Studies on phosphoproteins of submandibular gland nuclei isolated from isoproterenol-treated rats

Rat submandibular gland nuclei incubated with γ-³²P-ATP incorporated the label into histone and non-histone phosphoproteins. The latter was the predominantly radioactive fraction. After a single injection of isoproterenol (Ipr), the incorporation of ³²P into non-histone phosphoproteins decreased during the first few hours, followed by an increase at 4 h which reached its peak at 24 h at a higher level compared with normal controls. The values returned to the control level at 40 h after the injection. The changes were reflected in the initial rates as well as the total level of incorporation of ³²P into the phosphoproteins. Temporally, the onset of increase in the phosphorylation of non-histone phosphoproteins appeared to precede that in RNA synthesis, although peak activity of the phosphorylation coincided with the peak of RNA synthesis. The non-histone phosphoproteins which depicted maximal changes in response to Ipr were further characterized as phenol-soluble acidic phosphoproteins. The phosphorylation of histone phosphoproteins also declined after the injection of Ipr, but the recovery of the rate of phosphorylation was not observed until 16 h after the injection, reaching the control levels at 24 h. Treatment of rats with actinomycin D or cycloheximide, prior to Ipr, abolished the increase in phosphorylation of non-histone phosphoproteins observed at 24 h after Ipr. Further, the changes in the phosphorylation of nuclear phosphoproteins induced by Ipr were blocked by prior treatment of the animals with dichloroisoproterenol. The results suggest that the phosphorylation of the non-histone phosphoproteins plays an important role in the events controlling the synthesis of RNA which precedes the replication of DNA and cell. In addition, the regulation of the metabolism of nuclear phosphoproteins may be controlled through a function of the cytoplasmic membrane.     

Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia

The tissue distribution and the effects of starvation and streptozotocin-induced diabetes on insulin B chain-degrading neutral peptidase activity in the rat have been studied. The neutral peptidase activity in tissue extracts was determined by measuring the formation of trichloroacetic acid-soluble radioactivity from ¹²⁵I-labeled B chain of insulin in 0.1 m Tris buffer (pH 7.2). Inhibition by several different compounds (EDTA, dithiothreitol, and potassium phosphate) which are known to inhibit the purified enzyme and the effects of pH suggest that the B chain-degrading activity measured in each of 12 tissue extracts may be similar to the neutral peptidase recently purified from rat kidney (P. T. Varandani and L. A. Shroyer, 1977, Arch. Biochem. Biophys., 181, 82–93). Neutral peptidase activity was observed in all tissues examined and varied in the order kidney ? intestine > pancreas, testis > liver > thymus > heart, skeletal muscle, diaphragm > lung, spleen > fat. Neutral peptidase activity in kidney, liver, fat, and skeletal muscle from diabetic animals was significantly depressed when compared with the levels in these tissues from normal animals. Insulin treatment of diabetic animals raised the neutral peptidase activity in kidney, liver, and fat to levels equivalent to or even exceeding normal levels; however, activity in skeletal muscle persisted at depressed levels. Heart muscle neutral peptidase activity was not significantly affected in either diabetes or starvation. In the liver, starvation reduced the level of neutral peptidase activity while subsequent refeeding raised the activity to a level exceeding the control. Opposite effects were observed in kidney: starvation increased neutral peptidase activity while refeeding brought the activity back to normal levels. Only small decreases in neutral peptidase activity were observed in fat and skeletal muscle after 24 h starvation, but were not evident after 64 h starvation. The changes in neutral peptidase activity correlated well with the changes in glutathione-insulin transhydrogenase activity previously reported in liver and kidney.     

Physiochemical characterization of lymphocyte inhibitory factor (LyIF) isolated from avian B and T cells

Thymic (T) or bursal (B) lymphocytes from chicks sensitized to Mycobacterium tuberculosis produce an avian lymphocyte inhibitory factor (LyIF). The physiochemical properties of both T and B LyIF were established by ultrafiltration which yielded four fractions with molecular weight ranges of greater than 100,000; 50,000-100,000; 10,000-50,000; and less than 10,000; enzymatic treatment with chymotrypsin and neuraminidase; varying pH; and heat exposure. These studies demonstrated that the maximum activity for both T and B LyIF was within a molecular weight range of 10,000-50,000. Both were sensitive to chymotrypsin and neuraminidase treatment. Both were stable at 56 degrees C for 30 min and resistant to changes in pH from 5 to 9. T-Cell migration was inhibited equally by B or T LyIF, while B-cell migration was inhibited to a lesser extent by T LyIF and B LyIF. Further experiments should establish the reasons for these observed differences in cross-reactivity.     

