A qualitative and quantitative comparison of the fat in human, feline and canine kidneys.

While xanthogranulomatous pyelonephritis in humans tends to be rich in birefringent fat, this variety cannot be demonstrated microscopically in the feline morphological counterpart of the condition, which is, however, rich in readily stainable fat. Since normal feline kidney is rich in such lipid, a qualitative and quantitative comparison of the lipid in human, feline and canine kidney was carried out in an attempt to throw further light on the possible origin of the birefringent fat in the human disease. No significant difference could be found in the amount of cholesterol in the 3 species. Despite its visual prominence in feline kidneys, human kidney was richer in neutral fat and the percentage of total lipid formed by cholesterol was greater in humans than in the other species. The results suggested that cases of xanthogranulomatous pyelonephritis may occur in those kidneys unusually rich in cholesterol.


Tissue levels of (3(-14)C) coumarin in the rat: distribution and excretion.

The benzo-pyrones (including coumarin) are a very effective therapy for mild thermal oedema and cases of acute and chronic lymphoedema. In this preliminary report the distribution of a single injected dose of coumarin was followed in normal tissues of rats for 100 hours. Comparisons are to be made later with drug levels in thermally injured and lymphoedematous tissues. The resluts show 7-4% of the injected dose to remain in the tissues after 100 h. During this time 30-9% was excreted in the faeces and approximately 47% excreted in the urine. At any given time most of the dose was present in the gut, muscular tissues, skin and liver. For the gut tissues this was 33%, for the muscular tissues 28%, for the skin 18% and for the liver 16%. The highest concentrations per gram of tissue were however in the kidney and liver, representing the two organs of metabolism and excretion of the coumarin.


Skin stretching and epidermopoiesis.

In order to determine whether the heightened epidermopoiesis seen over dermal expansile lesions is due to stretch, attempts were made to induce similar degrees of skin stretching by implanting Silastic materials in the dermis of guinea-pigs. Silastic elastomer produced mild inflammation and thus, this model could not be used to evaluate the effects of stretch alone. Silastic testicular implants caused no inflammation and were found to initially stimulate a rise in thymidine autoradiographic labelling index, but no increase in epidermal thickness during the experiment. It is suggested that there is an increase in the germinative pool size caused by stretch but that the epidermal hyperplasia seen over dermal lesions is due to some other process.


The role of catecholamine in the effects of trauma on thermoregulation, studied in rats treated with 6-hydroxydopamine.

Rats in which central noradrenergic terminals have been damaged by injecting 6-hydroxydopamine (6-OHDA) into the cerebral ventricular system have been used to study the possible role of noradrenaline (NA) in the effect of trauma on thermoregulation. In non-injured rats the 6-OHDA treatment did not affect the threshold environmental temperature (Ta) for the onset of shivering or the slope of the regression lines relating intensity of shivering to Ta. It raised the core temperature (Tc) threshold for the opening of the A-V anastomoses in the tail. Whereas in normal rats ischaemic limb injury depresses the threshold Ta for the onset of shivering without altering the slope of the regression line, in 6-OHDA-treated rats no depression of the threshold Ta was observed during limb ischaemia but the slope of the regression line was lower. The Tc threshold for the opening of the A-V anastomoses in the tail was the same as in the 6-OHDA-treated controls. The injection of NA into a lateral cerebral ventricle of a normal rat in a 5 degrees environment inhibited shivering and lowered Tc. The 6-OHDA treatment did not induce hypersensitivity to the injection of NA by this route. It is thought that NA is concerned in the central inhibition of shivering during limb ischaemia.


Localization of the brain regions concerned in the inhibition of shivering by trauma.

The effect on the inhibition of shivering by limb ischaemia of small 6-hydroxydopamine (6-OHDA) lesions in the hypothalamus has been studied in rats. The injection of 6-OHDA into the posterior part of the hypothalamus in the neighbourhood of the N. dorsomedialis or into the ventral ascending catecholaminergic bundle caudal to that nucleus prevented the depression of the ambient temperature threshold for the onset of shivering which usually occurs during hind-limb ischaemia and lowered the slope of the regression line relating the intensity of shivering to ambient temperature. In rats not treated with 6-OHDA this slope is unaffected by limb ischaemia. These lesions reproduced the changes seen when 6-OHDA was injected into the lateral cerebral ventricle. Lesions in other parts of the hypothalamus were without effect. It is concluded that the inhibitory catecholaminergic synapses are situated in the posterior part of the hypothalamus and that the impulses reach them from nerve cells in the hind-brain via the ventral bundle. The lesions produced in these experiments had little effect on thermoregulation in non-injured rats.


Glomerular lesions in argyric NZB/NZW mice.

Glomerular basement membranes showed strong silver labelling on electron microscopy in NZB/NZW mice fed 12 mmol AgNO3 for 6 weeks at ages from 1-7 months. Although some silver granules were seen within mesangial immune deposits following their concurrent deposition, silver was usually deposited in electron-lucent areas around pre-existing deposits, or formed a boundary to an expanded mesangium when immune deposits subsequently developed. Immune deposits in capillary loops occurred on the outer or inner aspect of the zone of silver labelling.


An electron microscopical study of chronic ulcers of the colon in rats.

Chronic colonic ulcers in the rat were produced by the administration of a hydrogen peroxide enema. Lesions of up to 10 months' duration were obtained by this method and a selection was examined ultrastructurally. It was hoped that the study would reveal causes for the chronicity of the lesions. From previous work it appeared that there was no failure of epithelial migration from the edge of the wound. The majority of cells at the ulcer margins demonstrated features of regeneration similar to those seen in other tissues, and that there were no specific changes concomitant with chronic ulceration. The majority of epithelial cells at the ulcer edge showed features of differentiation which indicated that there was no obvious fault in this process so essential to the basic requirements of tissue repair. It is possible that chronicity is produced by a failure of the epithelio-mesenchymal interaction mechanisms which, potentiated by an ischaemia resulting from submucosal vascular damage, results in the consequential death of migrating epithelial cells. This hypothesis is supported by the appearance of a squamous metaplasia in the healing lesions, a process which is commonly attributed to a chronic inflammatory response which may be induced by a relative vascular insufficiency.


Response of pulmonary macrophages to lead.

The intratracheal instillation of moderate doses of PbO to Long-Evans strain rats resulted in a significant increase in the number of recoverable pulmonary alveolar macrophages. The viability of recovered cells was remarkably constant throughout the experimental period of 40 days. The results obtained indicate a low order of toxicity of PbO to alveolar macrophages and show that the mucociliary escalator is a significant route of excretion of PbO from the respiratory tract. The in vitro survival of macrophages from PbO-treated rats was significantly reduced. Survival of cells from both treated and control animals was somewhat enhanced by the addition of formalinized lymphocytes to the culture medium. Morphological changes and evidence of phagocytosis are discussed.


Family distribution of concomitant squint in Greece.

From a study of a large number of brothers and sisters suffering from concomitant squint, the following data were obtained: (1) 42-9% of the patients showed congenital strabismus. (2) In 96-5% of the sibs strabismus was of the same type. (3) The presence of a significant refractive error occurred in 82-8% of the patients. (4) In all twins strabismus appeared at the same age in both twins, and the squint and the refractive error were of the same type. (5) The distance of the patients' homes from Athens had no effect on either (a) the time intervening between the onset of squint and the first visit to the eye specialist, or (b) the effectiveness of treatment.


Stereopsis and aniseikonia in uniocular aphakia.

Uniocular aphakia in the adult when corrected with a contact lens is in most patients no obstacle to binocular function. The long-term result is a balance between binocular function obtained and the comfort of the contact lens, and in this respect the advent of the soft lens, and especially the continuous-wear lens, appears to be most promising.


Hydrophilic lenses for "continuous" wear in aphakia: definitive fitting and the problems that occur.

Patients have been very satisfied with these lenses (78% wearing at the end of six months), not the least those with reduced function of the macula. The problems of infection have been discussed, and apart from improvement in lens materials patient selection is of paramount importance. The problem of deposits is presented, but theri elimination will depend on more research into their nature and development, improvement of the material's resistance to this form of attack, and improved lens design to overcome corneal and tear flow problems.


The problem of screening children for visual defects.

All the cases of squint and amblyopia referred to both hospital and school clinics in one district during one calendar year have been reviewed in order to clarify when, where, and how these cases first present to the ophthalmologist. The types of case at present seen in "hospital" and "school" clinics are quite different, and effective screening methods are unlikely to be developed if these two services continue to be regarded as separate entities. Exotropia is much less frequent than esotropia (only 15% of all cases of squint). The concept of a single peak number of first attendances of children with esotropia at age 3 to 4 years is not substantiated. There is a second peak at age 5 years, when children first go to school. In fact 49% first attended after their fifth birthday. The majority (69%) of cases of amblyopia presented after the age of 5 years. Three-quarters of these (46% of the total) have no clinically detectable squint. Neither they, nor those children who have a strabismic amblyopia, but not a cosmetically noticable squint, will at present be detected until they have a sight test at school. This is a measure of the need to re-examine our long-established methods of screening children for amblyopia. One screening test designed to identify both the "squinter" and the "straight-eyed amblyope" is required.


Fluorescein angiography of the hereditary choroidal dystrophies.

The hereditary choroidal dystrophies are divided into (1) geographic choroidal dystrophies (central areolar, peripapillary, generalised), (2) gyrate atrophy, and (3) choroideremia. Each of these disorders is discussed with regard to mode of inheritance, age of onset, symptoms, fundus appearance, and visual function testing. A typical case history of each disorder is presented together with fluorescein angiography, and the fluorescein angiographic findings are related to our present understanding of these diseases. Fluorescein angiography was found to be most helpful in diagnosing the early cases, by confirming the absence of the choriocapillaris, and in demonstrating either a local or general abnormality. The role of fluorescein angiography in understanding the aetiology of choroidan dystrophies is discussed.


Management of small elevated pigmented choroidal lesions.

