Protein kinase C (PKC) is a family of serine/threonine kinases implicated in intracellular signalling events triggered in response to a large variety of agonists. Currently, 11 mammalian PKC isoforms have been identified which are divided into three groups, the calcium-dependent, the non-calcium-dependent and the atypical isoforms. Common to all members is the presence of an aminoterminal regulatory domain, which renders the kinase inactive by interacting with the carboxyterminal catalytic domain. Thus, intracellular PKC activation requires the release of this autoinhibitory restraint, which, as this review summarizes, may involve both interactions with lipids and proteins. Furthermore, post-translational PKC phosphorylation events, required to convert PKC to an activation competent state, are discussed.
Neutral cholesterol ester hydrolase from rat liver microsomes was inactivated in a dose and time-dependent manner by classical sulphydryl-reacting reagents such as p-hydroxymercuribenzoic acid, 5,5'-dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide, or iodoacetate. The concentrations at which half-maximal inhibition of the native microsomal cholesterol ester hydrolase occurred (IC50) were 15, 68, and 370 mumol/l and 68 mmol/l, respectively. Only partial reactivation of the enzyme was observed under excess dithiothreitol or mercaptoethanol treatment. The stimulation of cholesterol ester hydrolase by the metal ions Ca2+ and Mg2+ was dependent on the integrity of the thiol groups. Solubilization of cholesterol ester hydrolase from membranes preserved its sensitivity towards sulphydryl reagents and thiols, as well as its ability to be activated by Ca2+ and Mg2+. Dithiothreitol, mercaptoethanol, and Ca2+ and Mg2+ provided total protection of the enzyme against inactivation by thiol-reacting reagents. The results indicate that one or more thiol groups are either at the active centre of the native and solubilized forms of rat liver microsomal cholesterol ester hydrolase or are sufficiently near, to interfere with the catalysis when they are reacted.
A variant alkaline phosphatase (ALP), with heat-sensitivity characteristics similar to that of the bone type, was found in the serum of a patient suffering from lung cancer. In disc polyacrylamide gel electrophoretic studies most of this enzyme had migrated to the region corresponding to liver ALP, with the remainder affecting bone ALP. Like kidney ALP, this ALP was markedly inhibited by 0.5 mmol/l L-cysteine. The K(m) of this ALP for p-nitrophenylphosphate was 0.39 mmol/l, similar to that of kidney ALP. The sugar moiety of this enzyme bore greater resemblance to that of kidney ALP than liver or bone ALP. However, immunoprecipitation of this particular ALP was strong with a monoclonal antibody against liver ALP and moderate with an antibody against bone ALP.
Adenylate kinase (AK; EC 2.7.4.3, hAK1) catalyzes the reaction: MgATP(2-)+ AMP2- reversible MgADP-(+) ADP3-. To elucidate the catalytic and structural roles of threonine residues in human AK, Thr35 and Thr39 mutants were analyzed by steady-state kinetics. The K(m) values of T35P and T35Y were not changed for MgATP2- and AMP2-, and the kcat values were decreased by 1/39 compared to those of wild-type AK. Thr35 was suggested to be essential for catalysis. The K(m) values of T39S, T39V and T39P were increased 5.6- to 59.0-fold for AMP2-; however, the kcat values were not reduced. Although the K(m) values of T39F and T39L were unchanged, the kcat values were reduced by more than 1/57. Thr39 appears to play an important role in the binding of AMP2- and to be essential for catalysis. As noted above, a hydroxyl group of the Thr residue in human AK appears to be important.
We report on 2 children, brother and sister, who presented with cardiomyopathy and muscular hypotonia at the age of B months. They both excreted significant amounts of 3-hydroxy-3-methylglutaric acid (3-HMG) and 3-methylglutaconic acid (3-MGC) but no 3-methylglutaric acid (3-MG). Enzyme analysis in fibroblasts revealed normal activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase and of 3-methylglutaconyl hydratase and other enzymes of 3-HMG metabolism. Loading tests with leucine did not affect the excretion of 3-HMG and 3-MGC. The girl died as a result of her cardiomyopathy, while the boy recovered and was treated with cardiac supportive therapy. He showed a steady improvement during his clinical course with biochemical normalization of the urinary excretion of 3-HMG, concomitant with marked improvement in the hypertrophic cardiomyopathy. In cultured fibroblasts from both patients a reduced activity of complex II/III of the respiratory chain was measured which may be the cause of this new type of 3-HMG uria. Analysis of mitochondrial DNA heart muscle, liver and fibroblast culture of the patient did not reveal any major mitochondrial DNA rearrangements (deletion, duplication) or any point mutation that had been described in association with mitochondrial cardiomyopathy.
