3H-THYMIDINE INCORPORATION INTO CELL NUCLEI IN GERMINATING LETTUCE SEEDS

Lettuce seeds were soaked in a ³H-thymidine solution and autoradiogramof sections of these seeds, fixed prior to and after germination(root protrusion), were prepared. Labeled nuclei in intact,punctured, as well as broken seeds began to appear approximatelyat the time of root protrusion. In the nuclei of the radicle,the beginning of ³H-thymidine incorporation precedes the divisionof nuclei by one to two hours, and all the dividing cells incorporate³H-thymidine before their first division. Occasional dividingcells in the epidermis of the hypocotyl were not found to be³H-labeled. In intact seeds, the nuclei observed in the coneof the plumule showed radioactivity approximately 3–4hr after germination, while in punctured and broken seeds the³H-incorporation occurred some hours earlier. (Received December 3, 1962; )     

IMIPRAMINE-INDUCED CHANGES OF BRAIN ADENOSINE TRIPHOSPHATASE ACTIVITY

Abstract— Adenosine triphosphatase (ATPase) activities of brain microsomal and synaptosomal preparations are inhibited by imipramine [5-(3'-dimethylamino propyl)-10, 11-dihy-dro-5H-dibenz (b,f) azepine] in vitro , whereas microsomal ATPase activity is stimulated and synaptosomal ATPase activity remains unaltered under in vivo imipramine treatment. The inhibition of ATPase activity can to some extent be counteracted by spermine [N, N'-bis(3 aminopropyl)-1,4-butanediamine]. Determination of K_m values from double reciprocal plots (activity^-1 vs. ATP mM^-1) under drug and spermine-treated conditions appear to indicate that spermine can to some extent imparta stabilizing effect mainly on the microsomal membrane ATPase, preventing inhibition in presence of imipramine in vitro , although spermine has no effect on the already destabilized membrane ATPase. Spermine exerts a stabilizing effect on membrane ATPase possibly by increasing the affinity of the enzyme for the substrate.     

PROPERTIES AND CHANGES OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE IN GERMINATING LETTUCE SEEDS

Glucose-6-phosphate dehydrogenase is present in lettuce seedsand seedlings in a particulate, 20,000 g fraction and in a solublefraction of the cells. In both fractions the enzyme activityoccurs in the presence of either NAD or NADP. It seems probablethat an enzyme with two active sites is involved. Total activity does not change during 48 hours germination inwater. It is depressed by coumarin or thiourea treatment. Specificactivity rises in the soluble fraction when germination occurs,but not in the mitochondrial one. Coumarin treatment largelydepresses the increase in specific activity. In vitro 100 ppmcoumarin totally inhibit enzyme activity. 1,250 ppm thioureado not affect enzyme activity. The results are analysed and it is concluded that glucose-6-phosphatedehydrogenase is important, if at all, only in the very earlystages of germination. (Received August 13, 1965; )     

ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE MEMBRANES OF THE SQUID NERVE FIBER

This investigation deals with the localization of sites of ATPase activity, especially of transport ATPase, in nerve fibers of the squid Doryteuthis plei, at the subcellular level. Splitting of ATP liberates inorganic phosphate which reacts with lead to form a precipitate in the tissue. The reaction was made on nerve fibers fixed with glutaraldehyde. Frozen slices were incubated in Wachstein-Meisel medium containing ATP and Pb(NO₃)₂. Deposits of reaction product were found in the axolemma (towards its axoplasmic side), Schwann cell membranes (mainly at the channels crossing the layer), and mitochondria. Control experiments revealed that no deposits were observed in nerve fibers fixed in osmium tetroxide prior to incubation in the medium containing ATP, or in nerve fibers incubated without substrate or with adenosine monophosphate, adenosine diphosphate, glycerophosphate, or guanosine triphosphate as substrate. For evaluation of transport ATPase activity, these findings were compared with results obtained with nerve fibers treated with G-strophanthin or K-strophanthoside before or after glutaraldehyde fixation. The cardiac glycosides produced a disappearance or diminution of the deposits. The largest inhibitory effect was observed in the axolemma. The findings indicate that the highest ATPase activity is localized in the axolemma and may be due primarily to transport ATPase.     

