A membrane-bound adenosine triphosphatase (EC 3.6.1.3) that requires Mg++ and that is stimulated by monovalent ions has been purified 7-to 8-fold from homogenates of oat (Avena sativa L. Cult. Goodfield) roots by discontinuous sucrose-gradient centrifugation. The enzyme was substrate specific; adenosine triphosphate was hydrolyzed 25 times more rapidly than other nucleoside triphosphates. The membrane fraction containing adenosine triphosphatase was enriched in plasma membranes, which were identified by the presence of a glucan synthetase (EC 2.4.1.12), a high sterol to phospholipid ratio, and by a stain consisting of periodic acid, chromic acid, and phosphotungstic acid that is specific for plant plasma membranes. Oat-root plasma membranes and the associated adenosine triphosphatase were purified on either a 6-layer discontinuous sucrose gradient or on a simplified gradient consisting of only two sucrose layers.These results represent the first demonstration that plant plasma membranes contain an adenosine triphosphatase that is activated by monovalent ions, and this finding further implicates the enzyme in the absorption of inorganic ions by plant roots.Absorption of inorganic ions bylplant-root cells is an energyrequiring process dependent on aerobic respiration (1, 2). Furthermore, adenosine triphosphate (ATP) appears to be the energy source, since ion absorption by plant roots is inhibited by dinitrophenol (3,4), arsenate (4), and oligomycin (5-7). The mechanism of energy transfer from ATP to the ion-transport system is unknown, however, and this phenomenon represents one of the major unresolved aspects of the ion-absorption process in plants.We have suggested (8, 9) that the energy transduction process involved in ion transport of plant cells involves an adenosine triphosphatase (ATPase; EC 3.6.1.3) similar to the "transport" ATPase of animal cells (10). Plant ATPase is associated with membranes, requires MIg++, and is further activated by monovalent ions (8, 9, 11). A high correlation exists between the KCl-or RbCl-activated component of the ATPase and K+ or Rb+ absorption by root tissue (9). Also, the kinetics of monovalent-ion transport into roots and the kinetics of monovalent ion-stimulated ATPase are similar (8, 9). However, in order for this ATPase to be involved in energy transduction for ion transport, it should be associated with one or both of the membranes involved in active ion transport (i.e., either the plasma membrane or tonoplast), and this has not been demonstrated.It is difficult to isolate and identify the membrane system containing the ion-stimulated ATPase because of the ubiquity of membrane-associated ATPases in plants (12)(13)(14) and the paucity of known membrane "markers" for plant cells (14). We have recently found, however, that the membrane system containing the monovalent ion-stimulated ATPase can be separated from nearly all the other membranes on either continuous or discontinuous sucrose gradients (14). In this paper, we show that this membrane system has a high ste...

Plasminogen was found to be present in bovine milk by crossreactivity between rabbit antiserum to plasminogen and casein prepared from milk by acid precipitation. This result was further supported by recovery of intact 125I-labeled plasminogen from rabbit milk after its intravenous injection. Freshly isolated whole bovine casein was observed to undergo slow autoproteolysis at 37°C. Polyacrylamide gel electrophoresis revealed gradual disappearance of major caseins accompanied by appearance and increase in intensity of numerous electrophoretic bands. This autoproteolysis was inhibited by low concentrations of &aminocaproic acid (0.1 mM) and diisopropyl fluorophosphate (1 mM); catalytic amounts of urokinase accelerated the process. Autoproteolysis of isolated bovine ,B-casein was shown by both urea and sodium dodecyl sulfate gel electrophoresis to result, in formation of Irl-and -Y2-caseins. Similar electrophoretic bands were formed when V-asein was degraded by plasmin prepared from bovine blood serum. These results support the hypothesis that bovine plasmin occurs in milk and is identical to alkaline milk protease.Plasminogen is the zymogen of the proteolytic enzyme plasmin which is responsible for the dissolution of fibrin clots in blood. In humans and other species several organs have been shown to contain considerable amounts of plasminogen activator activity (1-3). However, plasminogen itself is normally considered to be a constituent only of mammalian blood plasma (4). The presence of a naturally occurring protease in milk was first reported in 1897 (5). Casein, prepared by acid precipitation of skim milk, contains most of the proteolytic activity found in milk (6). Kaminogawa et al. (7) Immunological Study. Antiserum to bovine plasminogen was raised by weekly subcutaneous injection of 1 ml of plasminogen (10 mg) in phosphate-buffered saline containing 50% Freund's complete adjuvant into a New Zealand White rabbit. Five weeks after the first injection, blood was collected by cardiac puncture, allowed to coagulate for 30 min at 23°C, and centrifuged at 1500 X g for 10 min (16). The globulin fraction was prepared by two successive precipitations at 50% ammonium sulfate saturation (17) followed by washing with phosphate-buffered saline. After extensive dialysis against distilled water at 4°C, the globulin fraction was recovered by lyophili- Rabbit plasminogen was prepared as described above. Sodium dodecyl sulfate (NaDodSO4) gel electrophoresis of rabbit plasminogen was conducted in 7.5% acrylamide slabs (20) and gels were stained with 0.05% Coomassie blue. Gels were destained electrophoretically, dehydrated under reduced pressure, and exposed to x-ray film for 7 days.Abbreviations: EACA, E-aminocaproic acid; iPr2P-F, diisopropyl fluorophosphate; UKase, urokinase; NaDodSO4, sodium dodecyl sulfate. 2244 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicat...

