High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrane and of the whole platelet components established in this system.     

Tryptic peptide map analysis of the major human blood platelet membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point ($\text{p}\text{I}$), apparent molecular weight ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}$), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins (GP) Ia, Ib, IIa, IIb, GP_132–135^4–4.5 IIIa, IIIb and IIIc were all different. Glycoproteins with the same $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ but different $\text{p}\text{I}$ were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins.     

Analysis of Staphylococcus aureus cytoplasmic membrane proteins by isoelectric focusing

Cytoplasmic membranes were isolated from late-exponential phase Staphylococcus aureus 6538 P and the membrane proteins examined under non-denaturing conditions by thin-layer isoelectric focusing (TLIEF) in a pH 3.5–9.5 gradient. Isolated membrane preparations retained protein integrity as judged by the demostration of membrane bound adenosine triphosphatase (ATPase) activity in addition to four solubilzed membrane enzyme markers. Membranes were effectively solubilized with 2.5% Triton X-100 (final concentration). Examination of Triton X-100 solubilized membrane preparations established the presence of 22 membrane proteins with isoelectric points between 3.7 and 6.0. The focused proteins displayed the following enzymatic activities and isoelectric points by zymogram methods: ATPase (EC 3.6.1.3), 4.20; malate dehydrogenase (EC 1.1.1.37), 3.90; lactate dehydrogenase (EC 1.1.1.27), 3.85; two membrane proteins exhibited multiple bands upon enzymatic staining: NADH dehydrogenase (EC 1.6.99.3), 4.25, 4.35; succinate dehydrogenase (EC 1.3.99.1), 4.85, 5.10, 5.35.     

A new purification procedure for Clostridium difficile enterotoxin

$\text{Clostridium difficile}$ produces two toxins, an enterotoxin and a cytotoxin. The enterotoxin was purified using fast methods (tangential flow filtration, fast protein liquid chromatography). The purified enterotoxin is composed of two subunits (A₁ = 41,500, A₂ = 16,000) and its pI is 3.5.     

Presence of glycerol and fatty acids in the C-terminal end of a variant surface glycoprotein from Trypanosoma equiperdum

The composition of the C-terminal end of a variant surface glycoprotein from $\text{Trypanosoma equiperdum}$ (BoTat-1 VSG) has been examined. It has been reported for two $\text{Trypanosoma brucei}$ VSGs (Holder, A.A., Biochem. J. (1983), $\text{209}$, 261–262) that ethanolamine was involved in binding the C-terminal amino acid to an oligosaccharide side chain. Tryptic glycopeptides were prepared from BoTat-1 VSG and analyzed. One of them was found to contain ethanolamine and consequently was assumed to be C-terminal. It was shown that the glycopeptide also included phosphate, glycerol and fatty acids. The fatty acid composition was representative of that of glycerolipids. All the results suggest that the end of the molecule is a core of phosphatidylethanolamine.     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.     

Decreased in vivo protein and phospholipid methylation after in vivo elevation of brain S-adenosyl-homocysteine

The administration of adenosine together with homocysteine resulted in a dose-related elevation of cerebral S-adenosyl-L-homocysteine without concomitant perturbation of S-adenosyl-L-methionine levels. The adenosine + homocysteine treatment also decreased the incorporation of labile and stable methyl groups into brain proteins. Brain [³H]-phosphatidyl N,N-dimethylethanolamine and [³H]-phosphatidylcholine were also significantly decreased while [³H]-phosphatidyl-N-monomethylethanolamine remained unchanged. The data indicate that elevated brain S-adenosylhomocysteine can markedly and selectively inhibit the $\text{in vivo}$ methylation of brain proteins and phospholipids.     

Electrokinetic properties of (Na+, K+)-ATPase vesicles as studied by laser doppler spectroscopy

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (Na⁺,⁺)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a $\text{p}\text{K = 3.9}$; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na⁺ and K⁺; (3) Mg²⁺ binds with an association constant of $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^2\text{M}{}^{\mathrm{?1}}$ while ATP binds with an apparent $\text{K}{}_{\mathrm{<mtext>a</mtext>}}\text{= 1.1 · 10}{}^4\text{M}{}^{\mathrm{?2}}$ for 1 mM Nacl, 0.2 mM KCI, 0.1 mM MgCl₂, 0.1 mM Tris-HCI (pH 7.3). The binding is weaker at higher Mg²⁺ concentrations. There is no ATP binding in the absence of Mg²⁺. In addition, the average vesicle size derived from the linewidth of the quasi-elastic light scattering spectrum is $\text{203.7 ± 15.2}\text{nm}$. In the presence of ATP a reduction in size is observed.     