SODIUM-STIMULATED ADENOSINE TRIPHOSPHATASE ACTIVITY OF RAT BRAIN MITOCHONDRIA

Abstract The endogenous ATPase activity of rat brain mitochondria was stimulated 30-50 per cent by 15-50 m m concentrations of NaCl or Na acetate. The Na stimulation was completely abolished by small amounts of oligomycin but unaffected by ouabain. The differential effects of these inhibitors indicated that the Na-induced ATPase activity did not result from microsomal or synaptosomal contamination of mitochrondria. Sodium salts decreased the stimulatory effects of DNP, gramicidin, or Ca, but not that of Mg on the endogenous ATPase activity. These interactions were specific for Na⁺ as the corresponding salts of K⁺ did not affect the endogenous ATPase or inhibit the DNP-stimulated ATPase activity except at high concentrations. The Na-induced increases in ATPase activity and respiration were more sensitive to aging of the mitochondria than were ADP/O and respiratory control ratios, or the DNP-induced ATPase activity. These results suggest that Na⁺ may interact in brain mitochondria with the same high-energy intermediate of oxidative phosphorylation proposed for DNP and Ca.     

ADENOSINE TRIPHOSPHATASE LOCALIZATION IN AMPHIBIAN EPIDERMIS

The localization of ATPase¹ activity has been studied by light and electron microscopy in the epidermis of Rana pipiens, Rana catesbiana, and Bufo marinus. The reaction was carried out on skin (glutaraldehyde-fixed or fresh) sectioned with or without freezing. Best results were obtained with nonfrozen sections of fixed tissue. The incubation mixture was either a Wachstein-Meisel medium, or a modification which approximates assay systems used in biochemical studies of transport ATPases. The reaction product was found localized in contact with the outer leaflet of all cell membranes facing the labyrinth of intercellular spaces of the epidermis. It was absent from: (a) membrane areas involved in cell junctions (desmosomes, zonulae and maculae occludentes); (b) cell membranes facing the external medium (i.e., those on the distal aspect of the ultimate cell layer in s. corneum); (c) cell membranes facing the dermis (those on the proximal aspect of cells in s. germinativum). In the presence of (Na⁺ + K⁺) the localization did not change, but the reaction was not appreciably activated. A similar though less intense reaction was obtained with ITP, but not with ADP, AMP, and GP as substrates. The results are discussed in relation to available data on transport ATPases in general, and on the morphology and physiology of amphibian skin in particular. Assuming that the ATPase studied is related to transport ATPase, the findings suggest a series of modifications to the frog skin model proposed by Koefoed-Johnsen and Ussing. The salient feature of this modified model is the localization of the Na⁺ pump along all cell membranes facing the intercellular spaces of the epidermis.     

A STUDY OF THE ADENOSINE TRIPHOSPHATASE ACTIVITY OF MYOSIN AND ACTOMYOSIN

1. An experimental study was made on the adenosine triphosphatase action of crystalline myosin and actomyosin preparations under different conditions. 2. No enzymatic activity was found in the absence of salts. Activation was given by KCl and CaCl₂, whereas MgCl₂ in the presence of other ions inhibited. 3. The effect of pH is complex. In stabilizing buffers or at low temperature, there are two optima (pH 6.2 to 6.5 and pH 9.2) provided Ca is present. Without Ca only the acid optimum is found. The highest activities are reached in glycine buffer at pH 9.2 in the presence of Ca. 4. The study of the Mg-Ca antagonism revealed that the inhibition due to Mg is fully developed with Mg:Ca ratios less than 1, the inhibition usually exceeding 90 per cent. 5. It is shown that in the muscle the myosin-ATPase is most probably also subjected to the inhibitory action of the Mg ions. 6. From data in the literature it is calculated that the liberation of inorganic phosphate during muscular activity takes place at a rate of at least 0.200 mg. P per mg. myosin per minute. 7. From the results of the present study it is found that the myosin in the muscle can liberate inorganic phosphate from ATP at a rate of at most 0.003 mg. P per mg. myosin per minute. 8. It is concluded therefore that myosin-ATPase cannot be responsible for the liberation of the main part of the phosphate in contracting muscle, and therefore cannot have the rôle in muscular metabolism ascribed to it in recent hypotheses and discussions.     