The diagnosis of small choroidal melanomas may be difficult, and the natural history of these small tumours is poorly understood. We followed up 20 patients with the diagnosis of small choroidal melanomas for from 2 to 20 years. In 11 of these patients the melanoma was observed to grow. Nine of these patients have undergone enucleation. Eight of the eyes contained spindle B melanomas and one contained a melanoma of mixed cell type. None of the 20 patients has developed detectable metastatic disease. From this series and others reported it appears that close serial examinations without therapeutic intervention is safe in patients with small melanomas until growth is observed, and that there is little evidence to suggest that photocoagulation or radiotherapy is of greater benefit in those patients than close serial examination.


Orbital cyst due to Histoplasma duboisii.

A cystic swelling in the orbit due to Histoplasma duboisii, clinically thought to be a dermoid cyst, is described.


Refraction as a basis for screening children for squint and amblyopia.

+2-00 to +2-75 dioptres of spherical hypermetropia in the more emmetropic of a pair of eyes is significantly associated with esotropia (P less than 0-001) and the presence of amblyopia (P less than 0-01). Anisometropia is not significantly associated with esotropia (P = 0-31) unless there is spherical hypermetropia of +2-00 dioptres or more in the more emmetropic eye (P less than 0-001). Hypermetropic anisometropia of +1-00 DS or +1-00 D.Cyl. is associated with the presence of amblyopia (P less than 0-001). In the absence of esotropia there is also a significant association between the amount of anisometropia and the initial depth of amblyopia (P less than 0-01). The additional presence of esotropia increases the depth of amblyopia further (P less than 0-05) but not the incidence of amblyopia (P greater than 0-30). The level of significance of the association of refractive errors with squint/amblyopia was itself significantly higher (P less than 0-01) than that between a family history of squint or "lazy eye" on the one hand and squint and/or amblyopia on the other hand. 72 +/- 3% of all cases of esotropia and/or amblyopia in this sample of children had a refractive error of +2-00 DS or more spherical hypermetropia in the more emmetropic eye, or +1-00 D. or more spherical or cylindrical anisometropia. Since there is a close association between the refraction and how, when, and whether a child presents with squint and/or amblyopia, it would seem reasonable to reconsider refraction as a basis for screening young children for visual defects.


Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to tetrameric and unmodified or derivatized dimeric concanavalin A: equilibrium studies.

The binding of 4-methylumbelliferyl alpha-D-mannopyranoside (MUM) and concanavalin A, composed of intact polypeptide chains, was studied by equilibrium dialysis, difference spectroscopy, and fluorescence titration (Dean, B.R., and Homer, R.B. (1973), Biochim. Biophys. Acta. 322, 141-144), measured either at a fixed wavelength or above 350 nm. Dimeric and tetrameric concanavalin A samples were used under conditions of apparently full metal saturation. The results are consistent with a single carbohydrate-specific site per protomer, without interaction between sites; no indication for additional unspecific binding could be obtained. The values of the association constant are independent of the method or of the saturation range used and 4-methylumbelliferyl alpha-D-mannopyranoside, bound at a fractional saturation of 0.91 can be totally displaced by methyl alpha-D-mannopyranoside. The thermodynamic binding parameters for acetylated or succinylated concanavalin A, composed of intact polypeptide chains, were obtained by titration of total MUM fluorescence in the temperature range 9-39 degrees C. For unmodified dimeric concanavalin A at 25.0 degrees C, the values are K = (3.36 +/- 0.04) 10(4) M-1 with delta H degrees = -8.3 +/- 0.1 kcal mol-1 and delta S degrees = 7.2 +/- 0.3 eu; for tetrameric concanavalin A, the affinity is increased by 25% and within experimental error the values of delta H degrees and delta S degrees are identical to those for the dimeric protein. Derivatized concanavalin A shows binding characteristics that are entirely comparable to those of the native protein.


Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to dimeric concanavalin A: fluorescence temperature-jump relaxation study.

The kinetics of saccharide binding to the dimer form of concanavalin A (con A) has been studied at pH 5.5 with the fluorescence temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside, a fluorescent carbohydrate derivative which is quenched upon binding to con A, was used as the ligand. Three relaxation effects were seen. The major relaxation (r = 20-400 ms) was investigated at four different temperatures. The behaviour of this relaxation as a function of reactant concentrations is consistent with a simple one-step bimolecular association reaction. These conclusions result from the analysis of both the relaxation times and amplitudes, and from the comparison of the kinetically determined equilibrium parameters (Kass = 3.5 x 10(4) M-1 at 18.5 degrees C, delta H degrees = -(6-7) kcal/mol) to those obtained from a parallel series of equilibrium experiments (Loontiens, F.G., Clegg R.M., and Jovin, T.M. (1977), Biochemistry 16, preceding paper in this issue). The association and dissociation rate constants are in the range of (6-15) x 10(4) M-1 s-1 and (1.5 - 5.6) s-1, respectively, within a temperature range of 13.5-28.1 degrees C. The activation energies for the forward and reverse reactions are approximately 10 and approximately 15 kcal/mol, respectively. The two additional relaxations which are also present in the absence of saccharides result from changes in the protein fluorescence and are attributed to protein conformational changes which are not affected by the binding of saccharides. These effects were further studied using succinylated, acetylated, and demetallized con A. The faster relaxation (13 ms at 18.5 degrees C) was independent of the concentration of the protein and was not present in the derivatized con A samples. The two derivatized forms of con A show almost identical carbohydrate binding parameters as the underivatized protein. A limited series of stopped-flow experiments yielded results which were fully compatible with those from the relaxation measurements.


Isolation of type III collagen from human adult parenchymal lung tissue.

The isolation and characterization of type III collagen from adult human lung parenchyma are described. The identity of this molecule as type III collagen has been established on the basis of (a) demonstration of intramolecular disulfide cross-links in the helical portion of the molecule, (b) amino acid analysis characteristic for type III collagen, and (c) composition and size of isolated cyanogen bromide peptides alpha1(III)-CB3, alpha1(III)-CB5, and alpha1(III)-CB8. The molecular weight of lung alpha1(III) was determined as 93 000 by Agarose chromatography, but its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels was slower than that of type I alpha chains which also have a molecular weight of 93 000.


Bovine serum albumin and aqueous guanidine hydrochloride solutions. Preferential and absolute interactions and comparison with other systems.

The partial specific volume, upsilon20, of bovine serum albumin at 25 degrees C was found to be 0.728 +/- 0.001 ml/g in solutions of guanidine hydrochloride (GuHC1), 0.01 M dithioerythritol (DTE), independent of GuHC1 concentration (3-6 M). The volume decrease upon denaturation is about 400 ml/mol (upsilon20 in water at the same temperature was found to be 0.734). From the reduced density increments at constant chemical potential of diffusible solutes, The apparent volumes, phi, were found to increase from 0.693 ml/g at 3 M GuHC1 to about 0.725 ml/g at 7 M GuHC1. The phenomenological interaction parameter, xi3 (grams of GuHC1 "bound" per gram of protein), was found to decrease from about 0.2 at 3 M GuHC1 to about 0.07 at 6.4 M GuHC1. The phenomenological interaction parameter, xi1 (grams of water "bound" per gram of protein), is negative and become less negative with increase in GuHC1 concentration. The relation between xi3 and xi1 and physical binding and exclusion of low-molecular-weight components are discussed in terms of simple model consideration. It is concluded that over the range of GuHC1 concentrations studied about 0.2 g of water as well as 0.28 g of GuHC1 are bound per gram of protein. This corresponds on the average to 1.3 molecules of water and 0.35 molecule of GuHC1 per amino acid residue. Similar results were found by recalculating some previous results for aldolase. These results on proteins in GuHC1 solution are in marked contrast to the behavior of DNA at high concentrations of NaCl and CsCl, which is analyzed on the basis of earlier work.


A novel approach to water proton relaxation in paramagnetic ion-macromolecule complexes.

The use of the proton relaxation enhancement (PRE) technique in solutions of macromolecules and paramagnetic ions to obtain information on molecular motion and ion-hydration numbers can be hazardous with the possibility of large errors in the derived parameters. Here we describe a method based on solvent proton-deuteron comparative relaxation measurements at a limited number of different magnetic fields for obtaining, reasonably accurately, rapidly, and unambiguously, certain of the parameters normally obtained from a PRE study. In the non-immune rabbit immunoglobulin G(IgG)-Gd(iii) system, we are able to obtain values for the ion hydration number (q), the residence lifetime of water molecules in the hydration sphere of the ion (tauM) and a correlation time of 5 ns, which can be associated to a large part with a rotational correlation time (tauR) for the Fc portion (C-terminal half of heavy-chain dimer) of the IgG molecule. This shows that there is a rapid internal motion in the Fc region. The limitations and possible applications of the method are discussed.


Role of hydrophobic forces in membrane protein asymmetry.

M13 virus coat protein is an integral cytoplasmic membrane protein at all stages of viral infection. The pure virus coat protein can also be incorporated into synthetic lecithin vesicles near the lipid-phase transition temperature (Tm), spanning the bilayer with its N terminus exposed on the outside and its C-terminus inside (Wickner, W. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 1159-1163). The assembly of coat protein into vesicles in this asymmetric fashion has a sharp maximum near the phase-transition temperature of the lipid fatty acyl chains. At temperatures well below the Tm, coat protein assembles into lecithin vesicles with both termini exposed on the exterior vesicle surface. In contrast to this important role of lipid physical state, asymmetry of assembly is unaffected by parameters which govern polar interactions such as pH, ionic strength, lipid polar head group, or prior proteolytic removal of either the N or C terminus. It is proposed that the orientation of this membrane protein, and perhaps others, is determined by interactions of the hydrophobic portion of the protein with the lipid hydrocarbon core.


Kinetics of dithionite ion utilization and ATP hydrolysis for reactions catalyzed by the nitrogenase complex from Azotobacter vinelandii.

