A histochemical study of adenosine triphosphatase and other nucleotide phosphatases in young root tips

Summary The distribution of ATP-ase and other nucleotide phosphatases has been studied in young root tips of maize, barley and broad bean using frozen and paraffin sections stained by standard lead sulphide precipitation procedures. High ATP-ase activity was found at the root and cell surface which is in agreement with previous biochemical studies using excised roots and cell wall preparations. Staining was also found in the nuclei and at particulate sites in the cytoplasm. Differences were observed between the present work and the staining pattern obtained for -glycerophosphatase, and between ATP-ase staining in the three roots studied. These results are discussed in relation to the possible physiological activity of the enzymes and to the differences found between earlier histochemical studies of ATP-ase activity.Abbreviations F.L. frozen longitudinal - F.T. frozen transverse     

Changes of enzyme activity in the rat liver at different age in circadian cycle

Most of the biological processes in the living organisms of both animals and man are known to be of rhythmical nature. Variability of enzymatic activity in circadian cycle depends on many factors among other on age, sexual maturity, diet as well as medication. The results obtained in our studies indicate, that the activity changes of acid phosphatase and ATP-ase Mg++ dependent in the liver of all the examined age groups were of 24 hour circadian rythm. As to the acid phosphatase activity the results of this experiments showed that in circadian cycle in all examined age groups there is only one peak of elevated activity. ATP-ase Mg++ dependent showed two activity peaks appearing at the same hour both in 30 and 60 days old animals. It should be noticed that the activities of ATP-ase Mg++ dependent in 100 day old animals were two times higher than in 30 and 60 days old rats.     

ATPase activity of the smooth and rough microsomal subfractions of the heart and liver in rabbits with a short-term microcirculatory disorder]

Short-time disturbances of the microcirculation in rabbits had a different influence on the ATP-ase activity of smooth and rough microsomes of the liver and heart. Mg+2ATP-ase activity in the liver decreased in both microsome fractions; as in the heart, the enzyme activity increased only in the smooth (light) microsomes.     

Activity of Ca(+2)Mg(+2) --ATPase in erythrocyte membranes of women with diabetes mellitus type I]

The activity of Ca(++)-Mg++ ATP-ase present in erythrocyte membranes was determined in basal conditions and following stimulation with calmodulin in 8 women with insulin-dependent diabetes and in 9 healthy women. The isolation of erythrocyte membranes and the determination of activity of Ca(++)-Mg(++)-ATP-ase were carried out according to the method of Gietzen et al. A decrease in the activity of Ca(++)-Mg(++)-ATP-ase in basal conditions was found in fractions with the highest erythrocyte content obtained from diabetic patients. After stimulation with calmodulin the activity of Ca(++)-Mg(++)-ATP-ase in all the fractions was lower in diabetic patients than in the controls. Low activities of the enzyme were accompanied by high values of HbA1c. The results suggest that glycosylation of the ATP-ase or/and calmodulin may be the main cause of the observed fall in the enzyme activity in diabetes. Also the disturbances concerning the cumulation of intracellular calcium may be related to the changes caused by glycosylation of Ca(++)-Mg(++)-ATP-ase or/and calmodulin.     

Protein Tyrosine Kinase Activity and Its Endogenous Substrates in Rat Brain: A Subcellular and Regional Survey

The rat CNS contains high levels of tyrosine-specific protein kinases that specifically phosphorylate the tyrosine-containing synthetic peptide poly(Glu80,Tyr20). The phosphorylation of this peptide is rapid and occurs with normal Michaelis-Menten kinetics. Using this peptide to assay for enzyme activity, we have measured the protein tyrosine kinase activity in homogenates from various regions of rat CNS. A marked regional distribution pattern was observed, with high activity present in cerebellum, hippocampus, olfactory bulb, and pyriform cortex, and low activity in the pons/medulla and spinal cord. The distribution of protein tyrosine kinase activity was examined in various subcellular fractions of rat forebrain. The majority of the activity was associated with the particulate fractions, with enrichment in the crude microsomal (P3) and crude synaptic vesicle (LP2) fractions. Moreover, the subcellular distribution of pp60csrc, a well-characterized protein tyrosine kinase, was examined by immunoblot analysis using an affinity-purified antibody specific for pp60csrc. The subcellular distribution of pp60csrc paralleled the overall protein tyrosine kinase activity. In addition, using an antibody specific for phosphotyrosine, endogenous substrates for protein tyrosine kinases were demonstrated on immunoblots of homogenates from the various regions and the subcellular fractions. The immunoblots revealed numerous phosphotyrosine-containing proteins that were present in many of the CNS regions examined and were associated with specific subcellular fractions. The differences in tyrosine-specific protein kinase activity, and in phosphotyrosine-containing proteins, observed in various regional areas and subcellular fractions may reflect specific functional roles for protein tyrosine kinase activity in mammalian brain.     

