An inexpensive insoluble chromogenic substrate for the determination of proteolytic activity

Summary A new insoluble chromogenic substrate for the determination of proteolytic activity was developed. This substrate was prepared by incorporating black drawing ink into casein and heating this complex at 200°C for 4 h. It is especially suitable for determining the activity of alkaline bacterial proteinases.     

Phosphorylation of Delta Sleep-Inducing Peptide (DSIP) by casein kinase II in vitro

A phosphorylated analogue of DSIP at Ser⁷ has been shown to exist endogenously by immunochemical studies. An enzyme which could phosphorylate DSIP has not yet been identified. In the present study, we examined DSIP as a substrate for in vitro phosphorylation by casein kinase II. DSIP was phosphorylated by the enzyme with apparent K_m and V_max values of 20 mM and 90.9 nmol/min/mg protein, respectively. Both ATP and GTP were utilized as phosphoryl donors. Phosphorylation of DSIP was inhibited by heparin and enhanced by spermine. These results demonstrate that DSIP can serve as a possible substrate for casein kinase II in vitro.     

Autophosphorylation of type 2 casein kinase TS at both its α- and β-subunits: Influence of different effectors

Rat liver casein kinase TS (Ck-TS) having quarternary structure α₂β₂, autophosphorylates at its 25 kDa, β-subunits, incorporating up to 1.2 mol P/mol enzyme. According to their effects on the autophosphorylation pattern the effectors of Ck-TS activity can be grouped into 3 classes: (i) inhibitors, like heparin, which also prevent the autophosphorylation of the β-subunit; (ii) stimulators possessing several amino groups (like spermine) which increase the autophosphorylation at the β-subunit; (iii) stimulators possessing several guanido groups, like protamines and related peptides, which prevent the phosphorylation of the β-subunit, while promoting the autophosphorylation of the 38 kDa α-subunit. In the presence of such polyarginyl effectors the 130 kDa Ck-TS is converted into forms with higher sedimentation coefficient.     

The secretory nature of the excretory gland cells of Strephanurus dentatus. 3. Proteinase inhibitors

A proteinase inhibitor(s) was found in extracts of the excretory gland cells, intestines, esophagi, reproductive organs, and body walls from Stephanurus dentatus adults. The specific activity of the inhibitor(s) in the excretory gland cell extract was 45–175 times greater than in the other tissues. It is heat stable at pH 5.0 and inhibits the esterolytic activity of trypsin and chymotrypsin using p-toluenesulfonyl-l-arginine methyl ester hydrochloride (TAME) and benzoyl-l-tyrosine ethyl ester (BTEE) as the substrates, respectively, and also the proteolytic activity of both chymotrypsin and trypsin using casein as the substrate. S. dentatus adults maintained in NCTC 109 medium, secreted a trypsin inhibitor.     

Phosphorylation of Tubulin by Casein Kinase II Regulates Its Binding to a Neuronal Protein (NP185) Associated with Brain Coated Vesicles

We recently described a new protein associated exclusively with neuronal clathrin-coated vesicles (CCVs), and characterized two monoclonal antibodies that react with it (S-8G8 and S-6G7). In this report, the association of neuronal protein of 185 kilodaltons (NP185) with CCV kinases and its interaction with tubulin are described. The affinity of NP185 for tubulin is significantly enhanced when tubulin is phosphorylated by CCV-associated casein kinase II. In contrast, phosphorylation of tubulin by a kinase activity associated with purified brain tubulin decreases its affinity for NP185. Together, these data suggest that the interaction of NP185 with tubulin is modulated by protein phosphorylation. Recent evidence has suggested that tubulin is phosphorylated by casein kinase II during neurite development. The enhanced affinity of NP185 for tubulin phosphorylated by casein kinase II could be important for proper intracellular sorting of this protein in the developing neuron.     