The kinetics of S2O42-utilization and ATP hydrolysis during the nitrogenase-catalyzed H2 evolution and acetylene and nitrogen-reducing reactions were studied using a polarographic technique to monitor-S2O42-concentration. Rate constants for both S2O42-utilization and ATP hydrolysis were determined as a function of temperature and corresponding activation energies determined. The activation energy for ATP hydrolysis differs from that for product formation or S2O42-utilization by 5 kcal/mol above 20 degrees C and by 25 kcal/mol below 20 degrees C. The rate law for S2O42-utilization was determined and describes the enzyme catalyzed rate over a 1000-fold variation in S2O42-concentration and at least a 100-fold change in ATP concentration. The rate law for S2O42-utilization under N2-reducing conditions at 25 degrees C is given by -d([S2O42-]/dt = (2.3 x 10(-3) ET[S2O42-]1/2-[ATP]2)/([ATP]2 + K1[ATP] + K2), where ET is total enzyme concentration in mg/ml and K1 and K2 are equilibrium constants for ATP binding to nitrogenase. The half-order dependence of the rate on S2O42-concentration in interpreted in terms of the equilibrium S2O42- = 2SO2-, in which SO2- is the actual electron donor to nitrogenase. A partial mechanism incorporating these results is presented.


Metabolism of histones in avian erythroid cells.

The synthesis and enzymatic modifications of histones by phosphorylation, acetylation, and methylation during erythroid cell maturation have been studied. All newly synthesized histones, H1, H5, H2a, h2b, h3, and H4 undergo phosphorylation; histones H2a, H2b, H3, and H4, are acetylated and histones H3 and H4 are methylated. This type of histone metabolism is common to all dividing cells and therefore may be related to the assembly of histones into chromatin subunits. In the nondividing reticulocytes, the synthesis of histone H5 continues, while all the other histones show negligible incorporation of [3H]amino acids. Furthermore, the reticulocytes show a unique pattern of enzymatic modification: phosphorylation of histone H2b, acetylation of histones H2a, H2b, H3, and H4, and methylation of histones H3 and H4. These "differentiation-linked" modifications are not dependent on histone synthesis, nor related to RNA synthesis, but may be related to the reorganization of chromatin in preparation for genomic inactivation.


Phosphorylation and dephosphorylation of histone (V (H5): controlled condensation of avian erythrocyte chromatin. Appendix: Phosphorylation and dephosphorylation of histone H5. II. Circular dichroic studies.

During avian erythropoiesis, the blast cells of the bone marrow mature into polychromatic erythrocytes (late stages knwon as reticulocytes) and then into mature red blood cells. When chickens are made anemic, the proportion of immature cells in the anemic bone marrow increases dramatically. The level of the lysine-rich histones. H1 and H5, has been found to be constant in the blood and bone marrow of normal and anemic chickens. This implies that H5 replaces H1 quantitatively. Urea-aluminum-lactate starch gel electrophoresis of H5 from these sources show that the degree of phosphorylation of H5 is proportional to the number of immature cells. About 70% of the H5 from the most immature bone marrow is phosphorylated, while 50% of the H5 from anemic blood is phosphorylated and H5 in normal blood is almost completely devoid of phosphate. When immature cells of the anemia bone marrow are incubated in the presence of inorganic 32P and [3H]lysine and [3H]arginine, extensive 32P incorporation is found in the phospho species. A minimum of nine phosphorylated components have been demonstrated by starch gel electrophoresis. The incorporation of 3H is time dependent. After 1.5 h of labeling, 3H is found in H5 containing 0, 1, 2, and 3 phosphates. tthe combined data suggest that newly synthesized H5 becomes progressively phosphorylated and that at the terminal stage of development, the phosphorylated H5 is completely dephosphorylated. These events may be important in controlling the timing of chromatin condensation.


The structure of chromatin: interaction of ethidium bromide with native and denatured chromatin.

The binding of ethidium bromide, as monitored by fluorescence enhancement, to chromatin prepared by nuclease digestion has been compared with the binding of the dye to sheared chromatin. The nuclease preparation (native chromatin) is characterized by a high affinity region of the Scatchard plot (r = 0-0.025, K1 = 1 X 10(6) M-1), a transition (r = 0.025-0.05), and a low affinity region (r = 0.05-0.12, K2 = 3 X 10(5) M-1). The final amount of ethidium bromide bound per base is 0.12 as compared with 0.20 for free DNA. Sheared chromatin has the two regions of high and low affinity (K1 = 2 X 10(6) M-1, K2 = 5 X 10(5) M-1) as originally shown by Angerer and Moudrianakis (1972), but the transition is much reduced or absent. Binding of the dye to native chromatin is independent of salt at concentrations ranging from 0.2 mM EDTA to 10 mM Tris-Cl, 10 mM NaCl, 0.2 mM EDTA, while sheared chromatin and DNA both bind ethidium bromide electrostatically as well as by intercalation at the low salt concentration, leading to extensive energy transfer. Thus the phosphate groups in native chromatin are unavailable to external cations even at very low salt. Polarization of fluorescence of ethidium bromide intercalated into native chromatin at low r is very high, indicating a highly rigid structure. As r approaches 0.02, there is a very rapid depolarization; at r = 0.03, the polarization is no greater than that of the dye intercalated into DNA. Depolarization is not due to energy transfer. The Scatchard plot derived for the bulk preparation of native chromatin is very similar to the one derived for the monomer nu body. These results indicate that the DNA in native chromatin is in a very rigid form, with its phosphate anions neutralized by structural components, not by free salt. Ethidium bromide intercalation appears partially to disrupt this structure, perhaps by unwinding, leading to slight changes in its properties.


Binding sites for calcium on tubulin.

Calcium ions can inhibit the in vitro assembly of microtubules and, therefore, may play a role in the regulation of microtubule formation in vivo. In order to test the validity of this hypothesis; the interaction between calcium and pruified brain microtubular protein has been investigated by standard binding assays. We have detected and characterized two classes of binding sites for calcium on tubulin, the major component of cytoplasmic microtubules. There is a single high-affinity site per tubulin molecule, characterized by a dissociation constant of 3.2 X 10(-6) M. That site is inhibited by magnesium (k1 = 5 X 10(-5) M) and potassium chloride. There are approximately 16 low-affinity sites which have a dissociation constant of 2.8 X 10(-4) M, and which are also inhibited by potassium chloride. Binding at the low-affinity sites is slightly enhanced by low magnesium concentrations. Both classes of sites are distinguishable from the colchicine binding site, and are apparently also distinct from the vinblastine and guanine nucleotide sites. The characteristics of the calcium binding activity of tubulin are similar to those found for the calcium-binding proteins of sarcoplasmic reticulum. The results are consistent with a physiological role for calcium in the regulation of microtubule assembly.


Synaptic plasma membrane glycoproteins: molecular identification of lectin receptors.

Synaptic plasma membranes isolated from rat cerebral cortex reacted with concanavalin (con A), wheat germ agglutinin (WGA), Lens culinaris phytohaemagglutinin (LcH), and Ricinus commounus agglutinin (RCA). Competition studies indicated that specific topographical relationships exist between receptors for con A and LcH and for WGA AND LcH. Reaction of [125I]con A with synaptic membrane proteins following polyacrylamide gel electrophoresis identified eight distinct molecular weight classes of glycoproteins possessing receptor activity for con A, ranging in apparent molecular weight from 27 000 to 165 000. Each of these also reacted to varying degrees with LcH, WGA, and RCA, indicating that a diverse population of carbohydrate moieties is associated with each molecular-weight class of glycoprotein. Following gel electrophoresis, competition between lectins did not occur, suggesting that each lectin reacts with a distinct group of carbohydrates and that specific relationships between these groups are destroyed by the solubilization and electrophoretic procedure. Synaptic junctional complexes isolated by Triton-X-100 extraction of synaptic membranes exhibited a simplified glycoprotein composition with only three major molecular weight classes of glycoproteins possessing receptor activity for con A being present.


Serine transhydroxymethylase: evidence for a sequential random mechanism.

Initial velocity patterns in the presence of product and dead-end inhibitors suggest that in reaction 1 the addition of substrates and release of products occur by a sequential random mechanism: L-serine + tetrahydrofolate in equilibrium glycine + 5,10-methylenetetrahydrofolate. This interpretation is supported by equilibrium isotope-exchange studies. The relative maximum rates of exchange of L-serine in equilibrium glycine and L-serine in equilibrium 5,10-methylenetetrahydrofolate in reaction 1 were not inhibited by high levels of substrates. The relative rates of these two exchange reactions were similar but were not identical. These results suggest that the catalytic interconversion and dissociation of substrates are of the same order of magnitude. Reaction 1 represents the transfer of a one-carbon group from the third carbon of L-serine to tetrahydrofolate. Inhibition studies showed that abortive enzyme ternary complexes are formed with L-serine and tetrahydrofolate compounds, which also contain a one-carbon group, e.g., 5-methyltetrahydrofolate and 5,10-methylenetetrahydrofolate. This suggests that the one-carbon binding site can accomodate two one-carbon groups simultaneously without serious steric hindrance.


Enzymatic oxidation of cobalt protoporphyrin IX: observations on the mechanism of heme oxygenase action.

Studies on the enzymatic mechanism of microsomal heme oxygenase were made utilizing various porphyrins and metalloporphyrins of different ring substituents and central metal ions. Co-heme (cobalt protoporphyrin IX) was shown to be a substrate for the enzyme and the product of its oxidative metabolism was identified as the natural bile pigment, biliverdin IXalpha isomer. Metalloporphyrins, which do not bind molecular oxygen (Ni, Mn, and Sn protoporphyrin IX), were not substrates for heme oxygenase, although they could competitively inhibit oxidation of reactive substrates for the enzyme. The presence of lipophilic substitutents on pyrrole rings I and II, as well as a central metal atom, were required for the heme oxidation reaction to occur. The oxidative cleavage of Co-heme displayed typical characteristics of an enzyme-mediated reaction, and the oxidation of this substrate, as well as that of Fe-heme (iron protoporphyrin IX), could be supported with either reduced nicotinamide adenine dinucleotide phosphate or reduced nicotinamide adenine dinucleotide. A hypothesis is proposed on the mode of action of heme oxygenase in which the enzyme and its substrate are considered to form a "transitory" hemoprotein which can activate molecular oxygen for cleavage of the heme tetrapyrrole ring. In this formulation, heme as substrate for heme oxygenase is synonymous with heme as prosthetic group for the enzyme.


