Tyrosine protein kinase activity in purified rat Leydig cells

Tyrosine protein kinase activity has been estimated in purified testicular cells with the synthetic peptide substrate NH₂-GLU-ASP-ALA-GLU-TYR-ALA-ALA-ARG-ARG-ARG-GLY-COOH. High levels of enzyme specific activity (56–165 pmol/mg/min) were found in the two populations of Leydig cells isolated by Metrizamide gradient centrifugation. Some activity was also detected in germinal cells, red cells and seminiferous tubules from testis but at levels 6–20 times lower than those found in the Leydig cell fractions. Higher levels of tyrosine protein kinase specific activity were found in population I than in population II Leydig cells.     

Multiple forms of uridine kinase in normal and neoplastic rat liver

Two species of uridine kinase with molecular weights of approximately 120000 (I) and 30000 (II) have been identified in the rat liver system. Species I predominates in the 7-day postnatal and adult rat liver and increases in the regenerating remnant of the latter after partial hepatectomy; the concentration of species II is low in these tissues. Species I also predominates in the slow-growing hepatomas 5123D and 7800. In contrast, II is the predominant form in the foetal rat liver and accounts for 40% of the total activity in the rapidly growing Novikoff ascites hepatoma. In contrast to species II, which was stable, species I was inactivated by preincubation for 30min at 37 degrees C, before assay at 23 degrees C.     

Tyrosine protein kinase activity of rat spleen and other tissues

Using a synthetic peptide (Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly) as a substrate, various normal tissues from the rat were probed for tyrosine protein kinase activity. Spleen was shown to contain much higher tyrosine protein kinase activity than other rat tissues (lung, brain, testes, liver, kidney, heart, and thymus, in decreasing order of specific activity). Most of the tyrosine protein kinase activity of the various rat tissues (greater than 80%) was present in the particulate fraction, and Nonidet P-40, a nonionic detergent, could activate the particulate form of the enzyme 2-20-fold in many of the tissues. Epidermal growth factor (1 microgram/ml), cyclic AMP, cyclic GMP, or Ca2+ did not increase spleen tyrosine protein kinase activity. Half-maximal enzyme activity was observed at 60-80 microM MgATP and at 2.2 mM peptide, and both Mg2+ (10 mM) and Mn2+ (0.5-1.0 mM) were effective divalent metal ions for the expression of activity. When the particulate fraction of spleen was incubated with [gamma-32P]ATP followed by polyacrylamide gel electrophoresis in the presence of Na dodecyl SO4, a number of alkali-stable bands were identified by autoradiography. Two major bands at Mr = 53,000 and 56,000 were shown to contain phosphotyrosine. Two similar alkali-stable bands containing phosphotyrosine but with lower amounts of 32P labeling were also observed in the particulate fractions of various other tissues (lung, brain, kidney, and testes). The particulate form of tyrosine protein kinase of rat spleen could be solubilized by using high concentrations of Nonidet P-40 (5%) at an alkaline pH (pH 9.0). Partial purification and subsequent filtration on Sephacryl S-200 yielded a peak of tyrosine protein kinase activity with an apparent molecular weight of 55,000. The two major phosphorylated bands of Mr = 53,000 and 56,000 co-migrated with the peak of enzyme activity. The solubilized and partially purified enzyme preparation phosphorylated only tyrosine residues when either endogenous proteins or casein were used as substrates. These results suggest that relatively high activities of tyrosine protein kinase exist in a normal tissue (rat spleen). Major endogenous substrates of the enzyme(s) appear to be represented by two proteins of Mr = 53,000 and 56,000; one or both of these substrates may be the tyrosine protein kinase itself.     

Tyrosine protein kinase assays

Protein kinases form a large family of enzymes that play a major role in a number of live processes. The study of their action is important for the understanding of the transformation mechanisms and of the normal and pathological growth events. The quality of an enzyme assay is often the key point of an enzymatic study. It must be flexible and compatible with various experimental conditions, such as those for the purification process, the screening of inhibitors and the substrate specificity studies. As will be shown in the present review, two categories of substrates, peptidic and proteic, should be distinguished. The use of peptide substrates facilitates the determination of the recognition requirements of the enzyme and of the kinetic effects of even minute variations in their sequence. These linear peptide structures are assumed to mimic a complex interaction between the enzyme and a proteic substrate in which distant amino acids in the sequence are vicinal in the folded substrate. Less amenable to a systematic study, but probably more adequate to investigate the natural substrate of a given kinase, are the proteic substrates. Obviously the tools to measure protein kinase activities are not the same in these two cases. The main difficulty in assaying protein kinases is the use of labelles γ-ATP, mostly at large excess concentration, since the final product of the reaction has to be separated from the non-reacted labelled ATP. In the case of peptide substrates, the difficulty is to separate them from ATP basing on differences of molecular mass. Despite the efforts of many investigators to rely upon differences in solubility, in charges or in “affinity”, this separation, which is crucial for the assay, is still an unsolved experimental problem. Chromatographic, as well as electrophoretic assays appeared relatively late in this domain, and more work in assessing new methodologies might bring new breakthroughs in the next few years. Specific, simple and reliable kinase assays are still a major challenge. Their improvement will help to conduct specificity studies, to elucidate complex growth mechanisms in which they are involved and to discover more selective potent inihibitors.     

