isolated from the phytopathogenic fungus Verticillium dahliae (the parasite of cotton). The enzyme consists of three subunits with molecular masses of 53, 41, and 38 kDa. Highly specific immune serum against casein kinase 2 has been obtained. By means of immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunochemical isolation on protein A-Sepharose, it is shown that the amount of casein kinase 2 increases under heat shock conditions (at least in part due to the synthesis de novo), while the synthesis of the majority of other proteins falls. The activity of casein kinase 2 is supressed during heat shock and so does not correlate with its content. The results give an evidence for the two-step model of casein kinase 2 regulation during heat shock.

pH-dependent changes in structure and RNA-binding activity of casein kinase 2 from Rana temporaria oocytes.

It is demonstrated by filter-binding assay that casein kinase 2 from Rana temporaria oocytes binds rRNA in vitro with high affinity. Ligand-blotting shows that rRNA-binding activity is inherent to alpha and alpha' subunits of the enzyme. Increase of pH from 6.5 to 7.5 has little effect on casein kinase but completely suppresses rRNA-binding activity of the enzyme. Sedimentation coefficient of casein kinase 2 also depends on pH: at pH 7.5 it is mainly 10 S, and at pH 6.5-18 S. At pH 6.95 the amounts of both forms are equal. The heavy form of casein kinase 2 practically lacks rRNA-binding activity.

The decreased level of casein kinase 2 in brain cortex of schizophrenic and Alzheimer's disease patients.

The content of casein kinase 2 is considerably decreased in ribosome-free extracts of the frontal cortex of schizophrenic and Alzheimer's disease patients in comparison to normal brains as has been demonstrated by means of immunoblotting. The activity of casein kinase 2 towards endogenous substrates and casein is also diminished in the cases of mental pathologies examined. This phenomenon may explain the well-known aberrations in the phosphorylation of structural proteins of human brain which are intrinsic for the mental diseases.

[A comparative analysis of chromatographic, kinetic and antigenic properties of casein kinase type 2 from bovine liver and the phytopathogenic mushroom Verticillium dahliae]. / Sravnitel'nyi analiz khromatograficheskikh, kinetichskikh i antigennykh svoistv kazeinovykh kinaz tipa 2 iz pecheni byka i fitopatogennogo griba Verticillium dahliae.

Homogeneous preparations of casein kinases 2 have been isolated from bovine liver and the phytopathogenic fungus Verticillium dahliae. The isolation procedure was similar in both cases and included consecutive chromatography on heparin-Sepharose, phosphocellulose PII and monoQ. The bovine liver enzyme consists of two subunits with molecular masses of 38 kDa and 27 kDa, while the fungus kinase has three subunits with Mr of 53 kDa, 41 kDa and 38 kDa. Self-phosphorylation of casein kinase 2 in vitro resulted in phosphate incorporation into the smaller subunit of the mammalian enzyme and into all the three subunits of the fungal enzyme. The Km value of casein kinase 2 from bovine liver for ATP is 3.1 microM; that for GTP is 22.0 microM. The corresponding parameters of the fungal kinase are 14.3 and 18.2 microM, respectively. Specific antibodies to each kinase have been raised. The lack of antigenic cross-reactions between the two enzymes has been demonstrated by ELISA.

[A new type of Clostridium thermocellum endoglucanase produced by the recombinant strain of E. coli. Some properties and identification in donor cells]. / Novyi tip éndogliukanazy Clostridium thermocellum, obrazuemoi rekombinantnym shtammom E. coli. Svoistva i identifikatsiia v organizme donora.

The properties of endoglucanase produced by the recombinant strain of E. coli carrying plasmid pCU 104 with a 2.9 kb insert of chromosomal DNA of C. thermocellum encoding the multiple forms of the 35.5 kD polypeptide (pI 4.3-4.7) were studied. The enzyme has a broad pH optimum of activity (6.0-7.5). The half-inactivation time for different forms of the enzyme at 65 degrees C is similar and is equal to 25-30 minutes. The enzyme is related to endoglucanases weakly adsorbed on cellulose (Kp = 0.065 1/g). Hydrolysis of microcrystalline cellulose is completed within 7 days (7-9%) and is accompanied by the formation of cellobiose and cellotriose. The enzyme splits dyed lichenan (mixed 1,3-1,4-beta-glucane) at a higher rate than the dyed CM-cellulose. A guinea pig antiserum to enzyme isoforms with a pI of 4.46-4.54 was obtained. Using direct solid phase immunoenzymatic analysis, it was demonstrated that all the enzyme isoforms under study (pI 4.3-4.7) are immunologically related (serum titers for different enzyme isoforms vary from 1:20,000 to 1:50,000). In the original culture fluid of C. thermocellum, the antigen related to the enzyme isolated from the recombinant strain was unobserved. However, SDS-PAAG electrophoresis of SDS- and mercaptoethanol-treated culture fluids revealed among 11 protein bands at least 4 antigens interacting with antibodies (Mr = 107, 76, 67 and 37 kD), although their antibody titers were far lower and did not exceed 1:300-1:500. The cumulative data suggest that the endoglucanase under study is not identical to the earlier described enzymes encoded by the cel A- and ceI B-genes of C. thermocellum.