Thyrotropic activity of salmon pituitary glycoprotein hormones in the Hawaiian parrotfish thyroid in vitro

The thyrotropic activities of salmon pituitary extract, thyroid-stimulating hormone (TSH), gonadotropins (GTH), and glycoprotein fractions obtained during purification of salmon TSH and GTH were measured using the parrotfish thyroid culture system. Purified salmon TSH was approximately 1,000 times more potent than bovine TSH in stimulating thyroxine release into the culture medium. Most of the forms of salmon GTH had no thyrotropic activity. One of the forms of salmon GTH (GTH-F) and three chromatofocusing fractions (CF-B, -C, and -E) that were devoid of activity in the coho salmon in vivo had some thyrotropic activity in the parrotfish thyroid culture. Whether the activity of these fractions was due to contamination with TSH, less potent forms of TSH, or inherent thyrotropic activity of a form of GTH is discussed. These results indicate that the parrotfish thyroid culture system can be used to detect thyrotropic activity of fractions obtained during the purification of teleost TSH.     

Structuro-functional features of different links in the microhemocirculatory bed of the myocardium

Morphofunctional peculiarities of various elements of the microhemocirculatory bed (MHCB) of the myocardium performing correction of the nutritive blood stream, structures and mechanisms of the microvessel permeability have been studied. The investigation has been performed on 20 mature rabbits and 5 mongrel dogs, transmissive electron microscopy and certain cytochemical techniques applied for revealing ATP-ase, lactate-, malate-, succinate-dehydrogenases, cytochrome-C-reductase and glycosamine glycans. Structural differences in microvessels of various range are described. Together with smooth muscle cells in arterioles and precapillary sphincters, specialized endotheliocytes and valve-like formations, situating in the orifices of blood capillaries, participate in regulation of hemoperfusion of the MHCB functional units. The data have been obtained characterising cytolemma of the endotheliocytes as a complex system, where plasmolemma, being the bearer of ATP-ase and a complex of oxidoreductase, regulates physical-chemical properties of the paraplasmolemmal layer and ensures the control of all the forms of the transcapillary transport. Micropinocytic vesicles are proved to differentiate into two functionally asynonymous populations: one of them performs the energy-dependent transport, while the other reserves the plasmolemma.     

Further Properties of Adenosine Triphosphatase and {beta}-Glycerophosphatase from Barley Roots

Cell wall preparations from barley roots contain ATP-ase activitythat is stimulated by monovalent cations at alkaline pH values,above that obtained with calcium or magnesium ions. Sodium isthe most effective cation followed by potassium, lithium, andrubidium. Similar activation is obtained with a soluble enzymefraction and with excised root tips. ß-Glycerophosphataseshows no stimulation by calcium and sodium or potassium haveonly a small stimulatory effect. Disc electrophoresis demonstratesthe group character of ATP-ase and ß-glycerophosphataseactivities which consist of multiple forms either specific toone or other substrate or hydrolysing both.     

Fractionation of Jerusalem artichoke phenolase by immobilized copper affinity chromatography

Phenolase from tubers of Jerusalem artichoke was fractionated by metal chelate affinity chromatography using copper conjugated to iminodiacetic acid Sepharose 6B. Four fractions obtained after chromatography showed various specific activities, with an increase in activity of 160-fold for the first unbound enzymatic fraction, and 18-fold for a fraction eluted with glycine buffer. Electrophoresis of phenolase fractions in gradient polyacrylamide gel resulted in a pattern consisting of three major groups of bands differing in relative mobilities.     

Effect of vasopressin on the ATP-ase activity of microsomal fractions of rabbit heart and liver]

It was shown that intravenous injection of vasopressin in a dose of 5 pressor units per 1 kg of body weight led to changes in the ATP-ase activity of the heart and liver microsomes in one hour. These changes coursed in a different direction, i.e. ATP-ase activity of the heart microsomes increased, and ATP-ase activity of the liver microsomes decreased.     

Cytochemical identification of the leukocytes of torpedoes (Torpedo marmorata Risso and Torpedo ocellata Rafinisque)

The authors subjected peripheral blood smears of Torpedoes to cytochemical analysis of lipids, protein, neutral and acid polysaccahrides and of some enzymatic activities, i.e. adenosine triphosphatase (ATP-ase), acid and alkaline phosphatase, aliesterase and peroxidase. It was found that neutrophilic granulocytes are intensely PAS and aliesterase positive and weakly ATP-ase positive. Eosinophilic granulocytes show the presence of neutral polysaccharides in the matrix (which is PAS positive) and strong ATP-ase and acid phosphatase activities in the granules. Lymphocytes sometimes contain weakly PAS and aliesterase positive granules. Monocytes show some small PAS positive granules and weak acid phosphatase and aliesterase activities. Thrombocytes contain some peripheral granules which are PAS positive and slightly ATP-ase positive. There are no transitional forms between the various cellular types. The results confirm the classification of leukocytes of Torpedoes into neutrophilic granulocytes, eosinophilic granulocytes, lymphocytes, monocytes and thrombocytes and contribute some informations about the histoenzymatic content of Elasmobranch leukocytes.     