Calcium and the release from dormancy of freshwater sponge gemmules.

Divalent cations (Zn, Mn, Ba, Sr) inhibit the development of dormant gemmules of the freshwater sponge Spongilla lacustris. This inhibition is overcome by calcium which can be interpreted to mean that this divalent ion is essential for germination (cell division) in this system. Inhibitory divalent cations have different effective concentrations which indicate differing binding affinities for sites which may normally bind calcium. Ethylene glycol bis(β-aminoethyl ether)N,N-tetraacetic acid does not effect gemmule development at 15°C but stimulates it at 4°C, indicating that a dislocation of endogenous calcium stimulates release from dormancy. Magnesium will only partially substitute for calcium in overcoming divalent cation inhibition implying a different specificity for this ion in gemule development. Calcium is also indicated as being essential for hatching (cell motility) in this system.     

Action of neocarzinostatin on cell nuclei: release of specific chromatin

We report the first complete sequence of a P450 monoxygenase cytochrome. The P450_CAM from $\text{Pseudomonas}\text{putida}$ is a single polypeptide of 412 residues as determined from the isolated tryptic, clostripain, CNBr, and mild acid cleavage fragments. Significant molecular features, including secondary structure, are discussed.     

Synthesis of RNA containing polyadenylic acid sequences in preimplantation rabbit embryos

Messenger RNA synthesis has been estimated by assaying polyadenylic acid (poly A)-rich sequences in heterogeneous RNA from preimplantation rabbit embryos. Poly A containing RNAs are synthesized at least as early as the 16-cell stage and continue to be made through blastocyst formation and maturation. Sixty to 78% of the heterogeneous polysomal RNA in blastocysts contain poly A sequences. The portion of the heterogeneous RNA containing poly A sequences does not appear to change markedly between cleavage and blastocyst stages of development. Poly Arich sequences are greater than 4 S and consist of at least 84% adenine residues. RNA molecules ranging from 6 S to greater than 28 S contain poly A sequences.     

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side.

In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21 °C.

In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds.

Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds.

The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.     

Light-difference detector for reading Rayleigh fringe patterns from the ultracentrifuge

We have developed a dual-photocell, light-difference detector, easily attached to a comparator screen, which provides rapid and direct location of fringe centers from Rayleigh interferograms without the need for digital micrometers or measurement of optical densities. The device provides a pulse for digital micrometers which triggers the printing of fringe centers during manual movement of the stage, providing a two-thirds saving in time. From evaluation of sedimentation equilibrium patterns it was found that the precision of (1) concentration measurement is 0.003 fringes and (2) molecular weight determinations is several parts per thousand.     

Mediation of beta-adrenergic stimulated amylase release from mouse parotid gland

Amylase released from mouse parotid fragments by the β-adrenergic agonist, isoproterenol, was associated with l) enhanced ⁴⁵Ca⁺⁺ efflux and 2) a dependence on the extracellular Na⁺ concentration. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on ⁴⁵Ca⁺⁺ efflux. In the absence of extracellular sodium isoproterenol and monensin failed to significantly release ⁴⁵Ca⁺⁺. Complete inhibition of isoproterenol stimulated amylase release occurred when 75 per cent or greater of the extracellular Na⁺ was replaced by sucrose; carbachol stimulated amylase release was not affected. Tetracaine (0.2 mM to 1.0 mM) inhibited both isoproterenol and carbachol stimulated amylase release and inhibited the ⁴⁵Ca⁺⁺ uptake induced by carbachol. Monensin, a sodium ionophore, mimicked the effects of isoproterenol on amylase release; this effect was significantly reduced in the absence of extracellular Na⁺. It is proposed that a primary step in the release of amylase form mouse parotid gland in response to β-adrenergic stimulation is an increased influx of Na⁺ followed by release of intracellularly stored calcium.     

The amino acid sequence of carbonic anhydrase I from the rhesus macaque

The complete amino acid sequence of carbonic anhydrase I (CA I) isolated from the red cells of the rhesus macaque ($\text{Macaca}$$\text{mulatta}$) is presented. This sequence was obtained by aligning peptides derived from various fragmentation procedures with the fully characterized sequence of human CA I. When the peptides of rhesus CA I were ordered in this manner, 13 of the 260 residues were found to differ from the human CA I sequence. The known markedly higher specific esterase activity of rhesus CA I compared to human CA I could not be correlated with any changes in residues postulated to be within 10 Å of the single zinc ion at the active site.     