Kinetics of product inhibition during firefly luciferase luminescence.

A theoretical and experimental analysis is made of the kinetics of product inhibition during firefly luciferase luminescence. Equations for competitive, noncompetitive, and uncompetitive inhibition are derived which are useful in determining inhibitory mechanism when the product inhibitor, or its concentration, is unknown and not subject to direct experimental manipulation. Comparisons of experimental data with predictions based upon the three inhibitory models show that product inhibition during luciferase luminescence is noncompetitive with respect to both luciferin and ATP as substrates. The competitive and uncompetitive models are inconsistent with experimental data. These findings provide the basis for using luminescence to measure ATP concentration continuously in in vitro biological systems such as isolated mitochondria.


Hydroxylamine oxidoreductase from Nitrosomonas: inactivation by hydrogen peroxide.

Incubation of hydroxylamine oxidoreductase of Nitrosomonas with hydrogen peroxide resulted in the rapid and irreversible loss of the ability to catalyze the dehydrogenation of hydroxylamine in the presence of electron acceptors, such as phenazine methosulfate. The rate of the reaction was dependent on the concentration of enzyme and H2O2. Inactivation occurred most rapidly at pH values between 9 and 10. Inactivation of the enzyme by H2O2 did not result in alteration of absorption spectrum of either the oxidized form of the enzyme or dithionite-reduced enzyme cytochromes with alpha maxima in the wavelength range 540-570 nm, indicating that those cytochromes were not directly involved in the dehydrogenase step. In contrast to the active enzyme, cytochromes with alpha maxima in the wavelength range 540-570 nm were not reducible by hydroxylamine in the inactivated enzyme. The dithionite-induced absorption maximum at 460 nm (cytochrome P 460), present in the active enzyme, was lost upon inactivation of the enzyme. This is the first direct indication of the involvement of cytochrome P 460 in the action of hydroxylamine oxidoreductase. Protection from inactivation was afforded by (a) substrates for the reduction of enzyme cytochrome, hydrazine, and N-methylhydroxylamine; (b) metal binding agents, KCN, 1,2-dihydroxybenzene-3,5-disulfonate, and hydroxyurea; (c) reductants, o-dianisidine, p-phenylenediamine, hydroquinone, pyrogallol, and dithiothreitol; (d) electron acceptors, phenazine methosulfate, and 2,6-dichlorophenolindophenol; and (e) the singlet oxygen trapping agent, 1,3-diphenylfuran. Scavengers of superoxide anion or hydroxyl radical did not protect the enzyme from inactivation.


Substrate proton exchange catalyzed by gamma-cystathionase.

Pulsed Fourier transform proton magnetic resonance was used to study the labilization of protons of various L-amino acids by the enzyme gamma-cystathionase. In the course of the normal reaction, the enzyme labilizes the alpha and beta protons of the substrate, L-homoserine, and promotes elimination of the gamma substituent. It was found that gamma-cystathionase also catalyzes the exchange of the alpha and beta protons of L-amino acids which cannot undergo elimination reactions, but are competitive inhibitors of the enzyme. Both beta protons of L-alpha-aminobutyrate, although not stereochemically equivalent, were exchanged at equal rates, whereas selectivity was shown for one of the beta hydrogens when the carbon length was increased. The data also show that beta-proton exchange cannot occur without alpha-proton exchange. The rate of alpha-proton exchange from amino acids containing a terminal hydroxyl group at the beta, gamma, or lambda carbon is greater than from the corresponding unsubstituted amino acid. Exchange rates of the alpha proton for the inhibitors examined vary from one-seventh that of the normal enzymatic reaction to approximately the same rate as that for the elimination reaction with homoserine. An active site with two areas of substrate-enzyme interaction is proposed. One site contains pyridoxal 5'-phosphate and the base or bases involved in alpha- and beta-proton exchange; the second site contains a base which normally facilitates removal of the gamma substituent and can interact with the gamma and lambda carbons of the substrate molecule.


Isolation and characterization of initiation fragments from lens 10S and 14S alpha-crystallin messenger ribonucleic acids.

The alpha-crystallin 10S and 14S messenger ribonucleic acids (mRNAs) for the B and A chains, respectively, were isolated from calf lenses. Initiation complexes were formed with both mRNAs after which the unprotected regions were digested with ribonuclease T1. A single fragment of approximately 45 nucleotides was obtained from both the 10S and 14S mRNAs. The fragments retained the ability to reform initiation complexes under standard conditions. Two-dimensional fractionation of ribonuclease T1 digests indicated considerable similarity between the 10S and 14S fragments. However, marked differences in the (U)G region were observed. The addition of the methylating agent S-adenosyl-L-methionine to the mRNA initiation system increases complex formation form two to five times, suggesting that methylation may be required for initiation.


Messenger RNA binding protein purified from reticulocyte polyribosomes.

One of the proteins in the 0.5 M KCl eluate of rabbit reticulocyte polyribosomes which bind poly(A)-rich mRNA has been purified to apparent homogeneity using ammonium sulfate fractionation and phosphocellulose, hydroxylapatite, and diethylaminoethylcellulose column chromatography. The protein appears to contain two subunits of 66 700 and 56 400 apparent molecular weights with a 1:1 stoichiometry, since an apparent molecular weight of 110 000 was determined using Sephadex G-200 chromatography and an s020,w of 5.6 was obtained with rate-zonal sedimentation. The mRNA binding activity banded at pH 5.2-5.5 on isoelectric-focusing polyacrylamide gel electrophoresis. Protein-dependent binding appeared to be specific, since other natural or synthetic RNAs, including tRNA, ribosomal RNA, and poly(riboadenylic acid), were 90- to 250-fold less effective than mRNA at competing for binding of [3H]poly(adenylic acid)-rich mRNA. Poly(riboguanylic acid), however, was even more efficiently bound by this protein than mRNA.


High resolution proton nuclear magnetic resonance investigation of the structural and dynamic properties of d(C15A15)-d(T15G15).

The high resolution proton nuclear magnetic resonance (NMR) spectra of the synthetic DNA block polymer d(C15A15)-d(T15G15) were studied in order to more completely understand telestability in DNA, and to provide fundamental NMR data on DNA helices and random coils. Spectra were measured in the spectral region from 0 to 15 ppm downfield from the usual standard, sodium 4,4-dimethyl-4-silapentane-1-sulfonate(DSS), at various temperatures (24-98 degrees C) in solution containing either moderate or high ionic strength. The effect of actinomycin binding to the block polymer also was studied. The major conclusions derived from this study are as follows: (1) The majority of base pairs in the AT helix of the block polymer have the same conformation as in d(A)n-d(T)25 and d(A)21-d(T)21. (2) The conformation of the GC helix in the block polymer is different from the AT helix and this perturbs the conformation of three or four A-T base pairs at the junction of the AT-GC helix. (3) The conformation of the AT helix is unaffected by salt over the range examined (approximately 0.04 - approximately 2 M), but the conformation of the GC helix changes. (4) There are subtle changes in the conformation of the AT helix as the temperature is increased and resonances characteristic of the random coil and the double-helical state can be simultaneously observed. (5) Binding of actinomycin, which is specific for the GC helix, induces quite large (over 1 ppm) upfield shifts of the resonances from the GC base pairs. This is consistent with an intercalation model in which actinomycin D (Am) is assymetrically sandwiched between two GC base pairs in such a manner that overlap with the guanosine residues is greater than with the neighboring cytidines. (6) The presence of the drug may also perturb A-T base pairs located near the AT-GC junction, but it has no effect on the majority of the AT pairs. However, as expected, Am elevated the Tm of the AT helix, even though it binds to the other end of the DNA.


Inhibition and alternate-substrate studies on the mechanism of malic enzyme.

A number of dead-end inhibitors and alternate substrates were examined to gain an understanding of the substrate specificity and mechanism of malic enzyme. Comparison of Ki values for competitive inhibitors suggested that binding of the l-carboxyl of L-malate is by ion pairing with lysine or arginine, while binding of the 4-carboxyl is weaker, and probably of the induced-dipolar type. The 2-hydroxyl hydrogen bonds to a catalytic group, which, when it is protonated, adsorbs the keto form of oxalacetate. Since the only molecule other than L-malate that is oxidized is L-malate-beta-amide, carbon 4 must be trigonal for substrate activity, although a tetrahedral carbon bearing one or two hydroxyl groups gives good binding. Hydroxy groups at carbon 3 contribute to binding, but prevent substrate activity. Hydroxy and ketomalonates are bound more strongly than any of the four carbon acids, suggesting that the latter are bound with some strain. In inhibition studies, pyruvate analogues were competitive vs. pyruvate but noncompetitive vs. malate, while malate analogues were competitive vs. malate and noncompetitive vs. pyruvate. These compounds thus bind to both enzyme-triphosphopyridine nucleotide (E-TPN) and enzyme-reduced triphosphopyridine nucleotide (E-TPNH), but only malate analogues prevent release of TPN, while pyruvate analogues prevent release of TPNH. Ketomalonate and oxalacetate, both of which are slowly reduced by the enzyme in the presence of TPNH and thus must combine in the keto form with E-TPNH,, appear to combine with E-TPN mainly in the gem-diol (or for oxalacetate, also the enol) form. The substrate for the decarboxylation of oxalacetate at pH 4.5 is the keto form.


Ligand modification of corpus luteum mitochondrial cytochrome P-450 spectra and cholesterol monooxygenation: an assay of enzyme-specific inhibitors.

