Kinetic studies on the activation of pyrophosphate-dependent phosphofructokinase from mung bean by fructose 2,6-bisphosphate and related compounds

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) was purified from the mung bean Phaseolus aureus. The enzyme is activated by fructose 2,6-bisphosphate at nanomolar concentrations. The enzyme exhibits Michaelis-Menten kinetics, and the reaction mechanism, deduced from initial velocity studies in the absence of inhibitors as well as product and dead-end inhibition studies, is rapid equilibrium random in the presence and absence of fructose 2,6-bisphosphate. In the direction of fructose 6-phosphate phosphorylation, saturating fructose 2,6-bisphosphate (1 microM) increases V congruent to 9-fold and increases V/KMgPPi and V/KF6P about 30-fold. In the reverse direction (phosphate phosphorylation), the same concentration of activator has little if any effect on V or the Km for inorganic phosphate (Pi) and Mg2+ but does increase V/KFBP about 42-fold. No changes were observed in any of the other rate constants. The binding affinity of fructose 2,6-bisphosphate to all enzyme forms is identical. The activator site of the mung bean PPi-PFK binds fructose 2,6-bisphosphate with a Kact of 30 nM with the 2,5-anhydro-D-glucitol 1,6-bisphosphate (the most effective analogue) 33-fold less tightly. Of the alkanediol bisphosphate series, 1,4-butanediol bisphosphate exhibited the tightest binding (Kact congruent to 3 microM). These and a series of other activating analogues are discussed in relation to the activator site.     

A protein kinase copurified with chick oviduct progesterone receptor

A magnesium-dependent protein kinase activity was copurified with both the molybdate-stabilized 8S form of the chick oviduct progesterone receptor (PR) and its B subunit. In each case, purification was performed by hormonal affinity chromatography followed by ion-exchange chromatography. The Km(app) values of the phosphorylation reaction for [gamma-32P]ATP and calf thymus histones were approximately 1.3 X 10(-5) M and approximately 1.6 X 10(-5) M, respectively, and only phosphorylated serine residues were found in protein substrates, including PR B subunit. Physicochemical parameters of the enzyme [pI approximately 5.3, Stokes radius approximately 7.2 nm, sedimentation coefficient (S20,w) approximately 5.6 S, and Mr approximately 200,000] were compared to those of purified forms of PR (B subunit, pI approximately 5.3, Stokes radius approximately 6.1 nm, and Mr approximately 110,000; 8S form, Stokes radius approximately 7.7 nm and Mr approximately 240,000). The results suggest that most of the protein kinase activity copurified with both oligomeric and monomeric forms of PR belongs to an enzyme distinct from currently known receptor components. Its physiological significance remains unknown.     

Metabolic changes of membrane lipid composition in Acholeplasma laidlawii by hydrocarbons, alcohols, and detergents: arguments for effects on lipid packing

The packing of lipids into different aggregates, such as spheres, rods, or bilayers, is dependent on the hydrophobic volume, the hydrocarbon-water interfacial area, and the hydrocarbon chain length of the participating molecules, according to the self-assembly theory [Israelachvili, J. N., Marcelja, S., & Horn, R. G. (1980) Q. Rev. Biophys. 13, 121-200]. The origin of the participating molecules should be of no importance with respect to their abilities to affect the above-mentioned parameters. In this investigation, Acholeplasma laidlawii, with a defined acyl chain composition of the membrane lipids, has been grown in the presence of three different classes of foreign molecules, known to partition into model and biological membranes. This results in an extensive metabolic alteration in the lipid polar head group composition, which is expressed as changes in the molar ratio between the lipids monoglucosyldiglyceride (MGDG) and diglucosyldiglyceride (DGDG), forming reversed hexagonal and lamellar phases in excess water, respectively. The formation of nonlamellar phases by A. laidlawii lipids depends critically upon the MGDG concentration [Lindblom, G., Brentel, I., Sj?lund, M., Wikander, G., & Wieslander, A. (1986) Biochemistry (preceding paper in this issue)]. The foreign molecules tested belong to the following groups: nonpolar organic solvents, alcohols, and detergents. Their effects on the gel to liquid crystalline phase transition temperature (Tm), on the order parameter of the acyl chains, and on the phase equilibria between lamellar and nonlamellar liquid crystalline phases in lipid-water model systems are known in several instances.(ABSTRACT TRUNCATED AT 250 WORDS)     