Protease-activated protein kinase C in rat liver

In regenerating rat liver, an elevated protein kinase activity was detected which phosphorylated ribosomal protein S6 and histones. The properties of this enzyme were closely similar with those of protease-activated protein kinase C with Mr 45,000. During the study of the mechanism of proteolytic activation, type III protein kinase C (encoding alpha-sequence) was shown to be subjected to limited proteolysis by trypsin-like protease and converted to protein kinase M in ionic strength- and pH-dependent manner. This reaction was stimulated in the presence of Ca2+ and phospholipid under slightly higher ionic strength condition than physiological level (greater than 140 mM NaCl) and alkaline pH (7.5-8.0). These results suggest that activation of Na+/H+ exchanger in plasma membrane may trigger this type of proteolytic activation of protein kinase C. In addition to protein kinase M, another type of protease-activated kinase with Mr 80,000 was detected when limited proteolysis of protein kinase C was performed on inactive form of this enzyme (in the absence of either Ca2+ or phospholipid or both activators) under lower ionic strength condition. The molecular mass of this active enzyme was slightly smaller (approximately 200) than that of native protein kinase C. However, it is not clear at this time whether this small fragment was released from amino-terminal or carboxy-terminal domain to make protein kinase C partially active in the absence of Ca2+ and phospholipid. Although it has been proposed that proteolytic degradation of protein kinase C is involved in down regulation of this enzyme, the physiological significance of these two types of protease-activated forms of protein kinases in liver has remained obscure.     

Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3′ region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.     

The distribution of acid and alkaline ribonuclease activities in preneoplastic and neoplastic rat livers.

Ribonuclease (RNase) activities revealed by the substrate film method were compared with reactions for acid and alkaline RNase obtained by lead precipitation technique in serial sections of preneoplastic livers and hepatomas. The preneoplastic parenchymal tissue giving positive reactions with ribonucleic acid films showed both acid and alkaline RNase activities by lead precipitation technique, and the area of hyperplastic nodules nonreactive against substrate films were deficient in acid and alkaline RNase activities. Preneoplastic hyperbasophilic foci and hepatoma gave weak or negative reactions by either method, but necrotic areas and stromal tissue showed appreciable RNase activities. Thus a good correlation was observed in these tissues between the RNase activities revealed by the film method and those demonstrated by lead precipitation.     

Effect of DNA synthesis on induction of preneoplastic and neoplastic lesions in rat liver by a single dose of methylazoxymethanol acetate

A single intravenous injection of methylazoxymethanol (MAM) acetate in doses of either 20 or 35 mg/kg body weight to male Sprague-Dawley rats induced altered liver cell foci and later, liver neoplasms in a dose related manner. Sequential observations in the rats given 35 mg/kg and thereafter fed an iron-loading diet revealed that the number of iron-excluding foci/cm2 increased with time. Partial hepatectomy (PH) before the high dose of MAM acetate resulted in 100% lethality while hepatectomy before the low dose carcinogen exposure lead to a higher incidence of neoplasms than in rats that received carcinogen alone. PH after either high or low dose carcinogen exposure did not result in a greater occurrence of liver neoplasms.     

Tyrosine protein kinase activity in the DMBA-induced rat mammary tumor: inhibition by quercetin

Tyrosine protein kinase activity was measured in membranes from DMBA-induced mammary tumors, with Angiotensin II as substrate. The apparent Km for the peptide was 3.3 mM. This enzymatic activity is inhibited by Ca+2; Mn+2 can replace Mg+2 with an increase in the Km for ATP from 47 /microM to 172 microM. The enzymatic activity was not affected by cyclic AMP but was inhibited in dose dependent manner by quercetin, a bioflavonoid which is known to inhibit proliferation of malignant cells in vitro.     

Altered transferrin gene expression in preneoplastic and neoplastic liver lesions induced in rats with N-nitrosomorpholine.