Modulation of proton efflux from chloroplasts in the light by external pH

The effects of external pH on the efflux of protons from illuminated spinach chloroplasts have been studied by monitoring the rates of proton-pumping electron transport under a variety of steady-state conditions. Phosphorylation-coupled proton efflux through the ATP synthase (CF₀-CF₁), determined from the rates of ATP formation and that portion of the total electron transport attributable to phosphorylation, is strongly dependent upon pH over the range 6–9, with little activity below pH 7 and half-maximal activity at pH ≈ 7.6. Noncoupled proton efflux through the ATP synthase, determined in the absence of ADP and phosphate, was also strongly pH sensitive, with little activity below pH 7.5 and half-maximal activity at pH ～- 7.9. When proton efflux via CF₀ was prevented by triphenyltin, the rate of passive proton leakage across the membrane was very low and practically insensitive to external pH indicating that the major pH-sensitive pathway(s) for proton efflux in the light involves CF₀ · CF₁. Modification of CF₁ sulfhydryls by Ag⁺ resulted in an apparent increase in proton efflux via the normally coupled CF₀ · CF₁ pathway (half-maximal activity = pH 7.6), whereas modification by Hg²⁺ resulted in an apparent increase in proton efflux via the noncoupled CF₀ · CF₁ pathway (half-maximal activity = pH 7.9).     

Similarity of the primary sequences of ribosomal RNAs isolated from vegetative and developing cells of Dictyostelium discoideum

Differentiating cell aggregates of Dictyostelium discoideum exhibit a pattern of rRNA metabolism quite different from that observed in the single-celled vegetative amoebas of this organism. We have examined whether the differences are related to a requirement for the production of new types of ribosomal RNA during development. Oligonucleotide maps and supplementary sequence data for 25 S, 17 S, and 5 S rRNAs from vegetative and developing cells have revealed no detectable alterations in primary sequence distinguishing any species of rRNA in developing cells from its vegetative cell counterpart.     

FSH receptors in isolated Sertoli cells: changes in concentration of binding sites at the onset of sexual maturation

Previous evidence has shown that isolated rat Sertoli cells have the capacity to metabolize C₁₉ and C₂₁ steroids and the steroidogenic activity is age-dependent and stimulated maximally by FSH in rats between 10 and 17 days of age. The purpose of the present study was to determine whether these age-related variations in sensitivity of Sertoli cells to FSH could be related to differences in FSH receptor concentrations. Sertoli cells were isolated at different ages from rats which had been irradiated in utero. Protein and DNA measurements of Sertoli cells from rats 6 to 65 days old indicated that protein content per Sertoli cell remained constant while DNA content progressively decreased up to 40 days of age. For quantitation of the FSH receptor, the 23,000 x g pellets of Sertoli cells were incubated with purified rat FSH which had been iodinated by the chloramine-T method. Sertoli cells isolated from rats 6, 10, 16 or 60 days of age, exhibited age-related differences in FSH binding activity: the concentration of FSH binding sites in Sertoli cells from 10 and 16 day old rats was significantly higher than in cells from 6 and 60 day old rats. This temporal pattern in FSH receptor concentration parallels the steroidogenic capacity and the FSH sensitivity of the Sertoli cells at the onset of sexual maturation.     

DNA-dependent RNA-polymerases from Artemia embryos. characterization of polymerases I and II from nauplius larvae.

Partial purification and characterization of DNA-dependent RNA-polymerases from nauplius larvae of the brine shrimp, Artemia salina, are described. Fractionation of solubilized RNA-polymerases on columns of DEAE-cellulose yielded partially purified preparations of RNA polymerases I and II. The properties of these enzymes were found to be similar to properties of corresponding enzymes from other animal sources. A significant change in the relative amounts of polymerases I and II occurs between 36 and 72 hr of development. Polymerase activity obtained from 36-hr nauplii consisted of approximately equal amounts of polymerases I and II, whereas polymerase II accounted for more than 80% of the activity recovered from 72-hr nauplii. Total polymerase activity was lower at 72 than at 36 hr. The significance of these changes in relation to the decrease in RNA synthesis in vivo that occurs after 36 hr is discussed.     

Polyamines can replace the dialyzable component from crude reticulocyte initiation factors.

The polyamines spermidine and spermine can replace the dialyzable component, previously indicated as “iRNA”, in restoring the activity of dialyzed initiation factors on messenger RNA translation in vitro. These results further sustain our earlier suggestions (1, 2) that the RNA nature of the dialyzable component (3) is questionable.