It has been previously reported that Bryostatin 1 (Bryo1) induces differentiation of the human acute lymphoblastic leukemia (ALL) cell line, Reh, to a monocytoid B-cell stage. In this study we demonstrate that a novel protein, ubiquitin COOH-terminal hydrolase (UCH-L1), is associated with this differentiation. Reh cells were treated with 200 nmol/l of Bryo1 for 72 h and analyzed for changes in morphology, surface immunophenotype, acid phosphatase and terminal deoxynucleotidyl transferase. Protein patterns of the parent and differentiated cells, by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), were studied. Bryo1-treated cells expressed morphologic, phenotypic and enzymatic features of the monocytoid B-cell stage. The UCH-L1 enzyme (MW-pl 34-5.3) was detected by 2 D PAGE in the differentiated, but not in parent cells. The presence of UCH-L1 in the Bryo1-treated cells was further confirmed by immunoblotting of 2 D PAGE using UCH-L1 polyclonal antibody. Ubiquitin expression was studied in parent and Bryo1-treated cells and was compared with 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells. Both agents, TPA and Bryo1, increased the level of ubiquitin expression as detected by flow cytometry. Sodium borohydride, an inhibitor of UCH-L1, inhibited the Bryo1-induced differentiating effect on Reh cells. To date, the mechanism by which Bryo1, exerts its B-cell differentiating effect is not fully understood. This study shows that UCH-L1 expression may play a major role in Bryo1-induced differentiation in pre-B-ALL.
Kinetic studies were conducted to examine the effects of K+, Na+ and Li+ on human erythrocyte pyridoxal kinase (PK) activity. A dialyzed hemolysate served as the PK source. The substrates used were pyridoxal (PL) and ATP. Determination of the enzymatic activity was based on HPLC separation and fluorimetric detection of PL and pyridoxal 5'-phosphate as semicarbazone derivatives. In comparison to the poor activity of PK assayed without monovalent cation, all tested cations are activators. Among them, K+ is the most effective, improving both PK affinity for the substrates and maximal velocity. Na+ increases maximal velocity and PK affinity for ATP but decreases it for PL. Li+ is a poor activator which seems to modify the enzymatic mechanism from a random to an ordered sequential pattern with ATP bound before PL. Results suggest that K+ and Na+ bind to PK on the same site while Li+ binds on another site. This hypothesis and the mechanism of monovalent cation-PK interaction are compared to other well-known K(+)-activated enzymes.
We have studied some characteristics of N-acetyl-alpha-D-galactosaminidase in human plasma using a sensitive and very simple fluorimetric (single tube incubation/fixation) micromethod. The enzyme has a pH optimum at 4.5, is linear on incubation during at least 6 h, is protected from inactivation at room temperature by acidification, and is stable on freezing (about 85% residual activity after 1 year at -20 degrees C). Enzyme kinetics indicate that the affinity for the substrate is low (K(m) value 7 mmol/l). By studying about 10 possible effectors, no activation was found by detergents. As expected, the colorigenic p-nitrophenyl derivative substrate, N-acetylgalactosamine and galactose are low-affinity inhibitors. A histogram of 108 control samples showed a unimodal distribution pattern with a slight bias to the right. No pseudodeficients were found on analysis of 220 control plasma samples. Patients with alpha-galactosaminidosis had residual activity between 0.7 and 2.1%. In patients with 17 different lysosomal storage diseases, no increase was found except in mucolipidosis II and III. The main advantages of the method are its simplicity sensitivity, short incubation time requirement and economy in substrate consumption. The method can be used either for screening or diagnostic purposes of genetic N-acetyl-alpha-D-galactosaminidase deficiency.
Extracts from the parenchymatous leaf gel and the rind of the Aloe vera plant (Aloe barbadensis Miller) were shown to contain seven electrophoretically-identifiable superoxide dismutases (SODs). The chromatographic elution profiles and the migration of these bands on native polyacrylamide gel electrophoresis (PAGE), for both the gel and rind, are quite similar. Two of these seven activities are insensitive to cyanide treatment, suggesting that they are mangano-SODs. The other five activities are sensitive to cyanide treatment, but insensitive to azide treatment and are presumed to be cupro-zinc SODs. All of the seven proteins appear to be homodimers with apparent native molecular masses centered at approximately 32 and 42 kD as indicated by SDS-PAGE and gelfiltration (FPLC) chromatography. The specific activities of SODs in the A. vera rind and gel are comparable to those of spinach leaves and of rabbit liver.
