The mitogenic effect of A23187 in human peripheral lymphocytes

The mitogenic action of the divalent ionophore A23187 was confirmed and shown to be very sensitive to changes in extracellular calcium ion concentration. At optimal calcium and ionophore concentrations, an increase in [³H]-thymidine incorporation was seen that was similar to that seen after phytohemagglutinin addition. A calcium-dependent stimulation of α-aminoisobutyric acid transport was also seen after A23187 addition.Studies with three inhibitors demonstrate a similarity between proliferation induced by phytohemagglutinin and by A23187. Isoproterenol (10^?4 M) and ouabain (10^?7 M) blocked the effects of phytohemagglutinin and A23187. A drug, D-600, that has been shown to block calcium channels in cardiac muscle, inhibited proliferation induced by either phytohemagglutinin or A23187. This concentration of D600 had no effect on either phytohemagglutinin- or A23187-induced ⁴⁵Ca²⁺ uptake. Furthermore, the (+) and (?) isomers separated from racemic D600, which have been shown to block sodium and calcium channels respectively in smooth muscle, had equal potency in blocking lymphocyte proliferation.     

Ca2+ transport against its electrochemical gradient in cytochrome oxidase vesicles reconstituted with mitochondrial hydrophobic proteins

Cytochrome oxidase vesicles have recently been shown to accumulate Ca²⁺ in an energy-dependent manner. Energization of these vesicles with internally trapped cytochrome c and externally added ascorbate and phenazine methylsulfate generated an internally positive membrane potential and prevented Ca²⁺ influx (R. N. Rosier and T. E. Gunter, 1980, FEBS Lett.109, 99–103). In contradistinction, when cytochrome oxidase vesicles were reconstituted with complex V, a mitochondrial protein fraction containing the uncoupler binding site (Y. Hatefi, D. L. Stiggall, Y. Galante and W. G. Hanstein, 1974, Biochem. Biophys. Res. Commun.61, 313–321), both Ca²⁺ uptake and generation of an internally positive membrane potential were observed. The uptake was specifically dependent on energization of electron transport. Control experiments verified that the energization conditions used produced appropriately oriented membrane potentials. Other partially purified hydrophobic mitochondrial protein complexes were found to be less effective than complex V. The reconstituted system showed cation selectivity since Ca²⁺, Mn²⁺, and Rb⁺ were transported, while Na⁺ was not. Low levels of uncoupler, which did not affect oxidation rates, were found to partially inhibit Ca²⁺ uptake regardless of the membrane potential polarity. Uncoupling levels of uncoupler markedly inhibited Ca²⁺ uptake in internally negative cytochrome oxidase vesicles; however, inhibition in internally positive cytochrome oxidase vesicles was less relative to that at lower levels of uncoupler. The uncoupling combination of nigericin, valinomycin, and K⁺ was inhibitory to uptake regardless of membrane potential polarity. A reconstituted system of oxidative phosphorylation, which contains a hydrophobic protein fraction, energized with cytochrome oxidase similarly accumulated Ca²⁺ despite formation of an internally positive membrane potential. The results suggest that cytochrome oxidase, when coupled to appropriate hydrophobic mitochondrial proteins, can act as an electrogenic Ca²⁺ pump deriving its energy directly from electron transport.     

Interactions of alginates with univalent cations

Interactions of alginate with univalent cations in solution have been investigated by circular dichroism (c.d.) and rheological measurements. Poly-l-guluronate chain-segments show substantial enhancement (～ 50%) of c.d. ellipticity in the presence of excess of K⁺, with smaller changes for other univalent cations: Li⁺ < Na⁺ < K⁺ > Rb⁺ > Cs⁺ > NH₄⁺. The maximum c.d. change is attained by 0.3m, with no further increase at higher concentrations of cation. No significant dependence on polymer concentration is observed. Spectral changes for poly-d-mannuronate and heterotypic chain-sequences are much smaller. For intact alginates, the magnitude of c.d. change varies almost linearly with poly-l-guluronate content. Difference spectra (c.d. with excess of univalent counterion minus c.d. in distilled water) can be fitted accurately to two Gaussian bands at 211 and 198 nm, assigned to carboxyl n → π^* and π → π^* transitions, respectively. The perturbations induced by Li⁺, K⁺, Rb⁺, Cs⁺, and NH₄⁺ show a clear family relationship, and are mainly in the π → π^* spectral region. With Na⁺, by contrast, c.d. change is largely confined to the n → π^* transition, and is similar to that previously reported for intermolecular (“egg-box”) binding of divalent cations, consistent with results of rheological studies which indicate Na⁺-induced association of poly-l-guluronate chain-sequences. These associations are further enhanced on freezing and thawing. This combined evidence is interpreted in terms of three modes of interaction between univalent cations and alginate chains in solution: (a) ion-pair formation with carboxyl groups of mannuronate and isolated guluronate residues; (b) specific site-binding to contiguous guluronate residues; and (c) co-operative “egg-box” binding, particularly of Na⁺, between poly-l-guluronate chain-sequences.     

