Dyeing of wool fibres with natural dyes: effect of proteolytic enzymes

In spite of the widespread use of proteins (casein, peptone, etc.) and protein fragments as a substrate for the proteolytic enzymes, a substrate prepared from dyes that adsorb onto appropriate materials, such as wool and cotton, are also used for enzyme activity determination. In the point of view of this thought, it was our aim to develop the substrates which are easily and economically obtainable and also environmentally safer for the frequently used proteolytic enzymes, such as subtilisin carlsberg, trypsin, chymotrypsin, and protease type XVI and, if possible, to prepare the specific substrate at least for one of these enzymes. For this aim, wool was dyed with natural dyes such as juglone, lawsone, berberine, and quercetin. The optimum pH, incubation time, and agitation rate were determinated. The results indicate that, of all the tested enzymes on wool-dye complex as an insoluble substrate, the most appropriate complex was found to be wool-lawsone complex.     

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.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Purification of substrate proteins of casein kinases from the cytosol fraction of AH-66 hepatoma cells

We have attempted to purify endogenous substrate proteins for casein kinases I and II from the cytosol of AH-66 hepatoma cells. Utilizing the fact that only a few substrates are concentrated in the fraction eluted from DEAE-cellulose between 0.3 and 0.6 M NaCl, two substrates were purified from this fraction by DEAE-cellulose chromatography, hydroxyapatite chromatography, and HPLC on a DEAE-5PW column. The purified substrate proteins had molecular masses of 30.5 kDa and 31 kDa. The 31-kDa protein substrate was markedly phosphorylated by casein kinase II, but only slightly by casein kinase I. The radioactive phosphate incorporated into 31-kDa substrate by casein kinase II was 0.2 mol/mol of the protein and phosphorylation occurred on both threonine and serine residues. The 30.5 kDa protein was only slightly phosphorylated by casein kinase II, but not at all by casein kinase I.     

Identification of the 90 kDa substrate of rat liver type II casein kinase with the heat shock protein which binds steroid receptors

It was recently reported that the type II casein kinase of rat liver cytosol co-purified with a major 90 kDa substrate when subjected to gel filtration at low ionic strength. The identity of the 90 kDa substrate was unknown. We have verified this report and have shown that the 90 kDa substrate is recognized by a monoclonal antibody prepared against the 90 kDa heat shock protein. This ubiquitous phosphoprotein is known to increase in abundance in cells subjected to heat stress and has been shown to complex steroid receptors and certain retrovirus tyrosine kinases.     

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Induction of a substrate for casein kinase II during lymphocyte mitogenesis

Particulate fractions prepared from concanavalin A-activated murine T lymphocytes contain an endogenous protein kinase that phosphorylates an endogenous protein substrate of Mr 112 000. The phosphorylation of 112 kDa protein is greatly reduced or absent in unstimulated T cells. Phosphoamino acid analysis indicates that 112 kDa protein is labeled on a serine. Add-back experiments using purified protein kinases indicate that 112 kDa protein serves as a substrate for casein kinase II. Phosphorylation of 112 kDa protein by the endogenous kinase is inhibited by heparin, a known casein kinase II inhibitor. The site or sites modified by the endogenous kinase and exogenous casein kinase II appear identical by peptide-mapping experiments. A time-course of the appearance of phosphorylated 112 kDa protein following stimulation with concanavalin A, measured in the presence or absence of added casein kinase II, suggests that 112 kDa protein is induced in activated T cells. Subcellular localization studies suggest that 112 kDa protein is a nuclear protein. Silver-binding and purification studies suggest that 112 kDa protein is of the nucleolar organizing region.     

Role of acidic residues as substrate determinants for casein kinase I

Sites phosphorylated by casein kinase I have been characterized by the presence of acidic amino acids NH2-terminal to the modified residue. Recently, phosphoserine was shown to be a particularly effective determinant for casein kinase I action when present in the motif -S(P)-X-X-S- (Flotow, H., Graves, P. R., Wang, A., Fiol, C. J., Roeske, R. W., and Roach, P. J. (1990) J. Biol. Chem. 265, 14264-14269). Nonetheless, nonphosphorylated substrates for casein kinase I are well documented. In this study, we examined the efficacy of Asp and Glu residues as determinants of casein kinase I action using synthetic peptide substrates. Peptides with runs of Asp residues in the motif Dn-X-X-S- were substrates for casein kinase I. Peptides with n = 3 or 4 were the most effective substrates, much better than n = 2. The peptide with n = 1, a single Asp residue, was a very poor substrate. A block of 4 Glu residues was a little less effective as a substrate determinant than 4 Asp residues in an otherwise identical peptide. The most effective substrate, with the motif -D-D-D-D-X-X-S-, was specific for casein kinase I and was not detectably phosphorylated by cyclic AMP-dependent protein kinase, casein kinase II, glycogen synthase kinase 3, or phosphorylase kinase and thus will be useful for the specific assay of casein kinase I. This peptide was nonetheless significantly worse as a substrate than peptides in which casein kinase I action was determined by phosphoserine in the -3 position. Still, the fact that Asp or Glu residues can specify a casein kinase I substrate suggests that acidic character has a role in substrate selection by this protein kinase.     

