Transbilayer distribution and mobility of phosphatidylinositol in human red blood cells

The present studies describe the distribution of phosphatidylinositol (PI) within the membrane bilayer of the human red blood cell (RBC) as well as its transbilayer mobility. The membrane bilayer distribution was determined by measuring the hydrolysis of PI in the exterior leaflet of the RBC membrane using a PI-specific phospholipase C and by extraction of PI from the exterior leaflet using bovine serum albumin. The transbilayer mobility of PI was measured by following the fate of radiolabeled PI which was first incorporated into the outer leaflet of the RBC membrane. Our results indicate that PI is asymmetrically distributed in the membrane, with approximately 80% located in the inner and 20% in the outer leaflet of the bilayer. The rate of transbilayer mobility of PI is similar to that for certain molecular species of phosphatidylcholine and much slower than that reported for the aminophospholipids in the RBC membrane.     

Induction of F9 cell differentiation by transient exposure to retinoic acid.

The F9 cell is a mouse embryonal teratocarcinoma which can be induced to differentiate into visceral endoderm by treatment with retinoic acid (RA). Treatment with RA in conventional studies was carried out in the constant presence of RA. Here we demonstrate that treatment with RA can be as short as 3 hrs to induce differentiation of F9 cells. Morphology, alpha-fetoprotein gene activity, and temporal patterns of F9 cell differentiation are the same with both short- and long-term treatment with RA.     

Intermediate Filaments from Bovine, Rat, and Human CNS: Mapping Analysis of the Major Proteins

Abstract: Intermediate filaments were isolated by an axon-flotation method from bovine, rat, and human CNS. Gel electrophoresis showed four major proteins, having molecular weights of about 50,000, 70,000, 160,000, and 210,000, to be present in filaments of all three species. Small differences in molecular weights and major differences in relative distribution of the filament proteins were observed among species. In bovine and rat brain the predominant protein was the 50,000 band, but in human brain the 70,000 band was present in greatest amount. Each filament protein of the three species was studied by peptide mapping using limited proteolysis and cyanogen bromide cleavage. Within the same molecular weight group, filament proteins from different species gave similar maps with both techniques. Some degree of heterogeneity was also observed. However, filament proteins of different molecular weights of the same species gave distinctly different maps. These studies rule out the possibility that filament proteins from different molecular weight groups are related to each other by oligomerization; nor is it likely that the lower molecular weight proteins are derived from the subunit of molecular weight 210,000.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

Measurement of surface potential and surface charge densities of sarcoplasmic reticulum membranes

Summary The binding of the anionic fluorescent probe 1-anilino-8-naphthalene-sulfonate (ANS^–) was used to estimate the surface potential of fragmented sarcoplasmic reticulum (SR) derived from rabbit skeletal muscle. The method is based on the observation that ANS^– is an obligatory anion whose equilibrium constant for binding membranes is proportional to the electrostatic function of membrane surface potential, exp(e₀/kT, where ₀ is the membrane surface potential,e is the electronic charge, andkT has its usual meaning. The potential measured is characteristic of the ANS^– bindings of phosphatidylcholine head groups and is about one-third as large as the average surface potential predicted by the Gouy-Chapman theory. At physiological ionic strength the surface potentials, measured by ANS^–, referred to as the aqueous phase bathing the surface, were in the range –10 to –15 mV. This was observed for the outside and inside surfaces of the Ca²⁺-ATPase-rich fraction of theSR and for both surfaces of theSR fraction rich in acidic Ca²⁺ binding proteins. The inside and outside surfaces were differentiated on the basis of ANS^– binding kinetics observed in stopped-flow rapid mixing experiments. A mechanism by which changes in Ca²⁺ concentration could give rise to an electrostatic potential across the membrane and possibly result in changes in Ca²⁺ permeability.The dependence of the surface potential on the monovalent ion concentration in the medium was used together with the Gouy-Chapman theory to determine the lower limits for the surface charge density for the inside and outside surfaces of the two types ofSR. Values for the Ca²⁺-ATPase richSR fraction were between 2.9×10³ and 3.8×10³ esu/cm², (0.96×10^–6 and 1.26×10^–6 C/cm²) with no appreciable transmembrane asymmetry. A small amount of asymmetry was observed in the values for the inside and outside surfaces of the fraction rich in acidic binding proteins which were ca. 6.6×10³ and ca. 2.2×10³ esu/cm² (2.2×10^–6 and 0.73×10^–6 C/cm). The values could be accounted for by the known composition of negatively-charged phospholipids in theSR. The acidic Ca²⁺ binding proteins were shown to make at most a small contribution to the surface charge, indicating that their charge must be located at least several tens of Å from the membrane surface. The experiments gave evidence for a Donnan effect on the K⁺ distribution in the fraction rich in acidic binding proteins. This could be accounted for by the known concentration of acidic binding proteins in thisSR fraction.The equilibrium constant for ANS^– was shown to be more sensitive to changes in the divalent cation concentration than to changes in the monovalent cation concentration, as predicted by the Gouy-Chapman theory. Use of these findings together with the stopped-flow rapid mixing techniques constitutes a method for rapid and continuous monitoring of changes in ion concentrations in theSR lumen.     