Interactions of a 5-nitroxide-labeled poly(uridylic acid) with poly(adenylic acid)

The physicochemical properties of a high-molecular-weight spin-labeled nucleic acid, (RUGT,U)_n, synthesized by enzymatic copolymerization, were evaluated by uv and ESR spectroscopy. It was shown earlier that spin labeling of nucleic acids by chemical modification to an extent which gives a nitroxide-to-nucleotide ratio greater than 0.002 can cause noticeable lattice perturbations (A. M. Bobst, A. Hakam, P. W. Langemeier, and S. Kouidou (1979), Arch. Biochem. Biophys. 187, 339–345). The presence of RUGT, a 5-nitroxide-labeled uridine residue, in a (U)_n lattice at a $\text{RUGT}\text{U}$ ratio of 0.01 is shown here not to affect the complexation with (A)_n, since the uv melting temperature (T₀^OD) of the 2 → 1 transition and the hypochromicity changes were the same for (RUGT,U)_n· (A)_n and (U)_n·(A)_n. ESR measurements indicated that the nitroxide radical reflects the transition accurately within the error limit, although a slight destabilization of the spinlabeled segment could not be excluded. Computer simulations showed conclusively that the spin melting temperature (T_m^sp) corresponds to the temperature at which half of the spin-labeled segments are no longer complexed, for the ESR spectrum at T_m^spcan be simulated with equal contributions from the line shapes of ESR spectra taken before and after the transition. Arrhenius plots obtained by using two different approaches for computing correlation times were qualitatively the same. Computer analysis also revealed that the formation of a (RUGT,U)_n·(A)_n complex can be described by a two-state model, in contrast to results obtained with chemically spin-labeled (U)_n. Thus, using (RUGT,U)_n over chemically spin-labeled (U)_n can offer distinct advantages.     

Bioautography of cell attachment proteins.

A simple method, termed bioautography, is described which permits the visualization of bands of biologically active collagen-dependent cell attachment protein (c-CAP) after gel electrophoresis. The principle of bioautography depends on the “staining” of electrophoretically separated c-CAP's by live mammalian cells. Bioautography demonstrates (a) two bands of cell attachment protein (c-CAP) in serum; and (b) electrophoretic mobility differences between c-CAP's derived from the sera of various mammalian species.     

Association of leucogenenol,a thymothyroid hormone,with carrier proteins in the thymus

Leucogenenol a heterocyclic enolic thymothyroid hormone (MW 383) whose concentration in the serum regulates the rate at which already committed cells of the bone marrow develop into functional cells, was found to be associated in the thymus with a carrier protein. The carrier protein for leucogenenol is not precipitated by heating to 80° but following this treatment leucogenenol is precipitated in association with proteins precipitated by acetone and then by saturated ammonium sulfate. On chromatography on Sephacryl G-200 it was found that leucogenenol was associated with proteins of MW approximately 38,000. Leucogenenol is not eluted from the chromatographic column if it is not associated with its carrier proteins. It is suggested that other hormones such as those associated with the reproductive cycle or compounds that result from tissue damage induce the liberation of leucogenenol from its carrier protein in the thymus to the circulation where it is associated as previously described, with a protein of approximately MW 300,000.     

Turbidity changes of lipid vesicles near the phase transition temperature as an indication of fusion

Sonicated liposomes of dipalmitoyl phosphatidylcholine show sharp turbidity changes on heating at two distinct temperatures. A decrease in turbidity at the lower temperature (approx. 37°C) is thought to be associated with the phase transition of small vesicles and a decrease at about 44°C with larger vesicles or multilayer. An increase of turbidity between 38 and 43°C is attributed to the fusion of small vesicles. The turbidity changes were studied under various modes of vesicle preparation to confirm the interpretation of the turbidity data. Alternate interpretations are discussed.     

Microfingerprints of proteins through labeling acetylation of their proteolytic peptides

Microgram amounts of proteins applied to polyacrylamide gel electrophoresis were subjected to a fingerprinting procedure using a combined proteolysis-acetylation method with the aid of ¹⁴C-labeled acetic anhydride of high specific activity. After staining, gel slices were partially dried and were resoaked in a solution of a protease. After elution and acetylation, the resulting peptides were resolved in fingerprints on cellulose thin-layer chromatography plates and subjected to autoradiography with or without sensitization. Yields, completeness of fingerprinting, and possible artefacts were investigated.     