Determination of proteolytic activity with magnetic dye-stained gelatine

A procedure for the determination of proteolytic activity with dyed magnetic gelatine as an insoluble chromolytic substrate is described. The magnetic nature of the substrate enables magnetic separation of unhydrolysed substrate from the hydrolysed dyed peptide fragments. Such type of substrates could enable the development of new automated protease assays based on the principle of Flow Injection Analysis (FIA).     

Insoluble chromolytic substrates for the determination of proteolytic activity

New insoluble chromolytic substrates for the determination of proteolytic activity were prepared by incorporation of dyed proteins into the structure of calcium sulphate dihydrate. The prepared substrates could detect approximately 0.25–1.0 μg of trypsin per ml. The spontaneous leakage of dyed substances in water solutions was negligible.     

Transglutaminase-mediated protein immobilization to casein nanolayers created on a plastic surface

An enzymatic method for covalent and site-specific immobilization of recombinant proteins on a plastic surface was explored. Using Escherichia coli alkaline phosphatase (AP) with a specific peptide tag (MKHKGS) genetically incorporated at the N-terminus as a model (NK-AP), microbial transglutaminase (MTG)-mediated protein immobilization was demonstrated. To generate a reactive surface for MTG, a 96-well polystyrene microtiter plate was physically coated with casein, a good MTG substrate. Successful immobilization of recombinant AP to the nanolayer of casein on the surface of the microtiter plate was verified by the detection of enzymatic activity. Since little activity was observed when wild-type AP was used, immobilization of NK-AP was likely directed by the specific peptide tag. When polymeric casein prepared by MTG was used as a matrix on the plate, the loading capacity of AP was increased about 2-fold compared to when casein was used as the matrix. Transglutaminase-mediated site-specific posttranslational modification of proteins offers one way of generating a variety of protein-based solid formulations for biotechnological applications.     

Effect of bivalent cations on rat liver cytosol casein (glycogen synthase) kinases 1 and 2. Influence of the protein substrate.

Rat liver cytosol casein kinases 1 and 2 were stimulated by free Mg2+, but the optimal concentration of cation varied with both the casein kinase and the protein substrate used. Mn2+, but neither Ca2+ nor Zn2+, could efficiently substitute for Mg2+ in forming the bivalent-cation-ATP complex used as substrate, but free Mn2+ was inhibitory. The magnitude of these effects depended on the type of casein kinase and the protein substrate used. These results support the idea that, besides the effects of Mg2+ as a component of the Mg-ATP complex, or through interaction with the protein substrate, free Mg2+ is an allosteric effector of both casein kinases.     

Purification and Some Properties of Crystalline Acid Protease from Acrocylindrium sp.

A crystalline acid protease produced by a strain of Acrocylindrium in a submerged culture was prepared by treatment with acetone (60%), salting out with ammonium sulfate (saturated) and, after chromatography on Duolite GS-101 column, dialysis against distilled water. This preparation was homogeneous on sedimentation analysis, starch-gel electrophoresis and gel filtration with Sephadex G-75. The optimum pH was 2.0 for milk casein digestion and the pH stability was for 2.0~5.0 at 30°C for one day. The crystalline enzyme was completely stable below 50°C, but lost the activity at 70°G in ten minutes. The acid protease was almost equal to pepsin on specific activity when milk casein solution (pH 2.0) was used as substrate.     

Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.     

Use of fluorescamine-labeled casein as a substrate for assay of proteinases.

Conditions have been investigated for the use of fluorescamine-labeled casein as a substrate for fluorometric assay of proteinases. Fluorescamine-labeled casein can be prepared simply by mixing solutions of casein and fluorescamine at pH 8.0 and used without removal of the excess reagent or its hydrolysis product. The fluorescence of the labeled casein and its enzymatic digest is moderately stable in the range of pH 7.0 to 10.0. Activities can be determined by measuring the fluorescence of the hydrolysis products soluble in 0.1 M trichloro acetic acid solution at pH 4.0 after adjusting the pH of the acid-soluble fraction to 7.7. This method is suited for assay of proteinases active at neutral to slightly alkaline pH values, and is capable of quantitating about 0.05 microgram of trypsin or 0.5 microgram of alpha-chymotrypsin or papain. The assay can be done in the presence of large amounts of contaminating amino acid, protein and/or exopeptidases which may interfere with the ordinary assay of proteinases.     