LOCALIZATION OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE RAT SPERM TAIL AS REVEALED BY ELECTRON MICROSCOPY

The epididymides of rat testis were fixed in glutaraldehyde and cut as frozen sections. The sections were incubated in lead nitrate solution containing as a substrate either ATP, AMP, creatinine phosphate, beta glycerophosphate, or phenyl phosphate. Then they were postfixed in osmium tetroxide, embedded, sectioned, and examined with the electron microscope. In the sperm tail, when ATP is used as a substrate the reaction product (lead phosphate) is observed both in the tail filament complex and on the surface membrane of the mitochondrial helix of the middle piece. In the tail filament complex, this product is seen near the nine paired peripheral and two central filaments, and in the matrix between the outer coarse fibers. But the product is not observed within these filaments and fibers. In longitudinal sections, no periodicity of the deposits in the complex is observed. When the other phosphate compounds are used as substrates the reaction products appear on the surface membrane of the mitochondrial helix, and are not found in the tail filament complex. No distinctly different localization of the reaction products is observed when substrates other than ATP are used. Possible relationships between the structure and the function of the sperm tail are discussed in the light of these findings.     

LOCALIZATION OF TRANSPORT ADENOSINE TRIPHOSPHATASE IN RAT CORNEA

Increasingly, physiological and biochemical data on the mammalian cornea have emphasized the importance of the endothelial rather than the epithelial layer in regulating the state of hydration of the cornea, via a transport ATPase. This view is supported by the observations reported here. With the Ernst method, the first cytochemical procedure to be readily responsive to ions and ouabain, the major sites of enzyme reaction product are the intercellular spaces of the endothelial layer. This localization was not found by us with the so-called ATPase method of Wachstein and Meisel.     

NEGATIVE STAINING AND ADENOSINE TRIPHOSPHATASE ACTIVITY OF ANNULATE LAMELLAE OF NEWT OOCYTES

Semi-isolated annulate lamellae were prepared from single newt oocytes (Triturus alpestris) by a modified Callan-Tomlin technique. Such preparations were examined with the electron microscope, and the negative staining appearance of the annulate lamellae is described. The annulate lamellae can be detected either adhering to the nuclear envelope or being detached from it. Sometimes they are observed to be connected with slender tubular-like structures interpreted as parts of the endoplasmic reticulum. The results obtained from negative staining are combined with those from sections. Especially, the structural data on the annulate lamellae and the nuclear envelope of the very same cell were compared. Evidence is presented that in the oocytes studied the two kinds of porous cisternae, namely annulate lamellae and nuclear envelope, are markedly distinguished in that the annulate lamellae exhibit a much higher pore frequency (generally about twice that found for the corresponding nuclear envelope) and have also a relative pore area occupying as much as 32% to 55% of the cisternal surface (compared with 13% to 22% in the nuclear envelopes). The pore diameter and all other ultrastructural details of the pore complexes, however, are equivalent in both kinds of porous cisternae. Like the annuli of the nuclear pore complexes of various animal and plant cells, the annuli of the annulate lamellae pores reveal also an eightfold symmetry of their subunits in negatively stained as well as in sectioned material. Furthermore, the annulate lamellae are shown to be a site of activity of the Mg-Na-K-stimulated ATPase.     

