Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney.

A study has been made to determine whether renal plasma membranes contain an HCO3 stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney. The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase. The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome c oxidase activity. These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Histiocytic sarcoma of the scrotum in a rat

A 225-day-old male fourth generation rat from a developing recombinant inbred line (Lewis x Brown Norway) had a bilaterally symmetrical enlargement of the scrotum. Palpation indicated the presence of a firm lobulated mass extending from the tip of the scrotum to the abdominal wall. Bilateral nodular masses totally occupied the scrotal sacs, surrounded the testicles, and extended along the spermatic cords into the abdominal cavity. Tumor nodules also were present in the intestinal mesentery, omentum, mesenteric lymph nodes, pancreas, and lung. Histologically, the neoplasm presented a spectrum of characteristics varying from that of a granuloma with giant cells to a diffuse proliferation of spindle-shaped mononuclear cells.     

The effects of decapitation on the distribution of cytokinins and growth of Phaseolus vulgaris plants

The growth of the primary leaves of Phaseolus vulgaris L. was enhanced greatly by decapitation of the rest of the shoot. This increased growth was manifested by an increase in leaf area, leaf weight, and in a higher synthesis of chlorophyll and soluble proteins. Within the roots and stems decapitation resulted in a detectable increase in the endogenous cytokinins within 2 days after the surgical treatment. In the primary leaves increased cytokinin levels were only detected after 16 days. At this time most of the recorded activity co-chromatographed with the cytokinin glucosides. When plants which were decapitated were left under normal growing conditions for 16 days and then transferred to continuous darkness for 8 days the senescence of the primary leaves of the decapitated plants, in which the cytokinins had increased, was delayed significantly when compared with that of the primary leaves of the intact plants. the significance of these findings is discussed.     

Fast freezing of cow embryos in French straws with an automatic program

Cow embryos between day 6.5 and 9 were frozen in 1.5M DMSO in PBS at 2 degrees C/min from seeding to -25 degrees C before being plunged into liquid nitrogen directly or after 10 min at -25 degrees C. Cooling rate from 20 degrees C to -5 degrees C was 9 degrees C/min. Seeding was induced automatically at -5 degrees C by injection of liquid nitrogen vapour. Embryos were subsequently thawed by direct transfer to water at 20 degrees C (group I) or at 37 degrees C (group II). Survival was assessed by culture in vitro and by transfer. In group I, 35.7% were degenerated after thawing (compared to 35.4% in group II). Survival rate after culture in vitro for 24h was not significantly different (48.3% vs 42.8%) and hatching rate after 96h culture was quite similar (33.3% vs 34.4%). In group II, four pregnancies were obtained from 10 embryos transferred. Time at -25 degrees C did not improve the results. Automatic seeding did not impair survival. These results show that the quality of the embryo is the determinant factor for survival after freezing and that the plastic straw is the most suitable vessel for freezing, storage and transfer of embryos.     

Heterotrophic Uptake Experiments with 14C-Labeled Histidine in a Histidine-Limited Chemostat

The histidine uptake by bacterial strain HIS 42 was determined with [U-¹⁴C]histidine and through oxygen uptake experiments on samples taken from a histidine-limited chemostat. The uptake of [U-¹⁴C]histidine was characterized by a saturation constant of 12.8 to 78.6 nM histidine. At higher growth rates, the measured maximum uptake rate of histidine was lower than the actual uptake rate in the culture. The percentage of respired substrate (76 to 93%) was about 30 to 40% higher than the comparable value for the culture. The uptake of histidine as analyzed through the measurement of oxygen uptake rates was characterized by a saturation constant of 1.7 to 10.5 μM histidine; the maximum uptake rate was always greater than the actual histidine uptake rate in the culture. By the application of the two cited methods, set up to determine the histidine uptake kinetics, two different uptake processes were analyzed. It appeared that the determination of the histidine uptake through measurement of the oxygen uptake rate showed a better reflection of the actual uptake process of histidine in the culture. With the available data it was impossible to assess a correlation between the uptake of histidine, as determined with [U-¹⁴C]histidine, and the actual metabolism of the bacterial population.     

Column chromatography of plant polyphenols on weak anion exchangers.

Mixtures of phenols and cinnamic acid derived plant polyphenols are separated on several WAX cellulose materials and on DEAE Sephadex. Reference mixtures are also split up into the following distinct groups: phenolic carboxylic acids--neutral o-dihydroxy phenolics--neutral non o-dihydroxy phenolics. Quantitative recoveries are obtained, while no isomerizations occur with the pH labile plant phenolics. It is suggested that these materials can be used for preparative scale purification of plant phenolics, or for cleaning up plant extracts prior to HPLC examination.     

Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: isolation of a functionally active chemically pure domain and evidence for subunit heterogeneity.

α-Hemocyanin of the vineyard snail, Helix pomatia, is a large oligomer composed of 20 subunits with a molecular weight of 360,000 ± 30,000. Limited proteolysis showed these subunits to be composed of about seven structural domains, each having one oxygen binding site (1). This paper describes the production of these structural domains by tryptic digestion of $\text{1}\text{10}$ hemocyanin molecules. The digestion pattern was followed as a function of time by examining the proteolytically obtained fragments electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels. Based on the molar ratio of each fragment present during digestion the first part of the reaction sequence for trypsinolysis could be deduced. This reaction scheme was simulated by means of an analog computer. Pseudo-first-order reaction rate constants of the various proteolytic cleavages were estimated from the computer generated time course of digestion. On the basis of these results it is postulated that Helix pomatia α-hemocyanin possesses at least two kinds of subunits which differ in their proteolytic susceptibility. These subunits occur in equimolar amounts. A functionally active domain with a molecular weight of about 50,000 has been isolated from a tryptic digest of hemocyanin subunits. This domain seems to be chemically pure, as suggested by the unique sequence of its first six amino acids, viz: Lys-Val-His-Leu-Asn-Lys.     

Nitrogen and ammonia assimilation in the cyanobacteria: regulation of glutamine synthetase.

Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA by use of established procedures and by affinity chromatography as a final step. No adenylylation system controlling glutamine synthetase activity was found. The enzyme shows a marked specificity for Mg²⁺ in the biosynthetic assay and Mn²⁺ in the transferase assay. Under physiological conditions, Co²⁺ produces a large stimulatory effect on the Mg²⁺-dependent biosynthetic activity. The enzyme is inhibited by the feedback modifiers l-alanine, glycine, l-serine, l-aspartate, and 5′-AMP. Inhibition by l-serine and l-aspartate is linear, noncompetitive with respect to l-glutamate with apparent K_i values of 3 and 13 mm, respectively. Cumulative inhibition is seen with mixtures of l-serine, l-aspartate, and 5′-AMP. The results indicate that, in vivo, divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and hence ammonia assimilation in nitrogen-fixing cyanobacteria.     

The Endoplasmic Reticulum of Mung Bean Cotyledons: ROLE IN THE ACCUMULATION OF HYDROLASES IN PROTEIN BODIES DURING SEEDLING GROWTH

The subcellular localization of two hydrolases (ribonuclease and vicilin peptidohydrolase) which are synthesized de novo in the cotyledons of mung bean seedlings was studied. Earlier experiments had shown that both enzymes accumulate in the protein bodies in the course of seedling growth. Two methods to fractionate subcellular organelles were used to demonstrate that a significant proportion of the enzymes is organelle-associated. This proportion is highest (up to 50% for vicilin peptidohydrolase and 15% for ribonuclease) when synthesis of the enzymes has just started. Evidence obtained with isopycnic sucrose gradients indicates that both hydrolases are associated with membranes rich in NADH-cytochrome c reductase, a marker enzyme for the endoplasmic reticulum (ER). The hydrolases band with the NADH-cytochrome c reductase under conditions where the ribosomes remain attached or are detached from the ER-derived vesicles. Treatment of the ER-derived vesicles with Triton X-100 shows that vicilin peptidohydrolase and vesicle membranes can be physically separated without dissolving the membranes, indicating that the proteinase is soluble within the vesicles. These data support the conclusion that the ER is involved in the transport of ribonuclease and proteinase to the protein bodies.     

Sequence heterogeneity in kinetoplast DNA: Reassociation kinetics

Melting and reannealing of purified kinetoplast DNA (kDNA) from Crithidia fasciculata, Trypanosoma mega, and T. brucei have been studied with an automated optical system. The slow reassociation rate of trypanosome kDNA is due neither to the formation of hyperpolymers nor to mispairing of bases and certainly reflects extensive sequence heterogeneity. Simulation of the reassociation kinetics indicates that the kDNA comprises essentially two kinetic components: a fast renaturing component which might be a common sequence present in all the minicircles and a slow renaturing component which is responsible for minicircle heterogeneity. The rapidly renaturing component is more abundant in Crithidia than in trypanosomes.