A fluorescence study of A23187 interaction with phospholipid vesicles

The fluorescence of the ionophore A23187 has been monitored in suspensions of egg yolk phosphatidylcholine (EYPC) and dipalmitoyl phosphatidylcholine (DPPC) vesicles. Both the protonated form of A23187 and the Ca²⁺ complex exhibit fluorescence enhancement when extracted into a hydrophobic environment. Measurements of fluorescence intensity versus lipid concentration were thus used to establish lower limits to the lipid/ water partition coefficients. Values obtained in this way were ? 50 ml water/mg phosphatidylcholine. Quenching of A23187 fluorescence by the spin labels 5NMS (methyl ester of 5-nitroxyl stearate), 12NMS, 16NMS, and TEMPO stearamide in EYPC and DPPC vesicles was also investigated. In EYPC all the labels yielded fairly linear Stern-Volmer plots, with TEMPO stearamide quenching about half as strong as the other probes. Quenching in DPPC was generally much stronger than in EYPC, but 12 NMS and 16NMS gave hyperbolic Stern-Volmer plots, apparently due to clustering of the labels. In all the cases the protonated form of A23187 was quenched approximately twice as efficiently as the Ca²⁺ complex, possibly due to a longer fluorescence lifetime for the former. Calculations based on measured spectral properties were performed which indicate that the Förster transfer mechanism extends the nitroxides' quenching range to ～- 10 Å.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

Measurement of nanogram quantities of protein by hydrolysis followed by reaction with orthophthalaldehyde or determination of glutamate.

Two methods are described for determining protein by measurement of amino acids released by acid or base hydrolysis. One achieves sufficient sensitivity to measure as little as 3 ng of protein by reaction of the amino acids with orthophthalaldehyde and mercaptoethanol in a small volume, followed by dilution in 0.5 n NaOH for fluorometric measurement of the product. The other method is somewhat more sensitive. Released l-glutamate is determined enzymatically by reaction with glutamic dehydrogenase (EC 1.4.1.2) and NAD⁺; the NADH produced is amplified by enzymatic cycling. To avoid the use of sealed tubes and minimize dangers of contamination, hydrolysis is carried out in small droplets in “oil wells.”     

Regulation of synthesis of the larval serum proteins of Drosophila melanogaster

We have determined the relative amounts of subunits of larval serum proteins (LSPs) 1 and 2 during larval development in Drosophila melanogaster. These results indicate that synthesis of polypeptide subunits of LSP-1 and LSP-2 is coordinate: the proteins are first detected at the same time; they accumulate in a coordinate fashion; their RNAs are first detected at the same time; the RNAs also accumulate in similar relative amounts. Analyses of fat body polypeptides and fat body RNA indicate that synthesis of LSP-1 declines at a time when there are still substantial quantities of LSP-1 RNA in the cytoplasm. Cessation of LSP-1 subunit synthesis occurs before cessation of LSP-2 synthesis, indicating that at late times the genes (or mRNAs) for these two proteins are subject to different "switch-off" controls.     

Phosphorylation of the myosin light chains and satellite proteins in detergent-skinned arterial smooth muscle

Isoelectric focusing of extracts prepared from detergent-skinned porcine carotid artery showed that contraction was associated with phosphorylation of the regulatory myosin light chains and two additional proteins of the same apparent molecular weight (20,000). These two proteins, previously described as satellites, did not appear to be artifactually derived from the phosphorylated light chains during electrophoresis. That is, each of the phosphorylated proteins migrated as separate and distinct proteins when subjected to a second cycle of isoelectric focusing. Moreover, relaxation of skinned fibers was associated with dephosphorylation of the light chains and both satellites. These findings suggest that the satellites may represent varients of the light chains per se, or another regulatory protein which is reversibly phosphorylated and dephosphorylated during contraction and relaxation of vascular smooth muscle.     

Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [³⁵S]methionine in vivo or in organ culure in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several α-tubulin subunits and one β-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)⁺ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte.     