Absorbance changes in the spectrum of cytochrome P-450 were related to the inhibition of [26-14C]cholesterol oxidation to [14C]isocaproate and pregnenolone in mitochondria from bovine corpus luteum produced by two types of ligands. Nitrogenous inhibitors, such as aminoglutethimide, elicit an absorption maximum at about 427 nm and a minimum at about 393 nm (type II), while steroidal inhibitors, such as (20R)-20-(p-tolyl)-5-pregnene-3beta,20-diol (20-tolyl-pregnenediol), cause difference spectra with maximum at about 420 nm and minimum at about 390 nm (reverse type I). The magnitude of spectral change and the amount of inhibition of pregnenolone synthesis by aminoglutethimide are closely correlated at concentrations ranging from 5 to 750 muM and by the model steroid, 20-tolyl-pregnenediol, at concentrations from 0.5 to 25 muM. The responses are concentration dependent and linear over the range of effective concentrations. The concentrations of inhibitors for the half-maximal inhibition of pregnenolone biosynthesis are identical with the concentrations producing half-maximal spectral changes within experimental error. Displacement of substrate from cytochrome P-450 and/or stabilization of the redox potential subsequent to to the ligation of heme iron is proposed as the specific mechanism of cholesterol side chain cleavage inhibition. Finding, together, the two procedures offer a sensitive, specific, and accurate means of screening inhibitors of the cholesterol side chain cleavage system.


Effects of detergents on the properties of 4-hydroxybenzoate. Polyprenyl transferase and the specificity of the polyprenyl pyrophosphate synthetic system in mitochondria.

The properties of 4-hydroxybenzoate:polyprenyl transferase and the system synthesizing polyprenyl pyrophosphate have been studied in mitochondria from rat and guinea pig livers. With solanesyl pyrophosphate and 4-hydroxybenzoate as substrates the formation of 3-nonaprenyl-4-hydroxybenzoate was linear with time, concentration of protein, and concentration of solanesyl pyrophosphate. Solanesyl monophosphate is inactive as a substrate and is noninhibitory. Conversion of solanesyl monophosphate to the pyrophosphate could not be detected. Detergents such as Triton X-100, Tween-80, and sodium deoxycholate activated the enzyme in mitochondria which were aged by freezing at -20 degrees C for periods ranging from 1 h to several days. Maximum activation also required Mg2+. In agreement with previous observation the effect of Mg2+ and Triton X-100 on fresh mitochondria was quite variable; however, activation with aged preparations was very consistent. Treatment with TritonX-100 causes al alteration in the biosynthetic pattern of rat liver mitochondria so that rather than nonaprenyl, decaprenyl, pyrophosphate is preferentially made in the presence of solanesyl pyrophosphate and isopentenyl pyrophosphate. In the presence of Triton X-100 and added pool of solanesyl pyrophosphate appears to exert a feedback inhibition on the incorporation of isopentenyl pyrophosphate into solanesyl pyrophosphate. In the case of guinea pig liver mitochondria a different pattern is observed with Triton X-100 in contrast to the rat. The de novo formation of decaprenyl pyrophosphate from isopentenyl pyrophosphate appears to be inhibited by Triton X-100, but the synthesis of decaprenyl pyrophosphate from isopentenyl pyrophosphate and nonaprenyl pyrophosphate is not inhibited. The data also indicate that in guinea pig liver in a system synthesizing decaprenyl pyrophosphate from isopentenyl pyrophosphate, there does not appear to be a detectable pool of nonaprenyl pyrophosphate. These results show that detergents can affect the specificity of the mitochondrial system synthesizing polyprenyl pyrophosphates.


Interaction of bovine neurophysin with oxytocin and vasopressin measured by temperature-jump relaxation.

The interaction between bovine neurophysins I and II and oxytocin and vasopressin was measured using temperature-jump relaxation. A single relaxation time in the 10 to 90 ms range was noted for each solution. This time depended upon the concentration of both neurophysin and hormone and increased with increasing pH. The formation rate constants (+/- SE) for the interaction of neurophysin I dimer with the protonated form of oxytocin and vasopressin at pH 7.4 in 0.1 M KNO3 and 25 degrees C were 2.8 (+/- 0.4) x 10(6) and 2.3 (+/- 0.5) x 10(6) M-1 s-1, respectively; the dissociation rate constants were 11 and 15 s-1, respectively.For neurophysin II dimer, formation rate constants were 6.0 (+/- 0.2) x 10(6) and 2.4 (+/- 0.3) x 10(6) M-1 s-1; dissociation rate constants were 24 and 16 s-1 for oxytocin and vasopressin, respectively. Formation rate constants for the interaction of neurophysin monomer with protinated hormone were approximatley an order of magniment with circular dichorism and pH titration studies which indicate that this interaction occurs between a negatively charged carboxyl group on the neurophysin and a protonated alpha-amino group on the hormone. Our results indicate that oxytocin and vasopressin bind with greater affinity to the neurophysin dimer than the monomer and that the binding of oxytocin and vasopressin in neurophysin dimer than the monomer and that the binding of oxytocin and vasopressin in neurophysin dimer at pH 7.4 and concentrations between 10(-4) and 10(-5) M is nearly identical for each hormone.


Hydrocarbon trans-gauche isomerization in phospholipid bilayer gel assemblies.

The vibrational Raman spectra of dimyristoyl (DMPC)-, dipalmitoyl (DPPC)-, and distearoylphosphatidycholine (DSPC)-water bilayer system were used to probe lipid hydrocarbon chain trans-gauch isomerization dynamics below the gel-liquid crystalline phase transition temperature. In addition to the 1090-1085 cm-1 vibrational transitions, which appear with increasing temperatures and are characteristic of gauche conformers within the acyl chains, a new feature arises in all three bilayer systems at approximately 1122 cm-1. This carbon-carbon stretching mode is associated with the formation of the gauche bond rotation of the terminal methyl group oriented toward the center of the bilayer. Estimates of the enthalpy differences (deltaH) between hydrocarbon chains in an all-trans conformation and chain configurations containing gauche forms may be made from peak height intensities of vibrational features associated with the appropriate rotational isomers. For the DMPC-H2O, DPPC-H2O, and DSPC-H2O assemblies, the Raman data yield enthalpy differences of 2.9 +/- 0.6, 3.4 +/- 0.5, and 9.9 +/- 1.2 kcal/mol, respectively. These values are interpreted to reflect approximately two gauche bonds per lipid molecule for the DMPC-H2O and the DPPC-H2O gels and six gauche bonds per molecule for the DSPC-H2O gels.


Secondary-structure predictions of calcium-binding proteins.

The known tertiary structure of carp muscle parvalbumin is consistent with an "EF-hand" architecture (helix-loop-helix) for each calcium-ion binding site. Primary-sequence alignments have indicated four EF hands in rabbit skeletal muscle troponin C and in rabbit myosin alkali light chains. Five secondary-structure prediction methods, based on amino acid sequence only, have been fully computerized and used to calculate joint prediction histograms for several calcium-binding proteins. The joint histogram can suggest directly the extent and sequence of the helical- and loop-structural elements, as well as any secondary structural distortions or evolutionary developments. Since the histogram predicted well the length and sequence of secondary structural elements in carp muscle parvalbumin, it seemed reasonable to calculate the joint distribution for other proteins that might bind calcium through the EF-hand configuration. The histograms indicated the four EF-hand regions speculated fro rabbit skeletal muscle troponin C but suggested only three such hands in bovine cardiac muscle troponin C and with a distorted fourth hand. Considerable secondary structural distortion is postulated for the alkali light chains. Possible EF configurations consistent with the histogram results are speculated for Escherichia coli acyl-carrier protein and bovine prothrombin fragment 1, which have been shown to bind calcium. The secondary-structure-prediction algorithms appear to be a useful adjunct to sequence-alignment techniques, especially in cases where the primary sequence homology is weak or the evolutionary distance is large.


Allosteric interactions between the membrane-bound acetylcholine receptor and chemical mediators: equilibrium measurements.

An approach to equilibrium dialysis measurements has been developed which enables one to study the interaction of chemical mediators with the membrane-bound acetylcholine receptor and to gain information of a type previously obtainable only with soluble proteins. Equilibrium dialysis experiments conducted at pH 7.0,4 degrees C, and mu = 0.18 M, with electroplax membrane preparations from Electrophorus electricus revealed apparently homogeneous binding isotherms for decamethonium with dissociation constants in the range of 0.2-0.4 muM. The following new information has been obtained. (1) The activators of neural transmission, decamethonium and carbamylcholine, occupy overlapping binding sites. (2) These activators and the inhibitors, alpha-bungarotoxin and d-tubocurarine, compete for only one-half of the sites available to them even through the stoichiometry of these is 1:1 as measured with decamethonium (a reversibly binding activator) and alpha-bungarotoxin (an irreversible specific inhibitor). Different receptor molecules, preexisting nonequivalent binding sites, or an allosteric mechanism involving ligand-induced conformational changes are often considered to account for such observations.


Allosteric interactions between the membrane-bound acetylcholine receptor and chemical mediators. Kinetic studies.

The kinetics of the specific irreversible reaction of a snake neurotoxin, alpha-bungarotoxin, with the acetylcholine receptor of electroplax membrane preparations have been investigated. The effects of activators (decamethonium, carbamylcholine) and inhibitors (alpha-bungarotoxin, d-tubocurarine) of neural transmission on this reaction have been measured and the following new information obtained. (1) The irreversible reaction is preceded by the reversible formation of toxin-receptor complexes. (2) Two types of receptor binding site exist. d-Tubocurarine directly competes with the toxin for one type of binding site. Decamethonium and carbamylcholine are noncompetitive inhibitors of the toxin reaction. (3) The data are inconsistent with binding sites on separate and distinct molecules or with preexisting nonequivalent binding sites. A simple model is proposed to explain both the kinetic data and equilibrium measurements which indicated that activators and inhibitors of neural transmission compete for only one-half of the receptor sites available to them. The model proposes that for the compounds investigated the binding sites of activators do not overlap with those of inhibitors and the ligand-induced conformational changes of the receptor result in changes in the affinities of the binding sites. The model is simple and is based on mechanisms which have been found to be valid for many well-characterized regulatory enzymes.