Phosphate groups as substrate determinants for casein kinase I action

Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase has been shown to enhance subsequent phosphorylation by casein kinase I (Flotow, H., and Roach, P. J. (1989) J. Biol. Chem. 264, 9126-9128). In the present study, synthetic peptides based on the sequences of the four phosphorylated regions in muscle glycogen synthase were used to probe the role of substrate phosphorylation in casein kinase I action. With all four peptides, prior phosphorylation significantly stimulated phosphorylation by casein kinase I. A series of peptides was synthesized based on the NH2-terminal glycogen synthase sequence PLSRTLS7VSS10LPGL, in which phosphorylation at Ser7 is required for modification of Ser10 by casein kinase I. The spacing between the P-Ser and the acceptor Ser was varied to have 1, 2, or 3 intervening residues. The peptide with a 2-residue spacing (-S(P)-X-X-S-) was by far the best casein kinase I substrate. When the P-Ser residue at Ser7 was replaced with P-Thr, the resulting peptide was still a casein kinase I substrate. However, substitution of Asp or Glu residues at Ser7 led to peptides that were not phosphorylated by casein kinase I. Phosphorylation of one of the other peptides showed that Thr could also be the phosphate acceptor. From these results, we propose that there are substrates for casein kinase I for which prior phosphorylation is a critical determinant of protein kinase action. In these instances, an important recognition motif for casein kinase I appears to be -S(P)/T(P)-Xn-S/T- with n = 2 much more effective than n = 1 or n = 3. Thus, casein kinase I may be involved in hierarchal substrate phosphorylation schemes in which its activity is controlled by the phosphorylation state of its substrates.     

Calcium and the assays of human plasma clotting Factor XIII

1. A continuous fluorimetric assay for blood-clotting Factor XIII based on the incorporation of dansylcadaverine into casein was investigated. 2. Hammarsten casein was fractionated to yield the beta-casein, which was dephosphorylated and acetylated to give a substrate which itself did not bind calcium and which was not cross-linked by the activated Factor. 3. The modified beta-casein was used as substrate for a continuous fluorimetric assay and as substrate for the incorporation of radioactive glycine ethylester. 4. A linked fluorimetric assay for the zymogen is described. 5. The K(m) for calcium was redetermined and at 0.2mm was in the physiological range and much lower than the values reported by others using substrates which interact with calcium. 6. The K(m) for casein is about 14mum.     

Activation of casein kinase II by sphingosine

Sphingosine activates casein kinase II in the presence of endogenous substrates as well as a synthetic peptide substrate. The activation response occurred between 12 and 25 micrograms/ml sphingosine and exhibited positive cooperativity with a Hill coefficient of 3.0. Sphingosine not only increased the Vmax of casein kinase II but decreased the Km(app) for the peptide substrate from 0.5 to 0.08 mM. In contrast, the Km(app) for MgCl2 was increased from 0.12 to 0.7 mM. Consequently, sphingosine altered significantly several parameters which determine casein kinase II activity. The effect of sphingosine was relatively specific, inasmuch as related lipids were less potent activators or largely ineffective in stimulating casein kinase II. On the other hand, the effect of sphingosine itself could be potentiated or inhibited by other lipids. Ceramide and sphingosylphosphorylcholine augmented the sphingosine effect. Phospholipids alone did not alter the activity of casein kinase II significantly, but abolished enzyme activation by sphingosine with different potencies (phosphatidylserine greater than phosphatidylethanolamine greater than phosphatidylinositol greater than phosphatidylcholine). Moreover, the sphingosine effect could be abrogated by KCI and NaCl, which alone are known to induce enzyme activation and dissociation of aggregated casein kinase II protein; LiCl and NH4Cl also inhibited the sphingosine effect. Polyamines, known activators of casein kinase II, partially mimicked the effect of sphingosine on endogenous polypeptide phosphorylation but failed to do so with the peptide substrate. These observations demonstrate that sphingosine is a potent activator of casein kinase II. The potential pharmacological and physiological modulation of casein kinase II by sphingoid bases is discussed.     

Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.     

Phosphoserine in peptide substrates can specify casein kinase II action

Casein kinase II is a ubiquitous serine/threonine protein kinase which utilizes acidic amino acid residues as recognition determinants in its substrates, the motif -S/T-X-X-D/E- being particularly important. To test whether a phosphoserine residue can act as a substrate determinant, a peptide was synthesized, containing the sequence -S-X-X-S, which was not phosphorylated by casein kinase II. However, upon phosphorylation at the +3 position, the peptide became a substrate for casein kinase II. With another peptide, a positive influence of more distal phosphorylations was found. The results indicate the potential for casein kinase II to participate in hierarchal phosphorylation schemes.