Seasonal decomposition analyses were applied to the statistical evaluation of an oscillating activity for a plasma membrane NADH oxidase activity with a temperature compensated period of 24 min. The decomposition fits were used to validate the cyclic oscillatory pattern. Three measured values, average percentage error (MAPE), a measure of the periodic oscillation, mean average deviation (MAD), a measure of the absolute average deviations from the fitted values, and mean standard deviation (MSD), the measure of standard deviation from the fitted values plus R-squared and the Henriksson-Merton p value were used to evaluate accuracy.Decomposition was carried out by fitting a trend line to the data, then detrending the data if necessary, by subtracting the trend component. The data, with or without detrending, were then smoothed by subtracting a centered moving average of length equal to the period length determined by Fourier analysis. Finally, the time series were decomposed into cyclic and error components. The findings not only validate the periodic nature of the major oscillations but suggest, as well, that the minor intervening fluctuations also recur within each period with a reproducible pattern of recurrence.

Preparations enriched in apparently intact secretory vesicles were isolated from homogenates of lactating rat and bovine mammary tissue by differential and density gradient centrifugation in isoosmotic media. Morphologically, these preparations consisted nearly entirely of vesicles of varying sizes, at least some of which containedcasein micelles. Endoplasmic reticulum vesicles, Golgi apparatus cisterna and dictyosomes, mitochondria, peroxisomes, lysosomes, and nuclei were not observed in secretory vesicle-rich fractions. Vesicle preparations were enriched in lactose relative to total membrane fractions from mammary gland. (11, 12). The pellets that collected in centrifuge tubes during secretory vesicle isolation on Ficoll gradients were collected and analyzed for lactose content and will be referred to as the "vesicle-depleted particulate fraction." Total particulate fractions were obtained by centrifugation of clarified homogenates at 120,000 X g (maximum) for 60 min at 2'. Analytical Methods. Protein was determined with the Folin phenol reagent with bovine serum albumin as standard (27). 5020The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

BRANEN, A. L., and T. W. KEENAN. 1970. Diacetyl reductase of Lnctobacilllts cnsei. Can. J. Microbial. 16: 947-951. Diacetyl reductase (diacety1:reduced nicotinamide adenine dinucleotide (NADH) oxidoreductase, EC. 1.1.1.5) has been isolated from Lactobacill~a casei. Cell sonication, ammonium sulfate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography, and alumina gel adsorption were used to obtain the partially purified enzyme. Both NADH oxidase and diacetyl reductase activity were associated with the same fraction at all stages in purification. Growth in media containing added pyruvate resulted in a 10-fold increase in the NADH oxidase activity and a 3-fold increase in the diacetyl reductase activity of crude cell extracts on a protein basis. Purified preparations showed maximal reductase and oxidase activities at pH 4.5 and 5.0, respectively. Lineweaver-Burke plots yielded intersecting lines when NADH and diacetyl concentrations were varied, suggesting a flavin-linked reaction. The absorption spectrum of the purified preparation was characteristic of that of a flavoprotein. The product of the reduction of diacetyl was identified as acetoin. Acetoin and methylene blue were inactive as acceptors.