A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S.

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.


The complete amino acid sequence of the major component myoglobin from the arctic minke whale, Balaenoptera acutorostrata.

The complete primary structure of the major component myoglobin from the Arctic minke whale, Balaenoptera acutorostrata, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the apomyoglobin at the two methionine residues with cyanogen bromide along with the four peptides resulting from the cleavage of the methylacetimidated apomyoglobin at the three arginine residues with trypsin. The further digestion of the central cyanogen bromide peptide with trypsin and S. aureus strain V8 protease enabled the determining of the remainder of the covalent structure. This myoglobin differs from that of the dwarf sperm whale, Kogia simus, at 16 positions, and the common dolphin, Delphinus delphis, at 14 positions, from that of the common porpoise, Phocaena phocaena, and the bottlenosed dolphin, Tursiops truncatus at 13 positions, from that of the Amazon River dolphin, Inia geoffrensis, at 10 positions, and from that of California gray whale, Eschrichtius gibbosus, at 3 positions- All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.


Differences in the solubilizing effectiveness of the sodium dodecyl sulfate complexes of various proteins.

The detailed reversible binding isotherms of sodium dodecyl sulfate (NaDodSO4) with 13 different initially native proteins are reported; the data were obtained at 20 degrees C and pH 7.1, ionic strength 0.033, with amounts bound with some proteins up to 1.1 g per g of protein. Although the isotherms of some of the proteins do not vary widely, extreme variations between certain classes are found. Thus, for example, hemoglobin and myoglobin both have high affinities and high binding capacities, while gamma-globulin, apoferritin, and transferrin have low initial affinities, and change drastically at higher concentrations. The protein-NaDodSO4 complexes solubilize the water-insoluble dye dimethylaminoazobenzene (DMAB) as effectively as micelles of pure NaDodSO4 when only small amounts (0.2 to 0.5 g/g of NaDodSO4) are bound. In most cases this effectiveness falls progressively as larger amounts are bound, and may even cease altogether at limits characteristic of the individual protein. With some of the latter, a second region of renewed solubilization occurs when substantially higher amounts of NaDodSO4 are present. In all cases, solubilization by ordinary micelles in normal amount occurs when the free NaDodSO4 concentration exceeds the critical micelle concentration, but the binding of NPADodSO4 to the protein also increases, in competition with formation of micelles. With some, but not all proteins the NaDodSO4 bound at concentrations above the cmc also solubilizes DMAB. In such cases the solubilizations by the protein-NaDodSO4 complexes and by the simple micelles are additive. The significance of the differences in binding and solubilizing encountered among these proteins is discussed in terms of surface structure, cooperativity of binding, and protein composition. No certain correlations with content of most amino acids, subunit structure, solubility, and hydrophobicity have been found, but there is a weak inverse dependence of solubilizing effectiveness on molecular size and indications of a strong dependence on content of cationic groups.


Proton magnetic resonance studies of actinomycin D complexes with mixtures of nucleotides as models for the binding of the drug to DNA.

The proton magnetic resonance spectra of actinomycin solutions with mixtures of deoxynucleotides have been investigated to determine the relative preference for the binding of guanine and adenine nucleotides to the two nucleotide binding sites of actinomycin D. An analysis of the chemical shifts of the actinomycin D resonances shows that adenine and guanine nucleotides competitively bind to the benzenoid portion of the phenoxazone ring of actinomycin D while guanine nucleotides bind stronger than adenine nucleotides to the quinoid portion of the phenoxazone ring. The chemical shift data for the titrations of actinomycin D with pdG-dG, pdC-dC, and an equimolar mixture of these complementary deoxydinucleotides show that: (1) pdG-dG forms a stacked complex much like dGMP; (2) pdC-dC does not bind to actinomycin D under the conditions used in these experiments; (3) in the titration of actinomycin D with the equimolar mixture of pdG-dG + pdC-dC, a miniature intercalated complex is formed.


Solubilized DNA-dependent RNA polymerase activities in resting and growing fibroblast.

The activities of RNA polymerases I and II have been measured in 3T6 during the transition from the resting to growing state by solubilization of the enzymes followed by chromatography on DEAE-Sephadex columns. The activity of RNA polymerase II remains unchanged during the first 12 h after serum stimulation while the activity of RNA polymerase I increases and closely parallels the increased activity seen in isolated nuclei. Compared to enzyme from resting cells. RNA polymerase I from serum stimulated cells elutes at a lower ammonium sulfate concentration on DEAE-Sephadex chromatography and its activity shows distinctly different dependencies on the concentration of ammonium sulfate and magnesium ion. These observations are discussed in relation to the possible mechanism by which 3T6 regulates the synthesis of preribosomal RNA.


Complete nucleotide sequence and properties of the major species of glycine transfer RNA from wheat germ.

The complete nucleotide sequence of wheat germ tRNA1Gly has been determined. It is a small tRNA, containing only 74 nucleotides of which 10 are methylated or otherwise modified. Several important features of this tRNA are: (a) a complete absence of ribothymidine which is the most common methylated nucleoside in tRNA; (b) a methylated sugar in the acceptor stem which is a region almost always free of modified residues; and (c) unusually weak base pairing in the dihydrouridine stem region consisting of only one standard A-U base pair in addition to G pi, AC, and GU. This tRNA functions in a wheat germ cell free protein synthesizing system directed by either natural mRNAs or poly(G,U). The complete nucleotide sequence of wheat germ tRNAlGly is: pG-C-A-Cm-CAGU-m1G-G-U-C-PSI-A-G-D-G-G-U-A-A-U-A-G-U-A-C-C-C-U-G-C-C-A-mC-G-G-U-A-C-A-C-A-G-A-mC-m5C-G-G-G-U-PSI-C-G-mA-U-U-C-C-C-G-G-C-U-G-G-U-G-C-A-C-C-AOH.


Organization of the photosynthetic membrane in maize mesophyll and bundle sheath chloroplasts.

The freeze-fracturing technique has been used to investigate membrane architecture in the mesophyll and bundle sheath chloroplasts of Zea mays. The structural organization of mesophyll chloroplasts is virtually identical to that of other species of higher plants which have been investigated with this technique. Characteristic distributions of particles of various sizes are seen on each fracture face after membrane splitting during the fracturing process, and these distributions indicate the differentiation of the membrane system into sacked (grana) and unstacked (stroma) regions, typical of grana-containing chloroplasts. Bundle sheath chloroplasts contain very few grana, and the thylakoids of these plastids are therefore largely unstacked. Analysis of artificially unstacked mesophyll chloroplasts indicates that this difference is not merely related to the presence or absence of adhesion between adjacent thylakoids, but reflects a substantial difference in membrane substructure between mesophyll and bundle sheath photosynthetic membranes. Bundle sheath thylakoids contain virtually the same number of small (P fracture face) particles as mesophyll thylakoids, but contain only 40% as many of the larger (E fracture face) tetrameric particles. These differences, together with biochemical data indicating the comparative deficiency of bundle sheath chloroplasts in Photosystem II activity, suggest that the E face particles are related to the presence or absence of Photosystem II activity.


Distribution of excitation energy among photosystem I and photosystem II in red algae. I. Action spectra of light reactions I and II.

Action spectra of light reaction I and light reaction II from red algae (marine members of Florideae and Bangiales) were measured with 550 nm (light 2) or 699 nm light 1) background light, using a Teflon-covered platinum electrode for O2 measurement. Care was taken to ensure that maximum enhancement was reached by the background light. The action spectra of light reaction I, we found under these conditions, are very similar to the thallus absorption, whilst the action spectra of light reaction II show, besides strong bands of the phycobilins, only minor bands of chlorophyll a, which account for only 10-20% of the total chlorophyll. The spectra are discussed on the basis of two main types of models of energy distribution over both photosynthetic systems. If this distrubution is considered to be invariable (models 1a and b), one has to assume that almost exactly half of the total chlorphyll is not involved in the supply of the non-cyclic electron transport with excitation energy. This part, however, has to be thought of as incorporated in the thylakoid membrane in a similar manner to the chlorophyll in photosystem I. However, if one supposes an almost complete equilibration in the energy distribution over both systems as long as the primary absorption in photosystem II prevails (models 2a and b), there is no need for the assumption of such photosynthetically 'inactive' or less active chlorophyll. Some evidence is shown that strongly supports model 2.


A one microsecond component of chlorophyll luminescence suggesting a primary acceptor of system II of photosynthesis different from Q.

The kinetics of the luminescence of chlorophyll a in Chlorella vulgaris were studied in the time range from 0.2 mus to 20 mus after a short saturating flash (t 1/2 = 25 ns) under various pretreatment including anaerobiosis, flashes, continuous illumination and various additions. A 1 mus luminescence component probably originating from System II was found of which the relative amplitude was maximum under anaerobic conditions for reaction centers in the state SPQ- before the flash, about one third for centers in the state S+PQ- or SPQ before the flash, and about one tenth for centers in the state S+PQ before the flash. S is the secondary donor complex with zero change; S+ is the secondary donor complex with 1 to 3 positive charges; P, the primary donor, is the photoactive chlorophyll a, P-680, of reaction center 2; Q- is the reduced acceptor of System II, Q. Under aerobic conditions, where an endogenous quencher presumably was active, the luminescence was reduced by a factor two. The 1 mus decay of the luminescence is probably caused by the disappearance of P+ formed in the laser flash according to the reaction ZP+ leads to Z+ in which Z is the molecule which donates an electron to P+ and which is part of S. After addition of hydroxylamine, the 1 mus luminescence component changed with the incubation time exponentially (tau = 27 s) into a 30 mus component; during the same time, the variable fluorescence yield, measured 9 mus after the laser flash, decreased by a factor 2 with the same time constant. Hereafter in a second much slower phase the fluorescence yield decreased as an exponential function of the incubation time to about the dark value; meanwhile the 30 mus luminescence increased about 50% with the same time constant (tau = 7 min). Heat treatment abolished both luminescence components. The 1 mus luminescence component saturated at about the same energy as the System II fluorescence yield 60 mus after the laser flash and as the slower luminescence components. From the observation that the amplutide is maximum if the laser flash is given when the fluorescence yield is high after prolonged anaerobic conditions (state SQ-), we conclude that the 1 mus luminescence is probably caused by the reaction PWQ- + hv leads to P*WQ- leads to P+W-Q- leads to P*WQ- leads to PWQ- + hv in which W is an acceptor different from Q. The presence of S+ reduced the luminescence amplitude to about one third. Two models are discussed, one with W as an intermediate between P and Q and another, which gives the best interpretation, with W on a side path.