The synthesis of guanidinosuccinic acid (GSA) increases in uremics, and GSA is implicated as a uremic toxin. The GSA synthesis increases roughly in proportion to the serum urea level that increases in patients with renal failure. Urea is a specific inhibitor of argininosuccinase, the fourth urea cycle enzyme, and might lead to the increase of argininosuccinate (ASA). We found that GSA is formed from ASA by reactive oxygen species in vitro. In this paper, we investigated GSA synthesis from ASA in isolated rat hepatocytes and the effect of reactive oxygen species on this synthesis. When isolated rat hepatocytes were incubated with 5 mmol/l ASA, GSA was formed linearly with time up to 6 h (16 nmol/g wet liver/6 h). GSA was formed depending on the ASA concentration up to 10 mmol/l. Dimethylsulfoxide, a hydroxyl radical scavenger, inhibited GSA synthesis by 65%. GSA was actively formed when the hepatocytes were incubated with 32 mmol/l urea. The GSA formation in the presence of urea was also inhibited by dimethylsulfoxide, although the inhibition was less marked. FeCl2, that increases the hydroxyl radical generation, increased GSA synthesis. These results indicate that GSA is formed from ASA in isolated hepatocytes. The results also suggest that reactive oxygen species are important for GSA synthesis in the cells.
Synthesis of guanidinosuccinic acid (GSA), a uremic toxin, has been suggested to relate to the urea concentration and synthetic rate. Among the urea cycle enzymes, inhibition of argininosuccinate (ASA) lyase by urea has been reported. Argininosuccinate which contains a GSA structure is a candidate of a GSA precursor. We found that another uremic toxin, methylguanidine, is formed from creatinine with reactive oxygen species. Therefore, we investigated in vitro whether GSA is formed from ASA with reactive oxygen species. GSA was measured by HPLC by a post-column-labeling method using 9,10-phenathrequinone. When 1 mmol/l ASA was reacted with the hydroxyl radical-generating system for 5 min at pH 7.4, 9 mumol/l GSA was formed. Dimethylsulfoxide, a hydroxyl radical scavenger, markedly inhibited GSA synthesis. The superoxide radical generated by xanthine and xanthine oxidase reaction also formed 1 mumol/l GSA from 1 mumol/l ASA and the GSA formation was inhibited by superoxide dismutase or catalase almost completely. Addition of FeCl2 to the xanthine/xanthine oxidase reaction further increased GSA synthesis. These results indicate that GSA is formed from ASA by reaction with the hydroxyl radical and the superoxide radical.
Previous studies from our laboratory demonstrate that polyamines, namely spermine and spermidine, stimulate adipose triacylglycerol formation from the sn-glycerol-3-phosphate pathway by activation of several enzymes from this pathway, including sn-glycerol- 3-phosphate acyltransferase. Mg^2+-dependent phosphatidate phosphohydrolase and diacylglycerol acyltransferase. Since obesity in Zucker rats was associated with increased accumulation of adipocyte triacylglycerols, we have examined the relationship between changes in the activities of various triacylglycerol synthetic enzymes and the endogenous concentrations of spermine and spermidine in the adipose tissues from lean and obese animals. As compared with lean rats, the adipocytes from obese rats showed a 4-fold rise in the concentration of spermine and spermidine which was accompanied by 4- to 14-fold increases in the activities of various triacylglycerol synthetic enzymes, including Mg^2+ dependent phosphatidate phosphohydrolase. These studies suggest that obesity in Zucker rats is associated with the activation of various adipose triacylglycerol synthetic enzymes resulting from increased concentrations of endogenous spermine and spermidine.
Overexpression and altered trafficking of cathepsin B characterize the malignant phenotype of tumor cells. Human cathepsin B is encoded by a single-copy gene located on chromosome 8p22. With its 13 exons, the gene encompasses at least 27 kb of DNA. Expression of cathepsin B can be regulated at transcriptional and posttranscriptional levels. Multiple cathepsin B mRNA species arising from alternative splicing may be related to tissue- and tumor-specific differences in expression. There is selective overexpression, increased activity, membrane association and secretion of cathepsin B in many etiologically different cancers. This suggests that cathepsin B may play a functional role in malignant progression. Recent clinical studies provide confirmatory evidence in that cathepsin B expression is a prognostic indicator in colon carcinoma.