Targeting of the antiviral protein from Phytolaccaamericana with an antibody

The antiviral protein (PAP) of $\text{Phytolacca}\text{americana}$ was conjugated with the Fab' fragment of IgG from a rabbit antiserum against murine leukemia L1210 cells via a disulfide bond employing $\text{N}$-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as the coupling agent. The conjugate showed a potent $\text{in}\text{vitro}$ cytotoxicity against L1210 cells which was competitively blocked by F(ab′)₂ directed against L1210 cells. PAP itself did not exhibit the cytotoxicity at the concentration corresponding to the PAP content in the conjugate concurrently tested.     

Block of sodium conductance by n-octanol in crayfish giant axons

The block of the Na⁺ current by $\text{n-}\text{octanol}$ was studied in crayfish giant axons under axial wire voltage-clamp conditions. Standard kinetic analysis of the Na⁺ currents was undertaken to test the hypothesis that the $\text{n-}\text{octanol-induced}$ block of the Na⁺ current could be accounted for on the basis of changes in the voltage dependence of the kinetic parameters. Alterations in the membrane dipolar potential arising from rearrangement of membrane lipids would be the anticipated source of changes in the voltage dependence. Although some changes in voltage dependence did evolve with the block by $\text{n-}\text{octanol}$, the changes were not of sufficient magnitude to account for the block. In conclusion, although higher concentrations of $\text{n-}\text{octanol}$ produced shifts along the voltage axis of the kinetic parameters, direct blocking action of $\text{n-}\text{octanol}$ on the channel appears to be the most important mechanism of the block.     

Allosteric regulation of serine hydroxymethyltransferase from mung bean (Phaseolusaureus)

Serine hydroxymethyltransferase, the first enzyme in the pathway for the interconversion of one carbon compounds was purified from mung bean seedlings by ammonium sulfate fractionation, DEAE-Sephadex, Blue Sepharose CL-6B affinity chromatography and gel filteration on Sephacryl S-200. The specific activity of the enzyme, 0.73 (u mol HCHO formed/min/mg protein) was 10⁴ times larger than the highest value reported hitherto. Saturation of tetrahydrofolate was sigmoid, whereas with serine was hyperbolic, with n_H values of 1.9 and 1.0 respectively. Reduced nicotinamide adenine dinucleotide, lysine and methionine decreased, whereas nicotinamide adenine dinucleotide, adenosine 5′-monophosphate and adenosine 5′-triphosphate increased the sigmoidicity. These results suggest that serine hydroxymethyltransferase from mung bean is a regulatory enzyme.     

Initial membrane reaction in peptidoglycan synthesis. Interaction of lipid with phospho-N-acetylmuramylpentapeptide translocase

The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by $\text{phospho}\text{-N-}\text{acetylmuramyl}$ (MurNAc)-pentapeptide translocase (UDP-MurNAc-Ala-γ dGlu-Lys-dAla-dAla undecaprenyl phosphate phospho-MurN Acpentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurN Ac-pentapeptide. Two distinct discontinuities are observed in the slopes of the Arrhenius plots of the exchange and transfer activities at 22 and 30°C for the enzyme from Staphylococcus aureus Copenhagen. Anisotropy measurements of perylene fluorescence and electron spin resonance measurements of $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}$ derivatives of 12-and 16-ketostearic acid intercalated into membranes from this organism define the lower ($\text{T}{}_1\text{= 16–22°}\text{C}$) and upper ($\text{T}{}_{\mathrm{<mtext>h</mtext>}}\text{= 30°}\text{C}$) boundaries of a phase transition. These values correlate with the discontinuities observed for the activity measurements. Thus, it is proposed that the physical state of the lipid micro-environment of phospho-MurN Ac-pentapeptide translocase has a significant effect on the catalytic activity of this enzyme.     

Studies on the fragmentation of erythrocyte ghost membrane with p-chloromercuribenzoate in the micromolar range

The effects of nonsaturating amounts (5–60 nmol/mg membrane protein) of $\text{p-}\text{chloromercuribenzoate}$ on the stability of unsealed erythrocyte ghosts were studied by turbidimetric measurements and direct observation by phase contrast microscopy. The organic mercurial provokes drastic disorganization of the membrane involving vesicle formation by inter- and externalization of the bilayer. These effects are not associated with a release in solution of membrane proteins which was shown in previous studies to occur at higher $\text{p-}\text{chloromercuribenzoate}$ concentration. Attempts have been made to identify the proteins involved in this phenomenon by the use of nonsaturating amounts of radioactively-labelled $\text{p-}\text{chloromercuribenzoate}$. Actin and band 3 protein which are the first to be labelled, represent plausible candidates as sensitive targets for the disrupting organic mercurial. Stroma obtained from spherocytes did not show significant differences with normocytes in their stability with regard to $\text{p-}\text{chloromercuribenzoate}$. Other reagents including $\text{N-}\text{ethylmaleimide}$, diamide and DNAase I were also studied. The results suggest strongly that the integrity of the sulfhydryl groups of actin, as well as those of band 3 protein, is essential for the stability of the erythrocyte membrane.     