NMR characterization of three forms of ferredoxin from Desulphovibrio gigas, a sulphate reducer.

A NMR and magnetic susceptibility study of the oxidized and reduced states of three different oligomers (forms) of a [4Fe-4S] ferrodoxin protein from Desulphovibrio gigas, FdI, FdI', and FdII was carried out. FdI and FdI' are different trimers and FdII a tetramer of the same basic subunit. A probable assignment of the contact shifted resonances is indicated. Since the temperature dependences of the contact shifted responances associated with each [4Fe-4S] are not all similar a delocalized model for the spin densities on the 4Fe does not apply. The exchange rate between oxidized and reduced states is slow on the NMR time scale. The three oligomers are not magnetically equivalent. Using the "three state hypothesis" terminology it is shown that FdIox is predominantly in the C2- state and changes upon reduction into the C3- state, while FdIIox is in the C- state and changes into the C2- state. FdI' does not easily fit into this classification. This study shows a similarity of magnetic behaviour between FdI and bacterial ferredoxins (e.g. Bacillus polymyxa) and between FdII and HiPIP from Chromatium sp. The influence of the quaternary structure on the stabilization of the different oxidation states of ferredoxins as well as on their redox potentials is discussed.


Bounds on rate constants and relative potentials in electron transport chains.

Minimization of free-energy losses requires that the rate constants for reaction in both forward and reverse directions be several times the net rate of a reaction. In an electron transfer between wound molecules, the forward and reverse rate constants contribute separate factors to the free-energy drop across the reaction. If such a reaction has a reverse rate constant which is much greater than the net rate of reaction, then the midpoint potential of the acceptor may economically be more negative than the midpoint potential of the donor.


Influence of membrane structure on the kinetics of carrier-mediated ion transport through lipid bilayers.

Charge-pulse relaxation experiments of valinomycin-mediated Rb+ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experiment data the rate constants of association (kR) and dissociation (kD) of the ion-carrier complex as well as the rate constants of translocation of the complex (kMS) and of the free carrier (kS) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants kS and kMS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C20 fatty acid from one to four resulted in an increase of kS by a factor of seven and in an increase of kMS by a factor of twenty-four. The stability constant K = kR/kD of the ion-carrier complex as well as the translocation rate constants kS and kMS were found to depend strongly on the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced kR, kMS and kS up to seven-fold.


Evidence for the preferential interaction of glycophorin with negatively charged phospholipids.

Glycophorin, extracted from the erythrocyte membrane after treatment with lithium-diiodo-salicylate, still contains a significant amount of phospholipid, consisting predominantly of phosphatidylserine. Methods are described which lead to a full delipidation of the protein. After treatment with neuraminidase, delipidated glycophorin shows a preferential interaction with monolayers of negatively-charged phospholipids. This lipid-protein interaction is decreased by the presence of cholesterol in the lipid film.


Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography.

The major platelet membrane glycoproteins have been solubilized in 1.0% sodium deoxycholate and subjected to affinity chromatography on the lectins from Lens culinaris, wheat germ and Abrus precatorius. Polyacrylamide gel electrophoresis in the presence and absence of a reducing agent together with the differential binding of the lectins to the glycoproteins permitted the distinction of at least seven separate glycoprotein entities. A new nomenclature for the glycoproteins is proposed to accomodate the additional data. Using combinations of lectin columns, glycoproteins Ia and Ib could be prepared in a pure state and IIb and IIIa could be greatly purified. The binding of lectins to glycoprotein Ib has been strongly implicated as a necessary step in the aggregation response of platelets to lectins.


The effect of cholesterol incorporation on the temperature dependence of water permeation through liposomal membranes prepared from phosphatidylcholines.

The permeation of water through liposomal membranes composed of phosphatidylcholine plus varying amounts of cholesterol was studied as a function of temperature. 1. Increasing amounts of cholesterol caused a gradual disappearance of the abrupt change in the rate of water permeation near the gel to liquid-crystalline phase transition temperature of dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine liposomes. At cholesterol concentrations above about 30 mol % there was no longer a discontinuity in the rate of water permeation. 2. The incorporation of cholesterol produces a steep change in the activation energy of the water permeation above the transition temperature of the saturated lecithin occurring at about 15 mol % of cholesterol. Below the transition temperature there was a gradual decrease in the activation energy of the water permeation in the region of 0 to 33 mol % of cholesterol. 3. In systems containing unsaturated phosphatidylcholines cholesterol also enhanced the activation energy of the water permeation although to a lesser extent. The results indicate that the position of the cis-double bond in the fatty acid chain is very important in this respect. 4. In systems in which cholesterol increased the temperature dependence of the water permeation there is also an enhancement of the temperature dependence of the isotonic glycerol and erythritol swelling by the same number of kcal/mol.


gamma-Aminobutyric acid-stimulated chloride permeability in crayfish muscle.

gamma-Aminobutyric acid selectively increased Cl- permeability in isolated strips of crayfish abdominal muscle. Muscle fibers incubated in Van Harrevald's solution at room temperature took up 36Cl- to the extent of 700 ml/kg wet weight with a halftime of 2.5 min. During 15-S incubations, the control 36Cl- uptake space was 131 +/- 4 ml/kg (n = 60) and this was significantly increased by gamma-aminobutyric acid at 200 muM or higher concentrations to 177 +/- 4 ml/kg (n = 48, P less than 0.05). This effect was specific for chloride since gamma-aminobutyric acid did not increase the uptake by crayfish muscle of radioactive sucrose, inositol, or propionate. gamma-Aminobutyric acid stimulation of 36Cl- uptake is mediated by receptor-ionophore function since the process shows pharmacological properties virtually identical to those observed by electrophysiological techniques. The gamma-aminobutyric acid stimulation of Cl- permeability is dose dependent with 50% of the maximal effect at 40 muM gamma-aminobutyric acid and the dose vs. response curve is somewhat sigmoid. The gamma-aminobutyric acid agonist muscimol causes the same maximal effect on Cl- uptake as gamma-aminobutyric acid, but acts at 5-fold lower concentrations, i.e. is more potent. However, the partial agonist gamma-amino, beta-hydroxybutyric acid produced little or no stimulation of 36Cl- flux. The response to gamma-aminobutyric acid was blocked by 2 mM beta-guanidinopropionate or gamma-guanidinobutyrate, 0.5 mM bicuculline, and 10 muM picrotoxinin. Picrotoxinin inhibition was dose dependent with 50% inhibition occurring at 4 muM. Antagonists did not affect control 36Cl- uptake. These results confirm electrophysiological observations that the postsynaptic response to the inhibitory neurotransmitter gamma-aminobutyric acid involves a rapid increase in membrane permeability to Cl-.


Enzyme therapy. VI: Comparative in vivo fates and effects on lysosomal integrity of enzyme entrapped in negatively and positively charged liposomes.

Entrapment of enzyme in liposomes, biodegradable lipid vesicles, offers an intriguing strategy for the intracellular delivery of these macromolecules to the lysosomal apparatus for enzyme replacement endeavors in selected lysosomal storage diseases. Therefore, the in vivo tissue and subcellular fate and effect on the subcellular distribution of endogenous lysosomal hydrolases was determined following intravenous administration of beta-glucuronidase entrapped in positively and negatively charged liposomes into C3H/HeJ beta-glucuronidase-deficient mice. Enzyme entrapped in negatively charged liposomes was rapidly cleared from the circulation (t1/2 approximately 4 min); maximal tissue recovery, 75% of dose, was detedtec in the liver at 1 h, was maintained fro 48 h and then gradually declined to non-detectable levels by 8 days. A similar circulatory clearance and reciprocal hepatic uptake was observed fro positively charged liposomes; however, the beta-glucuronidase was retained in murine liver for 11 days. Significant activity, 15% of dose, was found in the kidneys up to 1 and 4 days post-injection of positively and negatively charged liposomes, respectively. No activity was recovered in neural or other visceral tissues except in spleen and lungs (less than 5% of the dose). Exogenous beta-glucuronidase activity administered in negatively charged liposomes was primarily localized in the lysosomally-enriched hepatic subcellular fraction, compared to the predominantly soluble localization of exogenous activity entrapped in positively charged liposomes. Administration of negatively charged liposomes caused no detectable change in the subcellular localization of several endogenous lysosomal hydrolase activities compared to their distribution in untreated mice. In contrast, a marked but temporary translocation of these hydrolase activities into the soluble fraction was observed following the administration of positively charged liposomes, identifying possible deleterious effects on cellular physiology.


Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe.

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.


The effects of hypothyroidism and fasting on electrogenic amino acid transfer: possible evidence for multiple neutral amino acid carrier systems in rat jejunum.

The jejunal mechanisms for the electrogenic transfer of four neutral amino acids (alanine, leucine, methionine, valine) and for sarcosine were characterised by an electrical method in vitro. The values for apparent Km obtained electrically agree well with those assessed by conventional chemical techniques. Hypothyroidism and/or fasting rats for 3 days induced differential changes in the apparent Km and p.d.max for the various amino acids. These alterations were interpreted as indicating the presence of at least three mechanisms for neutral amino acid transfer and one for sarcosine. In euthyroid rats, only alanine showed changes in apparent Km (decrease) and p.d.max (decrease) after fasting for 3 days. With hypothyroidism the kinetic parameters of electrogenic transfer for alanine, valine and sarcosine were significantly altered while those for leucine and methionine were unaffected.