Effect of parathyroid hormone and thyrocalcitonin on the cell membrane Na, K-ATP-ase of rat brain and kidney]

The influence of parathyroid hormone (PH) and thyrocalcitonine (TCT) on the enzymatic activity of ATP-ase systems of the membrane specimens of the cerebral cortex and renal cortex was investigated in experiments on rats. It was found that parathyroid hormone increased the activity of Na, K-ATP-ase and Ca-activated ATP-ase transport of the membranes in the brain and the kidneys both in vivo and in vitro. TCT caused analogous, but less expressed changes of the ATP-ase activity. Both hormones showed no influence on the Mg-ATP-ase activity of the both organs. It is supposed that the PH hormone influenced the membrane structures with the ATP-ase activity directly, while the action of TCT on them was mediated.     

Intracellular distribution of molecular forms of acetylcholinesterase in rat brain and changes after diisopropylfluorophosphate treatment

The subcellular distribution of acetylcholinesterase activities was studied in the striatum and cerebellum of rat brain. The highest percentage of the enzyme activity was found in the crude synaptosomal (P₂) fraction, with striatum much higher than cerebellum. On sucrose density gradient centrifugation analyses all the particulate fractions (P₁, P₂, and P₃) showed a major peak of the 10 S form of acetylcholinesterase activity with very little activity of the 4 S form of the enzyme. The 10 S/4 S ratio was much higher in striatum than in cerebellum. In the soluble fraction (100,000g supernatant) the 10 S form was less than the 4 S form in the adult rat brain, but this was reversed in the 6-day-old rat brain. After diisopropylfluorophosphate administration the recovery of acetylcholinesterase molecular forms in various subcellular fractions differed at different recovery periods. These results indicate that the distribution of molecular forms of acetylcholinesterase in rat brain differs in various subcellular fractions, and also the pattern of distribution differs in different regions of the brain as well as in adult and developing brains.     

Biochemical properties of Hsp70 chaperone system from Meiothermus ruber

The guanidine-hydrochloride (Gdn-HCl) and thermally induced unfolding of Hsp70 from Meiothermus ruber (Mru.Hsp70) were analysed using tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. The ANS binding to Mru.Hsp70 showed both the increase in fluorescence intensity and a shift in emission maximum. Analysis of the unfolding profile of Mru.Hsp70 indicated that Gdn-HCl induced unfolding of Mru.Hsp70 occurred through intermediate species. The tryptophan and ANS fluorescence emission spectra revealed that ATP induced conformational change increased the thermal stability of Mru.Hsp70. The data obtained are similar to those of Escherichia coli DnaK. The ATP-ase activity of chaperones is fundamental for their biological activity. It this paper we demonstrate that, in contrast to Thermus thermophilus, both Mru.Hsp40 and Mru.Hsp22 co-chaperones affect the ATP-ase activity of Mru.Hsp70. The use of truncated Mru.Hsp40 proteins showed that full-length Mru.Hsp40 is required for stimulation of ATP-ase activity of Mru.Hsp70. E. coli GrpE could act as nucleotide exchange factor the in thermophilic Hsp70 ATP hydrolysis reaction. However, the role of E. coli DnaJ in the M. ruber ATP cycle needs further analysis. We selected the new substrate laccA suitable for determination of refolding activity of thermophilic chaperones.     

Na+ + K+] ATP-ase in liver and brain of obese mice

The activity of hepatic [Na+ + K+]ATP-ase showed a gene-dosage relationship in 6 week old mice. Before weaning hepatic [Na+ + K+]ATP-ase activity was normal in preobese mice but fell within 7 days of weaning to the low levels observed in older ob/ob mice. Brain [Na+ + K+]ATP-ase activity was unchanged in ob/ob mice although [3H]-ouabain binding was reduced. Arrhenius plots of [Na+ + K+]ATP-ase activity in liver and brain and of [3H]-ouabain binding to brain preparations showed breakpoints at lower temperatures in ob/ob than lean mice. These breakpoints were altered by pretreatment of tissue with deoxycholate. It is suggested that changes in membrane lipid composition might be an important factor regulating [Na+ + K+]ATP-ase in ob/ob mice.     