The expression of the gene for the iron transport protein transferrin was found to be altered in preneoplastic and neoplastic lesions induced in the rat liver by N-nitrosomorpholine. The total RNA of ten hepatocellular carcinomas (HCC) was investigated by Northern blot analysis using a cDNA-probe comprising 150 bp of the 3' region and compared with the total hepatic RNA in untreated rats. Seven hepatocellular carcinomas showed slight or pronounced reduction in transferrin expression. In situ hybridization of two additional hepatocellular carcinomas revealed marked reduction in the mRNA level for the transferrin gene compared with the surrounding tissue. In contrast, the majority of early preneoplastic lesions storing excess glycogen and tigroid cell foci expressed increased levels of transferrin mRNA. The loss of glycogen in mixed cell foci, which represent a later stage of hepatocarcinogenesis, was usually accompanied by a decrease in transferrin mRNA suggesting a close relationship between this change in gene expression and cellular dedifferentiation emerging during hepatocarcinogenesis.     

Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver

A new method for the histochemical demonstration of pyruvate kinase (PK) activity was developed using a semi-permeable membrane and ATP-dependent phosphorylation of glucose coupled with tetrazolium reduction via glucose-6-phosphate dehydrogenase (G6PD) in order to investigate normal liver tissue and neoplastic hepatic nodules induced by thioacetamide (TAA). A series of control reactions and comparison with microbiochemical analysis of microdissected lyophilised material were used to determine the specificity of the reaction. In agreement with earlier reports, an activity gradient in control liver decreasing from zone 3 to zone 1 was apparent both histochemically and after biochemical analysis. Liver neoplastic nodules induced by 25 weeks dietary thioacetamide administration and characterized by increased G6PD demonstrated a clear decrease in PK activity. In contrast, epithelial cells within areas of cholangiocellular tumour development were characterized by a strong increase. Comparison of the results with immunohistochemical and biochemical data from the literature indicate that the specific histochemical method described will be of great assistance in future assessment of disease and physiological alteration in activity of this key enzyme of glycolysis.     

Histochemical and microbiochemical demonstration of reduced pyruvate kinase activity in thioacetamide-induced neoplastic nodules of rat liver

Summary A new method for the histochemical demonstration of pyruvate kinase (PK) activity was developed using a semi-permeable membrane and ATP-dependent phosphorylation of glucose coupled with tetrazolium reduction via glucose-6-phosphate dehydrogenase (G6PD) in order to investigate normal liver tissue nd neoplastic hepatic nodules induced by thioacetamide (TAA). A series of control reactions and comparison with microbiochemical analysis of microdissected lyophilised material were used to determine the specificity of the reaction. In agreement with earlier reports, an activity gradient in control liver decreasing from zone 3 to zone 1 was apparent both histochemically and after biochemical analysis. Liver neoplastic nodules induced by 25 weeks dietary thioacetamide administration and characterized by increased G6PD demonstrated a clear decrease in PK activity. In contrast, epithelial cells within areas of cholangio-fibrosis thought to be direct precursors for cholangioccllular tumour development were characterized by a strong increase. Comparison of the results with immunohistochemical and biochemical data from the literature indicate that the specific histochemical method described will be of great assistance in future assessment of disease and physiological alteration in activity of this key enzyme of glycolysis.Supported by the Deutsche Forschungsgemeinschaft. Dr. Malcolm A. Moore is the recipient of a guest research scientist fellowship from the German Cancer Research Center     

Isozymic forms of protein kinase C in regenerating rat liver

The expression of multiple forms of protein kinase C (PK-C) was studied in regenerating rat liver using hydroxyapatite column chromatography. Two forms of the enzyme were found in the cytosolic as well as membrane fraction of livers from partially hepatectomized rats. The kinetic variation in the activation of these two liver isozymes by fatty acids, phosphatidylserine and diacylglycerol was similar to that reported for the PK-C subspecies from rat brain, designated types II and III. Intracellular redistribution of PK-C caused by phorbol 12-myristate 13-acetate (PMA) was concentration-dependent and was due to translocation of isozyme III, because type II was insensitive to 5 x 10(-8) M PMA. The activity ratio of the two isozymes in either the particulate or cytosolic fraction was the same at 22 h as compared to 4 h after partial hepatectomy.     

Cyclic nucleotide-independent protein kinase from rat liver. Purification and characterization of a multifunctional protein kinase

A rat liver cAMP-independent protein kinase that phosphorylates peptide b of ATP-citrate lyase (Ramakrishna, S., Pucci, D. L., and Benjamin, W. B. (1983) J. Biol. Chem. 258, 4950-4956) has been purified to apparent homogeneity. The molecular weight, determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, sucrose density gradient, and by gel filtration, was found to be 36,000. This protein kinase phosphorylates in vitro ATP-citrate lyase, acetyl-CoA carboxylase, and glycogen synthase and does not phosphorylate phosphorylase, phosphorylase kinase, histone, phosvitin, and casein. It has Fa (activity factor) activity stimulating the ATP X Mg-dependent phosphatase and is therefore named a multifunctional protein kinase. This kinase differs from glycogen synthase kinase-3 with regard to substrate specificity, kinetic parameters, and physicochemical properties.