In nondividing cells, histone H1(0) is synthesized and deposited onto chromatin without accompanying phosphorylation

The phosphorylation of H1 histone subfractions was measured in mouse neuroblastoma cells stopped from dividing by three treatments that block cell division: 5 mM butyrate, 2% dimethyl sulfoxide, and serum withdrawal. H1 histone phosphorylation decreased in response to all three treatments, but the response differed in its timing and its extent for the different H 1 subfractions. The different decreases in phosphorylation correlated well with the differential decreases in biosynthesis of the individual H1 subfractions; however, an exception to this parallel decrease in synthesis and phosphorylation was observed in the case of histone H1(0). Phosphorylation of H1(0) was absent in each of the three treatments after 2 days, despite the continued synthesis and deposit of H1(0) on the chromatin. Thus, despite the fact that H1(0) was being synthesized and that the other newly synthesized H1 subfractions were phosphorylated at this time, the phosphorylation of H1(0) became uncoupled from its synthesis after prolonged treatments blocking cell division.     

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 2. 1,6-Diphenyl-1,3,5-hexatriene in lipid bilayers

The application of a new spectroscopic tool [Knutson, J. R., Davenport, L., & Brand, L. (1986) Biochemistry (preceding paper in this issue)] for studying rotational microheterogeneity of probe location in lipid bilayer systems is described. Anisotropy decay associated spectra are derived from experimentally obtained polarized emission components. "Early" difference spectra (IV - IH) contain contributions from both fast and slow rotors, while "late" difference spectra predominantly reflect the emission from slowly rotating fluorophores. Anisotropy decay associated spectra have been used to resolve the emission spectra of 1,6-diphenyl-1,3,5-hexatriene (DPH) imbedded within a known rotationally heterogeneous mixture of two vesicle types (L-alpha-dimyristoyllecithin and L-alpha-dipalmitoyllecithin). At 29 degrees C, diphenylhexatriene within pure dimyristoyllecithin vesicles rotates rapidly, with a small r infinity, while diphenylhexatriene in dipalmitoyllecithin vesicles exhibits a large r infinity. Spectra for diphenylhexatriene imbedded in the two vesicle types show small but significant spectral differences. A spectrum of a mixture of the two vesicle types with DPH lies between these characteristic component spectra. The spectrum extracted for "immobilized" probes in the mixture correctly overlays the dipalmitoyllecithin spectrum. Further studies have shown that diphenylhexatriene exhibits more than one emission anisotropy decay associated spectrum in vesicles of a single lipid type, when that lipid is near its phase transition temperature. Diphenylhexatriene apparently inhabits more than one rotational environment even in these "homogeneous" vesicle preparations.     

Properties of the alkali light-chain-20-kilodalton fragment complex from skeletal myosin heads

We have developed a rapid and reproducible procedure widely applicable to the preparation of pure aqueous solutions of the complex between an alkali light chain and the COOH-terminal heavy-chain fragments of skeletal myosin chymotryptic subfragment 1 (S-1) split by various proteases. It was founded on the remarkable ethanol solubility of these complexes. A systematic study of the ethanol fractionation of the tryptic (27K-50K-20K)-S-1 (A2) showed the NH2-terminal 27K fragment to behave like a specific protein entity being quantitatively precipitated at a relatively low ethanol concentration. Only the 20K peptide-A2 complex remained in solution when the S-1 derivative was treated with exactly 4 volumes of ethanol in the presence of 6 M guanidinium chloride. At a lower ethanol concentration, a soluble mixture of 50K and 20K peptides together with the light chain was obtained. The isolated 20K fragment-A2 system containing a 1:1 molar ratio of each component was investigated by biochemical and 1H nuclear magnetic resonance (NMR) techniques to highlight its structure and the interaction of the 20K heavy-chain segment with F-actin and with the light chain. During the treatment of the complex with alpha-chymotrypsin, only the 20K peptide was fragmented in contrast to its stability within the whole S-1. The binding of F-actin to the complex led, however, to a strong inhibition of its chymotryptic degradation. 1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide cross-linking of F-actin to the complex produced covalent actin-20K peptide only, the amount of which was lower relative to that observed with the entire split S-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

In this paper we report the first 1H NMR study of the base-paired secondary structure of yeast 5.8S RNA. On the basis of a combination of homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz NMR spectrum, we are able to identify and assign eight of the nine base pairs in the most thermally stable helical arm: G116.C137-C117.G136-C118.G135- C119.G134-C120.G133-U121.G132- U122.A131-G123.C130. This arm contains an unusually temperature-stable (to 71 degrees C) segment of four consecutive G.C base pairs. This work constitutes the most direct evidence to date for the existence and base-pair sequence of the GC-rich helix, which is common to most currently popular secondary structural models for eukaryotic 5.8S ribosomal RNA.     