Characterization and purification of polymorphic arylalkylamine N-acetyltransferase from the American cockroach, Periplaneta americana.

We separated two forms of arylalkylamine N-acetyltransferase (AANAT) from various organs of the American cockroach, Periplaneta americana. Both forms of the enzyme had an equivalent molecular mass of 28 kDa. One form isolated from the testicular accessory glands had high enzyme activity at acidic pHs. The isoelectric point was 5-6 and the substrate specificity was wider than the other type. The other isolated form from female midguts had a higher level of enzyme activity at basic pHs. These findings suggested that P. americana contains polymorphic AANAT, as is the case in Drosophila melanogaster. These forms differed not only in pH specificity, and substrate specificity but in chromatographic behavior and kinetic properties. Most of the organs we examined contained a mixture of the two forms since two types of AANAT activity were separated in different chromatographic fractions when two pH conditions were used for activity measurement.     

Effects of Aldehyde Fixation on Adenosine Triphosphatase and Peroxidase Activities in Maize Root Tips

The effects of aldehyde fixation of excised roots and subcellularfractions of Zea mays have been studied in relation to the preservationof ATP-ase and peroxidase activities. Total peroxidase was littleaffected by either formaldehyde or glutaraldehyde whereas ATP-aseshowed considerable loss of activity, particularly with glutaraldehyde.The activity remaining after fixation was dependent on boththe concentration of fixative and the pH of fixation. Subcellularfractions differed in their response to fixation with the cellwall fraction generally showing higher retention of activitythan the mitochondrial or microsomal fractions.     

Developmental changes in the protein kinase activity of chick brain

The protein kinase activity in the postmitochondrial and postribosomal fractions from chick brain at various stages of development was examined. It has been found, that the overall level of protein kinases activity, assayable under the experimental conditions, increases during embryogenesis, sharply decreases at the hatch, and again increases thereafter. The subcellular distribution of protein kinases alters during ontogenetic development. The embryonal protein kinases of both subcellular fractions differ in the protein substrate specificity, cAMP- and salts-sensitivity from those of the adult ones. Thanks to use of ribosomal proteins as an exogenous substrates it was possible to visualize the developmental changes in the protein kinase pattern.     

THE SUBCELLULAR LOCALIZATION OF ACETYLCHOLINESTERASE AND ITS MOLECULAR FORMS IN PIG CEREBRAL CORTEX

Abstract— The distribution of acetylcholinesterase among the subcellular fractions of pig cerebral cortex was determined. The crude mitochondrial and microsomal fractions obtained by differential centrifugation accounted for 75% of the enzyme, with the remainder divided between the crude nuclear and soluble fractions.
The occurrence and distribution of the multiple molecular forms of AChE was the same in all four fractions with the dominant species of molecular weights 350,000, 270,000 and 60,000. Further purification of the mitochondrial fraction by density gradient centrifugation gave a series of membrane fractions with very similar multiple forms. The one possible exception was the fraction containing the purified synaptosomal membranes where one band of mol wt 270,000 predominated, although the other molecular weight entities were present. The electrophoretic pattern of AChE present in the fractionated microsomes was the same as in the crude preparation. The content and pattern of the multiple molecular forms of AChE was therefore the same in all fractions of pig brain, apart from that containing the purified synaptosomal membranes.     

Biochemical properties of Hsp70 chaperone system from Meiothermus ruber

The guanidine-hydrochloride (Gdn-HCl) and thermally induced unfolding of Hsp70 from Meiothermus ruber (Mru.Hsp70) were analysed using tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. The ANS binding to Mru.Hsp70 showed both the increase in fluorescence intensity and a shift in emission maximum. Analysis of the unfolding profile of Mru.Hsp70 indicated that Gdn-HCl induced unfolding of Mru.Hsp70 occurred through intermediate species. The tryptophan and ANS fluorescence emission spectra revealed that ATP induced conformational change increased the thermal stability of Mru.Hsp70. The data obtained are similar to those of Escherichia coli DnaK. The ATP-ase activity of chaperones is fundamental for their biological activity. It this paper we demonstrate that, in contrast to Thermus thermophilus, both Mru.Hsp40 and Mru.Hsp22 co-chaperones affect the ATP-ase activity of Mru.Hsp70. The use of truncated Mru.Hsp40 proteins showed that full-length Mru.Hsp40 is required for stimulation of ATP-ase activity of Mru.Hsp70. E. coli GrpE could act as nucleotide exchange factor the in thermophilic Hsp70 ATP hydrolysis reaction. However, the role of E. coli DnaJ in the M. ruber ATP cycle needs further analysis. We selected the new substrate laccA suitable for determination of refolding activity of thermophilic chaperones.