Localization of Sites in the Tail Domain of the Middle Molecular Mass Neurofilament Subunit Phosphorylated by a Neurofilament-Associated Kinase and by Casein Kinase I

Abstract: We have shown previously that a neurofilament (NF)-associated kinase (NFAK) extracted from chicken NF preparations phosphorylates selectively the middle molecular mass NF subunit (NF-M). Here we show that the major kinase activity in NFAK is indistinguishable from enzymes of the casein kinase I (CKI) family based on the following criteria: (1) inhibition of NFAK phosphorylation by the selective CKI inhibitor CKI-7, (2) the similarity in substrate specificity of NFAK and authentic CKI, (3) the correspondence of two-dimensional phosphopeptide maps of NF-M phosphorylated in vitro by NFAK with those generated by CKI under similar conditions, and (4) immunological cross-reactivity of NFAK with an antibody raised against CKI. We have also identified Ser⁵⁰², Ser⁵²⁸, and Ser⁵³⁶ as phosphorylation sites by NFAK/CKI in vitro, each of which is also phosphorylated in vivo. All three serines are found in peptides with CKI phosphorylation consensus sequences, and Ser⁵²⁸ and Ser⁵³⁶ and flanking amino acids are highly conserved in higher vertebrate NF-M sequences. Neither Ser⁵⁰² nor Ser⁵³⁶ has been identified previously as NF-M phosphorylation sites.     

YI-S, a Casein-free Medium for Axenic Cultivation of Entamoeba histolytica, Related Entamoeba, Giardia intestinalis and Trichomonas vaginalis

ABSTRACT. Pancreatic digests of casein are major ingredients of media used in the axenic cultivation of lumen-dwelling parasitic protozoa, especially Entamoeba, Giardia , and trichomonads. The digest used almost exclusively in the development of these media, Medo-Peptone (Trypticase® BBL), has not been available since 1981. Moreover, none of dozens of similar type digests tested since then in our laboratory has proved equal to Medo-Peptone, and in the last two years it has become increasingly difficult to obtain new batches which will support even modest growth of Entamoeba histolytica . In response to this problem we have developed a casein-free medium, YI-S, consisting of a nutrient broth, vitamin mixture and serum. We recommend it as a replacement for the casein-dependent medium TYI-S-33, currently the most widely used for axenic culture of Entamoeba histolytica and other lumen-dwellers.     

Identification and Characterization of Protein Kinase FA/ Glycogen Synthase Kinase 3 in Clathrin-Coated Brain Vesicles

Abstract: Mg-ATP-dependent protein phosphatase activating factor [kinase F_A/glycogen synthase kinase 3 (GSK-3)] has been identified in highly purified clathrin-coated vesicles (CCVs) isolated from pig brain. Kinase F_A was found to exist in an inactive state but can be activated by 1% Triton X-100 or [ M /Tris-HC] extraction in brain CCVs. Activation of kinase F_A in CCVs is due to disassociation of the kinase from CCVs as demonstrated on sucrose density-gradient ultracentrifugation and Sepharose CL-4B gel filtration. Using purified brain CCVs as substrates, kinase F_A enhanced the endogenous phosphorylation of assembly protein complexes in the molecular weight range of 100,000-130,000 severalfold, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Comparisons with well-defined brain CCV-associated endogenous protein kinases such as pp50 kinase/AP50 and casein kinase 2 provide evidence that kinase F_A/GSK-3 represents a third potent and unique CCV-associated protein kinase distinctly different from the previously described CCV protein kinases, suggesting the possible involvement of kinase FA in the regulation of CCV functions in the brain. The results also support the notion that protein kinase F_A is involved in cell surface signal transduction in the CNS.     

Casein Kinase II Activity in the Postischemic Rat Brain Increases in Brain Regions Resistant to Ischemia and Decreases in Vulnerable Areas

Abstract: Casein kinase II (CKII) is a protein kinase acting in the intracellular cascade of reactions activated by growth factor receptors, and that has a profound influence on cell proliferation and survival. In this investigation, we studied the changes in the activity and levels of CKII in the rat brain exposed to 10. 15 and 20 min of transient forebrain ischemia followed by variable periods of reperfusion. The cytosolic CKII activity decreased during reperfusion by ∼ 30 and ∼ 50% in the selectively vulnerable areas, striatum and the CA1 region of the hippocampus, respectively. In the resistant CA3 region of hippocampus and neocortex, the activity increased by ∼ 20 and ∼ 60%, respectively. The postischemic changes in CKII activity were dependent on the duration of the ischemic insult. The levels of CKII did not change after ischemia, suggesting that the enzyme is modulated by covalent modification or is interacting with an endogenous inhibitor/activator. Treatment of the cytosolic fraction from cortex of rats exposed to ischemia and 1 h of reperfusion with agarose-bound phosphatase decreased the activity of CKII to control levels, suggesting that CKII activation after ischemia involves a phosphorylation of the enzyme. The correlation between postischemic CKII activity and neuronal survival implies that preservation or activation of CKII activity may be important for neuronal survival after cerebral ischemia.