Mathematical model for human myeloma relating growth kinetics and drug resistance

We present a computer-based mathematical model that can simulate characteristic features of the clinical time course of human myeloma. It asserts that therapy resistance in myeloma cells is an inherited trait associated with the longer inter-mitotic times of some cells and that the strength of this trait affects tumour growth characteristics. These kinetic differences within the malignant cell clone may also influence therapeutic efficacy. In the model, the same total therapy, administered in different time-dose fractions, could be 'curative' or 'minimally effective' depending on kinetic properties. For example, as others have shown, in myeloma pulsed intermittent therapy is often more effective than low dose continuous therapy. According to our model this finding is compatible with a high coefficient of inheritability of resistance from one cell generation to the next. The model also suggests that if there are subclones of varying resistance, a therapy must have some effect on each of them if it is to be employed in a curative fashion. While many aspects of the model are not yet clinically testable, exploration of its concepts might increase knowledge about fundamental neoplastic mechanisms.     

Response of Peyer's patch lymphocyte subsets to Giardia muris infection in BALB/c mice. II. B-cell subsets: enteric antigen exposure is associated with immunoglobulin isotype switching by Peyer's patch B cells

The aim of this study was to quantify the response of Peyer's patch B cells, surface IgA-bearing (sIgA) B cells, and surface IgM-bearing (sIgM) B cells to Giardia muris infection. Following infection of a cohort of immunocompetent BALB/c mice with G. muris cysts, Peyer's patch cell suspensions were prepared at serial time points during the infection, incubated with fluorescein-conjugated monoclonal antibodies directed against murine leukocytes, B cells, sIgA B cells, sIgM B cells, or T cells, and analyzed by flow cytometry. Of total Peyer's patch leukocytes, the percentages of B cells, sIgA B cells, and sIgM B cells in uninfected BALB/c mice were 64.7 +/- 2.0% (mean +/- SEM), 30.3 +/- 1.5%, and 52.5 +/- 2.4%, respectively. The total number of Peyer's patch leukocytes increased significantly (1.8 X) during G. muris infection, and returned to control levels as the infection was cleared. The percentages of Peyer's patch T and total B cells did not change significantly during Giardia infection. However, sequential changes were observed in the percentages and numbers of sIgM and sIgA B cells during the infection. Peyer's patch sIgM B cells rapidly increased in percentage and number, reaching maximum levels 1 week after cyst inoculation. After remaining constant the first week, the number of Peyer's patch sIgA B cells increased during the second week of G. muris infection, reaching a maximum level 11-14 days after cyst inoculation. The data support the hypothesis that immunoglobulin isotype switching in Peyer's patches is induced by antigen exposure.     

Some aspects of the biology of the red bass, Lutjanus bohar (Forsskal), from the Tigak Islands, Papua New Guinea

The growth rate, food, reproduction and mortality rates of the red bass, Lutjanus bohar , are described for fish associated with coral reefs in the Tigak Islands of Papua New Guinea. Spawning intensity by month was investigated by examination of gonad maturation stages. The length–at–maturity of female L. bohar was estimated from the relationship between changes in gonad index with increasing size. Female fish matured at about 45–0 cm fork length. The Petersen method was used to obtain age–at–length data from length frequency data of L. bohar captured by hook–and–line fishing. The growth of L. bohar was described by a von Bertalanffy growth curve with the parameters AT=0.27 and L_∞ = 81.7 cm. There was a positive correlation between water depth (D) and the length of L. bohar (L) that was described by L = 318–8+ 3.050 D . The mortality rates ( Z ) of L. bohar from shallow (≤30m) and deep (≤80m) water were estimated from length converted catch curves and were 1–181 and 0–64 respectively. The high Z value for the shallow water population of L. bohar was ascribed to the migration of larger specimens into deeper water. The deep water population of L. bohar remains unfished and the estimate of Z was believed to be equivalent to the natural mortality rate ( M ). An empirical method of calculating M gave an estimate of 0.59. Lutjanus bohar is a carnivorous piscivore that feeds throughout the water column.