A BIOCHEMICAL AND CYTOCHEMICAL STUDY OF ADENOSINE TRIPHOSPHATASE ACTIVITY IN THE PHLOEM OF NICOTIAN A TABACUM

A biochemical and cytochemical study has been made of the distribution of ATPase in mature and differentiating phloem cells of Nicotiana tabacum and of the substrate specificity and effects of fixation on enzyme activity. Homogenates of unfixed leaf midveins and midveins fixed in formaldehyde-glutaraldehyde were assayed for enzyme activity by determining the amount of P_i, liberated per milligram of protein from various substrates in a 30 min period at pH 7.2. In fresh homogenates, hydrolysis of ATP was not significantly different from that of ITP, CTP, and UTP. Hydrolysis of GTP was slightly higher than that of ATP. ATP hydrolysis by fresh homogenates was 17% more extensive than that of ADP, 76% more extensive than that of 5'-AMP, and was inhibited by fluoride and p-chloromercuribenzoate (PCMB). There was little or no hydrolysis of the competitive inhibitors 2'- and 3'-AMP nor with the alternate substrates p-nitrophenylphosphate (PNP) or β-glycerophosphate (β-GP). In homogenates of material fixed in formaldehyde-glutaraldehyde for 1¼ h, ATPase activity was 13% preserved. Hydrolysis of ATP by fixed homogenates was not significantly different from that of ADP, 5'-AMP, ITP, CTP, and GTP. Hydrolysis of UTP was lower. Fluoride and PCMB inhibited fixed ATPase activity. The results of cytochemical localization experiments using a lead phosphate precipitation technique were in agreement with the biochemical results. Similar localization patterns were obtained with the nucleoside triphosphates ATP, CTP, GTP, ITP, and UTP. Activity was also localized with ADP and 5'-AMP but not with the competitive inhibitors 2'- and 3'-AMP, nor with PNP or β-GP. Little or no reaction product was deposited in other controls incubated without substrate or with substrate plus fluoride, PCMB, or N-ethylmaleimide. ATPase activity was demonstrated chiefly at the plasma membrane of mature and differentiating phloem cells and was associated with the P-protein of mature sieve elements. It is suggested that the phloem transport system derives its energy from the demonstrated nucleoside triphosphatase activity.     

EFFECTS OF ETHANOL ON THE ACTIVITY OF ADENOSINE TRIPHOSPHATASE AND ACETYLCHOLINESTERASE IN SYNAPTOSOMES ISOLATED FROM GUINEA-PIG BRAIN

Nerve ending fractions from guinea-pig cerebral cortex contained more than one-half of the Na-K ATPase activity present in the original homogenate. Ethanol at concentrations ranging from 0·043 to 2·57 m inhibited the Na-K ATPase to a significantly greater extent than the Mg-activated ATPase or AChE. The inhibition of membrane-bound Na-K ATPase by ethanol was of the non-competetive type and the activity of Na-K ATPase was increasingly inhibited by alcohols of increasingly longer chain length. The ability of various alcohols to inhibit membrane-bound Na-K ATPase activity was correlated with their lipid solubility.     

METABOLIC PARAMETERS OF ONTOGENESIS OF ELECTRICAL ACTIVITY IN THE BRAIN. SODIUM-POTASSIUM ACTIVATED ADENOSINE TRIPHOSPHATASE IN DEVELOPING CHICK EMBRYO BRAIN

—(1) The activity of the Na-K ATPase in the particulate fraction of the chick embryo brain has been assayed at different stages of development with the objective of finding whether or not changes in the activity of this enzyme bear any relation to the maturation of spontaneous and evoked electrical activity of the growing chick brain. (2) The specific activity of the enzyme is low on day 6 and it rises rapidly between days 10 and 12, at which time it attains a plateau and remains essentially unchanged from day 12 until day 20. Experimental evidence rules out the possible presence of an inhibitor of the enzyme in 8-day-old brain homogenates, suggesting that these developmental changes in the activity of the enzyme may represent new synthesis of enzyme rather than its activation. The period between days 10 and 12 does not represent a unique stage of general protein synthesis. (3) The chick brain particulate enzyme has an optimum activity at pH 7·4 and at 37°. It is optimally activated by a Na⁺ concentration of 100mm and K⁺ concentration of 20 mm . The enzyme is inhibited by ouabain and Ca²⁺. (4) The results have been discussed.