Matrix metalloproteinases (MMPs) are expressed in normal remodeling tissues in a generally tissue-restricted pattern. Transcripts for stromelysin-l and collagenase are expressed primarily in stromal fibroblasts, whereas transcripts for matrilysin are expressed primarily in glandular epithelial cells. These expression patterns are maintained at carcinoma tumor sites until the late stages of tumor progression at which point many epithelially-derived tumors begin to express stromal fibroblast MMPs. Coincidentally, late stage carcinomas take on other characteristics of stromal fibroblasts, indicating that these tumor cells have 'transdifferentiated', that is, they have begun to exhibit characteristics of cells from a separate developmental lineage. Despite their distinct expression patterns, many of the promoters for MMP genes show the same general arrangement of the nuclear proto-oncoprotein-binding sites, AP-1 and PEA3. However, the specific interaction between these cis-elements and different combinations of Fos, Jun, and Ets proteins which recognize these sites may be important in controlling both the positive and negative regulation involved in the tissue-restricted pattern of MMP expression in normal and neoplastic tissues.
Components of matrix-degrading protease systems are in human cancer often expressed by tumour-infiltrating stromal cells. The cellular pattern of expression of these molecules appears to be unique for each type of cancer. In several cases there are similarities with patterns observed in nonmalignant remodelling processes in the same tissue. These findings indicate that the stromal cells actively participate in the process of cancer invasion. The implications of this new paradigm for cancer biology and cancer treatment are discussed.
In the initial stages of capillary formation (angiogenesis) microvascular endothelial cells of preexisting blood vessels locally degrade the underlying basal lamina and invade into the stroma of the tissue to be vascularized. A consistent body of experimental evidence has shown that this process requires a wide array of dedradative enzymes. Components of the plasminogen activator (PA)-plasmin system and of the matrix metalloproteinase (MMP) family play important roles. PAs trigger a proteinase cascade that results is the generation of high local concentrations of plasmin and active MMPs. This increase in proteolytic activity has three major consequences: it permits endothelial cell degradation and invasion of the vessel basal lamina, generates extracellular matrix (ECM) degradation products that are chemotactic for endothelial cells, and activates and mobilizes growth factors localized in the ECM. In addition, urokinase-type PA modulates some endothelial cell functions, including proliferation and migration, with a mechanism independent of proteolytic activity. PA and MMP activities are modulated in endothelial cells by complex mechanisms, including transcriptional regulation by a variety of growth factors and cytokines with angiogenic activity, extracellular control of the proteolytic activities by tissue inhibitors, and interaction with binding sites on the cell membrane and ECM.
Cathepsin D (Cath D) overexpression in breast cancer cells is associated with increased risk of metastasis in patients according to several clinical studies. The amino acid sequence of Cath D in two breast cancer cell lines was normal, but glycosylation appears to be different with more acidic isoforms. Transfection of a human cDNA Cath D expression vector increases the metastatic potential of 3Y1-Ad12 embryonic rat tumorigenic cells when intravenously injected into nude mide. The mechanism of Cath-D-induced metastasis seems to require maturation of the proenzyme, mostly in large acidic compartments identified as phagosomes. Cath D is mitogenic in different cell types, and different substrates (growth inhibitors, precursors of growth factors, etc.) are proposed to mediate this activity.
Stromelysin-1 was one of the first proteinases found to be associated with cancer. In this review we describe the role of stromelysin-1 in normal mammary gland involution. When stromelysin-1 is overexpressed in transgenic mice the mammary gland undergoes precocious involution and is predisposed to forming a reactive stroma resembling that of a wound site or a tumor. Stromelysin-1 may act as an oncogene because transgenic mice expressing an active form of the enzyme develop mammary tumors. These observations suggest that stromelysin-1 and other matrix metalloproteinases may be useful targets for therapeutic intervention in cancer.
Several classes of extracellular matrix (ECM)-degrading proteases have been shown to play an important role in tumor invasion and metastasis. Among them, the matrix metalloproteinases (MMPs) and the plasminogen activator (PA)-plasmin system have been the focus of numerous studies. However, few of those have examined the interaction of these two classes of proteases during tumor progression and their specific roles in this complex process have remained unclear. In this article, comparative information on the structure, function, and regulation of these two classes of proteases is reviewed and their interaction on various levels is discussed. This review shows that MMPs and the PA-plasmin system closely cooperate to achieve optimal degradation of the ECM during the invasive and metastatic process.
