Restricted pH ranges and reduced yields for bacterial growth under pressure

Hydrostatic pressure was found to cause a marked narrowing of pH ranges for growth and reductions in growth yields for a variety of bacteria. In many cases, reduced yields under pressure could be directly related to increased sensitivities to metabolic acids that accumulated in the enclosed culture vessels used. Magnesium and calcium ions partially reversed increases in sensitivities of representative gram-positive and gram-negative bacteria to low, but not high, pH. Growth inhibition of these organisms at both extremes of pH was associated with enhanced loss of K⁺ from pressurized cells. Inhibited cells in alkaline media also lysed under pressure, but microscopically observable lysis was clearly a secondary phenomenon because it occurred slowly. Apparent volumes for growth-inhibitory protonation-deprotonation reactions were calculated on the basis of measured shifts in inhibitory pH with pressure. The values ranged from 99 to 431 ml/mole, and their magnitudes indicated that growth inhibition by acids or bases involves cooperative changes in polymeric interactions such as those which accompany protein denaturation.     

The polyadenylate polymerases from yeast.

Poly(A) polymerase activity was first detected in yeast extracts, primarily in association with the ribosomal fraction, by Twu and Bretthauer in 1971 (Twu, J. S., and Bretthauer, RK. (1971) Biochemistry 10, 1576-1582). This activity has now been separated into three distinct enzymes by chromatography on DEAE-cellulose. Each of the three enzymes can catalyze the incorporation of adenylate residues from ATP into a polyadenylate (poly(A)) tract at the 3' terminus of a primer RNA. Enzyme I elutes at 0.07 M ammonium sulfate from the DEAE-cellulose column, utilizes the mixed polynucleotide poly(A,G,C,U) or ribosomal RNA most efficiently in vitro, and may be responsible in vivo for the initiation of the poly(A) tracts found on yeast messenger RNA. Enzyme II elutes from the column at 0.20 M ammonium sulfate, requires poly(A) itself or an RNA primer containing a 3'-oligo(A) tract, and may be responsible in the nucleus for the elongation of tracts initiated by enzyme I. Enzyme III elutes from the column at 0.56 M ammonium sulfate and is present in low amounts in nuclear extracts. It may be involved in adding poly(A) tracts to messenger RNA in mitochondria. These enzymes also have the intrinsic capacity for the incorporation of cytidylate residues from CTP, which correlates with the finding of cytidylate residues in the poly(A) tracts present in the yeast RNA which is rapidly labeled in vivo. About 75% of the total poly(A) polymerase activity of yeast is enzyme I, most of which is present in the soluble protein fraction of the whole yeast extract. About 20% of the total poly(A) polymerase is enzyme II, and 1 to 5% is enzyme III. All three of the yeast poly(A) polymerases require an RNA primer with a free 3'-hydroxyl group, show no requirement for a DNA template, require Mn-2+ for optimal activity, have pH optima of 8.5, and are inhibited by GTP, CTP, UTP, and native yeast DNA. Polymerases I and II have similar molecular weights by gel filtration.     

Spectral studies of conformational changes induced in beta-glucuronidase by saccharo-1,4-lactone.

Ultraviolet difference spectroscopy has been used to study the binding of the transition state analog saccharo-1,4-lactone to purified rat preputial gland beta-glucuronidase. At pH 4.5 (the pH optimum), the inhibitor induces a difference spectrum indicative of a change in the environment of tryptophyl residues. Based on the magnitude of the induced difference spectrum as a quantitative measure of inhibitor binding, a titration curve for saccharo-1,4-lactone was obtained. A Scatchard plot of the titration data indicates that 4 molecules of inhibitor bind to the enzyme tetramer at a K-I of 4 times 10-7 M. The inhibitor also induces a similar difference spectrum at pH 7.5, although the binding is considerably weaker at this pH than at pH 4.5. When the native enzyme at pH 4.5 is compared with the native enzyme at pH 7.5, a difference spectrum, distinct from that of the binding of saccharo-1,4-lactone, is observed, indicating that the enzyme exists in different conformations at these pH values. The indication that tryptophyl residues are perturbed upon binding of saccharo-1,4-lactone was supported by studies carried out with N-bromosuccinimide. At pH 4.3, this reagent was found to oxidize 6 tryptophyl residues in the native enzyme but only three in the saccharo-1,4-lactone-inhibited enzyme. A spectrophotometric titration of the enzyme indicated that of the 33 tyrosyl residues per subunit, only 5 to 6 ionize at the pK expected for free phenolic groups.     

Proteins Specified by Herpes Simplex Virus: II. Viral Glycoproteins Associated with Cellular Membranes

Membranes prepared from HEp-2 cells infected with herpes simplex virus and free from soluble proteins, virus, ribosomes, and other cellular constituents were solubilized and subjected to electrophoresis on acrylamide gels. The electropherograms showed the following. (i) The synthesis of host proteins and glycoproteins ceases after infection. However, the spectrum of host proteins in membranes remains unaltered. (ii) Between 4 and 22 hr postinfection, at least four glycoproteins are synthesized and bound to the smooth cytoplasmic membranes. On electrophoresis, these glycoproteins form two major and two minor bands in the gel and migrate with proteins ranging from 50,000 to 100,000 daltons in molecular weight. (iii) The same glycoproteins are present in all membranes fractionated by density and in partially purified virus. The implications of the data are discussed.     

Der Einfluß von Li- und SCN-Ionen auf die Differenzierungsleistungen desAmbystoma-Ektoderms und ihre Veränderung bei kombinierter Einwirkung beider Ionen

Summary The development of the mouse oocyte during the primordial, primary and secondary follicular growth stages was studied by means of the electron microscope.During the early stages of oocyte maturation, mitochondrial multiplication takes place along with an apparent temporary transition from round to oval shape. The internal structure of many of the mitochondria is altered by separation of membranes of a crista to form a vacuole. This enlarges to pear-shaped configurations and gradually it forms so large a structure as to result in compression of adjacent cristae, thereby altering the entire appearance of the organelle.Dense round bodies encapsulated by a single membrane are found in the cytoplasm of oocytes of primary follicles near the periphery. The Golgi complex appears in primary follicle oocytes as an aggregation of vesicles. Gradually the number of lamellae in the complexes increase and these organelles become more peripherally located. The Balbiani yolk nuclei apparently is represented by a conglomeration of Golgi complexes and are present only in primordial and young primary follicle oocytes.The endoplasmic reticulum appears in the early stages only as rough-surfaced vesicles. At later stages individual cisternae become prominent. Apparently, a modified form of E. R. appears during maturation of the secondary follicle oocyte.Multivesicular complexes, each consisting of two components, small vesicles and larger vesicles enclosing microvesicles (multivesicular bodies), were commonly found during all stages of oocyte growth. The secondary follicle oocytes frequently contain multilamellar bodies. These are commonly found in juxtaposition to the multivesicular complexes and also near the egg periphery and occasionally near the nuclear envelope.This investigation was supported by a Public Health Service Research Career Program Award (5-K3-HD-5356-07) from the National Institute of Child Health and Human Development.