Divalent cation-induced aggregation of chromaffin granule membranes.

Divalent cations induce the aggregation of chromaffin granule ghosts (CG membranes) at millimolar concentrations. Monovalent cations produce the same effect at 100-fold higher concentrations. The kinetics of the dimerization phase were followed by light-scattering changes observed in stopped-flow rapid mixing experiments. The rate constant for Ca2+-induced dimerization (kapp) is 0.86-1.0 x 10(9) M-1sec-1, based on the "molar" vesicle concentration. This value is close to the values predicted by theory for the case of diffusion-controlled reaction (7.02 x 10(9) M-1sec-1), indicating that there is no energy barrier to dimerization. Arrhenius plots between 10 degrees and 42 degrees C support this; the activation energy observed, +4.4 Kcal, is close to the value (4.6-4.8 Kcal) predicted for diffusion control according to theory. Artificial vesicles prepared from CG lipids were also found to have cation-induced aggregation, but the rates (values of kapp) were less than 1/100 as large as those with native CG membranes. Also, significant differences were found with respect to cation specificity. It is concluded that the slow rates are due to the low probability that the segments of membrane which approach will be matched in polar head group composition and disposition. Thus large numbers of approaches are necessary before matched segments come into aposition. The salient features of the chromaffin granule membrane aggregation mechanism are as follows: (a) In the absence of cations capable of shielding and binding, the membranes are held apart by electrostatic repulsion of their negatively charged surfaces. (b) The divalent and monovalent cation effects on aggregation are due to their ability to shield these charges, allowing a closer approach of the membrane surfaces. (c) The major determinants of the aggregation rates of CG membranes are proteins which protrude from the (phospholipid) surface of the membrane and serve as points of primary contact. Transmembrane contact between these proteins does not require full neutralization of the surface charge and surface potential arising from the negatively charged phospholipids. (d) After contact between proteins is established, the interaction between membranes can be strengthened through transmembrane hydrogen bonding of phosphatidyl ethanolamine polar head groups, divalent cation-mediated salt bridging, and segregation of phosphatidylcholine out of the region of contact.     

Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA.

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.     

Histidine:pyruvate transamination and phyenylalanine:pyruvate transamination may be catalyzed by the same enzyme.

Some properties of histidine:pyruvate transaminase (HPT) and phenylalanine:pyruvate transaminase (PPT) in the cytosol of rat liver were studied. HPT and PPT activity could not be separated by DEAE-Sephadex A-50 or hydroxylapatite column chromatography, and the ratio of $\text{HPT}\text{PPT}$ activity remained constant during these purification procedures. The two enzyme activities also showed similar heat stability and responses to glucagon injection. Based on these findings, we suggest that a single enzyme may specifically catalyze histidine:pyruvate and phenylalanine:pyruvate transamination.