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 Å subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 Å CF₁ are attached by stalks to an aggregate of CF₀.The CF₁-CF₀ complex was incorporated into phospholipid vesicles; freezefracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; these averaged 96 Å in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF₀, was isolated from membranes by butanol extraction and was incorporated rated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 Å in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF₀ and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.     

Variant specific surface antigens from Trypanosoma equiperdum: Chemical and physical studies

Nine variant specific surface antigens were purified from clones of $\text{Trypanosoma equiperdum}$ and characterized by amino acid analysis, isoelectric focusing and circular dichroism. The molecules showed extensive differences in their isoelectric points, and by comparison with the corresponding amino acid compositions, this variation seemed to be due to different amide contents. Circular dichroism data allowed one to divide the molecules into 4 groups according to their respective percentages inα-helical and β-sheet structure.     

Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica

Bromoacetyl[methyl-³H]choline is a highly specific label for the reduced acetylcholine binding site on the acetylcholine receptor from $\text{Torpedo californica}$. Only one of two binding sites per receptor monomer is susceptible to labeling. The labeled site is on the α chain of the receptor.     

The metabolism of formycin B in Leishmaniadonovani

Formycin B, a pyrazolo(4,3-d)pyrimidine C-nucleoside, inhibited the growth of $\text{Leishmania}\text{donovani}$ promastigotes in culture with an ED₉₀ of 0.2 μg/ml. Promastigotes incubated for 24 hrs with Formycin B at 10 μg/ml were found to convert it to the ribonucleotide, formycin B 5′-monophosphate. The parasites were also capable of aminating formycin B 5′-monophosphate as evidenced by the appearance of formycin A di- and triphosphate. The RNA contained the formycin A moiety in 3′,5′-polynucleotide linkage. Succino-AMP synthetase from these parasites was able to use formycin B 5′-monophosphate as an alternate-substrate with a K'm of 26 μM and a V'm of about 1% the V'm IMP. Formycin B 5′-monophosphate was also a substrate for mammalian succino-AMP synthetase with a V_m' of 40% the V_m' of IMP.     

Incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into proteins and lipid intermediates in microsomal and golgi membranes from rat liver

Rough and smooth microsomes and Golgi membranes incorporate $\text{N-}\text{acetylglucosamine}$ from $\text{UDP}\text{-N-}\text{acetylglucosamine}$ into endogenous protein acceptors. A lipid intermediate of the dolichol phosphate type participates in this transfer reaction in the case of both microsomal subfractions, but the nature of lipid glycosylation is different in these two fractions. Glucosamine transfer in Golgi membranes does not appear to involve a lipid intermediate. In contrast to the results obtained under in vivo conditions, no glucosamine label is recovered in nascent ribosomal proteins or on luminal secretory proteins after incubation in vitro. Proteolysis of intact vesicles of the subfractions removes glycosylated dolichol phosphate and protein acceptors to various extents and interferes with transferase activities. This finding suggests the possibility that glycosylation at the cytoplasmic side of the membrane of the endoplasmic reticulum may involve a system separate from that acting at the luminal side of the same membrane.     

The binding of cytochrome b5 to plasma membranes of rat liver. Its implication for membrane specificity and biogenesis

The in vitro incorporation of cytochrome $\text{b}{}_5$ into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome $\text{b}{}_5$ than did microsomal preparations; 60% of this cytochrome $\text{b}{}_5$ could not be reduced by the NADH-cytochrome $\text{b}{}_5$ reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome $\text{b}{}_5$ clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome $\text{b}{}_5$. These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell.     

Sites of in vivo extraction and interconversion of estrone and estradiol in the dog

The interconversion and extraction of estrone and estradiol-17β across and within different tissues or areas have been studied in the dog by the constant infusion technique. The results were calculated using the ³H/¹⁴C ratios and radioactive concentrations of estrone and estradiol obtained from afferent and efferent blood and tissues at equilibrium. From these results it is concluded that: (1) there is no significant difference between metabolic clearance rates of estrone and estradiol, (2) blood transfer constants indicate a higher conversion of estradiol to estrone than of estrone to estradiol, (3) the transtissue interconversion favors the formation of estrone while the intratissue interconversion favors the formation of estradiol, (4) no interconversion of the two estrogens is observed in adipose tissue, (5) the extraction of estradiol entering a tissue was lower than the extraction of estradiol formed in these tissues, (6) calculation of the tissue metabolic clearance rates show that 63% and 61% of the total metabolism of estrone and estradiol, respectively, occurs in the splanchnic bed, and (7) the contribution of each tissue to the total interconversion of estrone and estradiol show that more than 90% of this interconversion occurs extrahepatically.