Control of yeast cell type by the mating type locus: I. Identification and control of expression of the a-specific gene BAR1

The MATα allele of the yeast mating type locus confers the α mating phenotype and contains two complementation groups, MATα1 and MATα2. The α1–α2 hypothesis proposes that MATα1 is a positive regulator of α-specific genes and that MATα2 is a negative regulator of a-specific genes. According to this hypothesis, matα2 mutants, which are defective in mating and in production of extracellular α-factor, express both a-specific functions (because they lack MATα2 product) and α-specific functions (because they contain MATα1 product). Failure to produce extracellular α-factor results from antagonism between these functions; in particular, because α-factor (an α-specific function) is degraded by an a-specific function. If this view is correct, matα2 mutants should acquire the ability to produce α-factor if they also carry a defect in the gene(s) responsible for α-factor degradation. We have isolated a derivative of a matα2 mutant that produces α-factor and have characterized the suppressor mutation in this strain. (1) This strain carries a mutation (bar1-1) tightly linked to HIS6 (on chromosome IX) that allows matα2 mutants to produce α-factor. (2) It does not allow matα1 mutants to produce α-factor. (3) Haploids of the a mating type bearing the bar1-1 mutation still mate, but are unable to act as a barrier to the diffusion of α-factor. MATa bar1-1 cells display increased sensitivity to α-factor. (4) A mutation (sst1?2) that causes increased sensitivity to α-factor is allelic to bar1-1 and also allows α-factor synthesis by matα2 mutants. The ability of matα2 bar1 double mutants to produce extracellular α-factor indicates that matα2 mutants do produce α-factor but that it is degraded by the Barrier function. These results suggest that BAR1 is normally expressed only in a cells, and is negatively regulated in α cells by the MATα2 product.     

Identification of external membrane proteins of the T lymphoblast cell line CCRF-CEM

The 70 membrane proteins of the T lymphoblast cell line CCRF-CEM were characterized by

1. 1. [³⁵S]methionine internal radiolabeling;

2. 2. [¹²⁵I]iodine labeling by a lactoperoxidase-mediated method;

3. 3. [³H]fucose internal labeling;

4. 4. binding to a lentil lectin adsorbant column;

5. 5. susceptibility to digestion with limited amounts of papain.

Of the three methods of radiolabeling membrane proteins, [³⁵S]methionine best displayed all proteins although some individual proteins were heavily iodinated or fucosylated. Thirty proteins were externally exposed as defined by susceptibility to lactoperoxidase-mediated radio-iodination and to digestion with minute amounts of papain. Thirtyfive proteins were bound to a lentil lectin absorbant column. p44 (HLA-A and -B antigens) were iodinated, fucosylated, susceptible to papain digestion and bound to the lectin column. β₂-Microglobulin was iodinated and bound to the lectin column. The identifications and functions of other membrane proteins were not known. In general, proteins of high molecular weight (100 000 to 250 000 D) were more heavily radio-iodinated and fucosylated than were proteins of lower molecular weights. p95 was the most heavily fucosylated protein, p110, which had been identified only on T lymphoblasts, was fucosylated and was iodinated. p65, which was found only on the T lymphoblast line CCRF-CEM and could represent a lymphocyte subpopulation-specific molecule, was iodinated and fucosylated. p15 and p18 were equally and densely labeled with [³⁵S]methionine but only p18 was fucosylated and it was heavily radio-iodinated. These experiments help to define the external membrane proteins of a T lymphoblast cell line in part for the selection of proteins for isolation in order to raise antisera for immunodiagnostic and functional studies.     

Characterization of Ehrlich ascites tumor cell messenger RNA specifying ribosomal proteins by translation in vitro

We have examined the messenger RNA which codes for the ribosomal proteins in Ehrlich ascites tumor cells. Poly(A)-containing mRNA was isolated from polysomes and fractionated into 11 size classes whose average molecular weights were between 1.8 × 10⁵ and 24 × 10⁵. These mRNAs were used to direct protein synthesis in a fractionated translational system that was derived completely from Ehrlich ascites tumor cells. More than 90% of the ribosomal proteins which we could identify were coded for by mRNAs averaging in size between Mr = 180 × 10³ and 320 × 10³. The small size of these mRNAs indicates that the cytoplasmic mRNAs which specify the ribosomal proteins are monocistronic. We could detect the synthesis of 36 of 48 ribosomal reference proteins as well as 20 additional polypeptides which had characteristics similar to ribosomal protein. The ribosomal proteins were identified on the basis of their positive charge, small size, electrophoretic properties on two-dimensional polyacrylamide gels and chromatographic characteristics on carboxymethyl-cellulose.     

Synthesis and quantitation of glucitollysine, a glycosylated amino acid elevated in proteins from diabetics

A conceptually simple method of vertical gel electrophoresis is presented. It involves the use of pliable plastic envelopes to house individual gels and their constituent buffer reservoirs and electrical systems completely submersed in coolant separate from other gels. In conjunction with a common cooling tank this envelope technique provides unusual versatility for running a variety of different type gels simultaneously.