Freeze-fracture electron microscopy of human erythrocytes lacking the major membrane sialoglycoprotein.

Human erythrocytes of blood group En (a-), a rare homozygous condition involving a complete lack of the major sialoglycoprotein of the cell membrane (glycophorin A), were compared with erythrocytes from normal (En (a+)) individuals by freeze-fracture electron microscopy. No decrease in number, or variation in morphology, of the intramembranal particles of En (a-) cells was detectable. The results show that the erythrocyte sialoglycoprotein is not essential for the maintenance of the integrity of the intramembranal particles of the human erythrocyte membrane.


The preferential interaction of cholesterol with different classes of phospholipids.

1. By differential scanning calorimetry a preferential affinity of cholesterol for sphingomyelin was established in mixtures of sphingomelin and phosphatidylcholine where sphingomyelin was either the higher or the lower melting phospholipid. 2. A preferential affinity of cholesterol for sphingomyelin was also found in mixtures of sphingomyelin and phosphatidylethanolamine where sphingomyelin was either the higher or the  lower melting phospholipid. The sphingomyelin used was isolated from beef erythrocytes or synthetic palmitoyl sphingomyelin. 3. In mixtures of phosphatidylserine with phosphatidylethanolamine, or phosphatidylserine with phosphatidylcholine, cholesterol showed the highest affinity for the lower melting phospholipid. 4. In a previous paper (van Dijck et al. (1976) Biochim. Biophys. Acta 455, 576-588) it was established that cholesterol has a higher affinity for phosphatidylcholine than for phosphatidylethanolamine. The affinity order of cholesterol for the neutral phospholipids which can be deduced form these experiments is sphingomyelin greater than phosphatidylcholine greater than phosphatidylethanolamine.


Choline influx across the brush border of guinea pig jejunum.

Choline uptake across the mucosal border of guinea pig jejunum was measured to determine the characteristics of this step in intestinal absorption. Unidirectional influx of [14C]choline appears to proceed primarily by a saturable, carrier-mediated process at low mucosal choline concentrations; at high concentrations (greater than 4 mM) the influx rate is approximately linearly related to the mucosal choline concentration, suggesting that absorption by passive diffusion predominates. Influx was only minimally reduced by elimination of Na+ from the mucosal test solution or by reduction of the intracellular Na+ concentration. Preincubation of tissue samples with metabolic inhibitors or with ouabain did not markedly reduce influx. These results are consistent with a model of choline transport across the brush border membrane by a carrier-mediated mechanism which is similar to that involved  in fructose absorption but different from the Na+-dependent mechanism which participates in active transport of sugars and amino acids. At low lumenal choline concentrations, influx into colonic mucosa is slower than in jejunum and appears to be attributed solely to simple diffusion.


The erythrocyte membrane site for the effect of temperature on osmotic fragility.

The osmotic fragility of human erythrocytes is well known to decrease as the temperature is elevated. The cellular site for the temperature effect was studied by assessing possible roles of hemoglobin and of membrane lipids and by taking advantage of the unique response of camel erythrocytes to temperature. It is concluded that the erythrocyte membrane is the site for the temperature effect on osmotic fragility. The human erythrocyte is likely to rupture in protein--lipid boundary regions in the membrane, from which cholesterol is apparently excluded.


Effects of different bile salts upon the composition and morphology of a liver plasma membrane preparation. Deoxycholate is more membrane damaging than cholate and its conjugates.

1. Rat liver plasma membrane  preparations were incubated with various bile salts at 0 or 37 degrees C. the bile salts caused the removal of various amounts of proteins, membrane enzymes and phospholipids; the extent and nature of these losses, and the morphological changes which accompanied them, varied with the detergent used. 2. Cholate, taurocholate and glycocholate removed appreciable amounts of protein from the saline-washed membranes, and considerable amounts of both phospholipids and the membrane enzymes, 5-nucleotidase, alkaline phosphatase, alkaline phosphodiesterase 1 and L-leucyl-beta-naphtylamidase. These losses were greater at 37 that at 0 degrees C. The material remaining contained membrane-like profiles, many of vesicular form, even when the preparation was almost completely devoid of phospholipids. 3. Deoxycholate, both at 0 and 37 degrees C, removed more protein, membrane enzymes and phospholipids than did cholate and its conjugates. The material remaining was mainly granular and unorganised and the only remaining features were structures resembling the nexus, and occasional desmosomes. 4. Deoxycholate, a dihydroxy bile salt, therefore appears to cause greater perturbation of membrane structure than the trihydroxy bile salt, cholate, and its conjugates. The results may have implications for the effects of bile salts upon the membranes of liver cells during bile salt secretion and the production of bile.


Effect of anoxia, 2,4-dinitrophenol and salicylate on xylose transport by isolated rat soleus muscle.

1. These studies examined the theory that ATP served to regulate muscle sugar transport by a feedback mechanism. Xylose uptake by isolated rat soleus muscle was determined over a 5-min period following preincubation at 37 degrees C for various times in the presence of insulin (0.1 unit/ml), 2,4-dinitrophenol (0.5 or 0.05 mM) or salicylate (5 mM) or under anaerobic conditions. 2. Xylose uptake, measured in freshly isolated soleus muscles, was approximately 3.5--4.0 mumol/g per h. When the muscles were preincubated at 37 degrees C, this rate fell by 50% during the first 30 min and then slowly increased. 3. The stimulatory effect of insulin was evident within 2 min in freshly isolated soleus muscle and increased on preincubation, reaching a maximum value (approx. 14 mumol/g per h) after 20 min. 4. There was a 10-min lag period before xylose uptake was stimulated by anoxia. This lag period was approximately doubled when the incubation temperature was lowered from 37 degrees C. The stimulatory effect of anoxia was promptly reversed when muscles were transferred from anaerobic to aerobic conditions. 5. There was a 5-min lag period before xylose uptake was stimulated by 2,4-dinitrophenol (0.05 mM) or by sodium salicylate (mM). At a concentration of 0.5 mM, 2,4-dinitrophenol stimulated xylose uptake in freshly isolated muscle. Whereas the stimulatory effects of insulin, anoxia and salicylate all tended to plateau with time, the effect of 2,4-dinitrophenol tended to peak and then decline. 6. There was no obvious relationship between total muscle ATP levels and xylose uptake. The stimulatory effect of anoxia, 2,4-dinitrophenol or salicylate on xylose uptake was not preceded by the fall in muscle ATP. Similarly, ATP levels did not change when xylose uptake was stimulated by anoxia at 27 degrees C, or when xylose uptake was restored to basal values by transferring muscles from anaerobic to aerobic conditions. 7. It was argued that the presence of the myofibrils could act as a permeability barrier, which would limit the access of ATP produced within the interior of the cell to a regulatory site on, or close to, the sarcolemma. On the other hand, it is conceivable that the ATP produced on the periphery of the fibre by the subsarcolemmal mitochondria could play a more specific role in the feedback regulation of sugar transport. 8. Insulin stimulated xylose uptake in the presence of 2,4-dinitrophenol (0.5 mM) when this was measured in freshly isolated muscle, but not after a period of preincubation. This suggested that there may be some ATP-dependent process involved in the stimulatory effect of insulin.


Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements.

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes larger than or equal to 4-10(-5). A minimal concentration of about 6-10(11) dipicrylamine ions per cm2 of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method Ntopt is compared with the concentrations Ntel obtained from simultaneous electrical relaxation meausurements. Ntopt and Ntel agreed at low dipicrylamine concentrations (10(-8)--10(-7) M in the aqueous phase) and showed saturation at higher concentrations (up to 5-10(-6) M). In the saturation range Ntopt was maximally four times higher than Ntel. The significance of this difference is discussed together with general aspects of the saturation phenomenon.


Outside-inside distribution and translocation of lysophosphatidylcholine in phosphatidylcholine vesicles as determinied by 13C-NMR using (N-13CH3)-enriched lipids.

1. The outside-inside distribution of palmitoyl lysophosphatidylcholine and dioleoyl phosphatidylcholine in mixed sonicated vesicles is measured with (N-13CH3)-labelled lipids using 13C NMR and Dy3+ as an impermeable shift reagent. 2. Palmitoyl lysophosphatidylcholine is preferentially localised in the outside layer of the vesicle membrane. Incorporation of cholesterol in the vesicle diminishes the extent of lysophosphatidylcholine asymmetry. 3. Palmitoyl lysophosphatidylcholine added to dioleoyl phosphatidylcholine vesicles is incorporated in the outer monolayer of the vesicle. Even after 40 h less than 2% of the lysophosphatidylcholine could be detected in the inner monolayer. Since in the cosonicated vesicles 17% of the lysophosphatidylcholine is present in the inner monolayer it can be concluded that the transmembrane movement of lysophosphatidylcholine across the lipid bilayer of these vesicles is an extremely slow process.


Transbilayer distribution and movement of lysophosphatidylcholine in liposomal membranes.

Single bilayer vesicles were prepared by sonication of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine. Incubation with lysophospholipase results in a fast hydrolysis of 80-90% of lysophosphatidylcholine. The remaining lysophosphatidylcholine is only very slowly hydrolysed. There results are interpreted as lysophosphatidylcholine being asymmetrically distributed over the two halves of the bilayer. The slow phase of lysophosphatidylcholine hydrolysis sets an upper limit to the rate of transbilayer movement of lysophosphatidylcholine. The half time of this process at 37 degrees C is estimated to be about 100 h. Incorporation of cholesterol in the vesicles reduces the distributional asymmetry of lysophosphatidylcholine to the extent of an outside-inside ratio of 60 : 40 [14C]Lysophosphatidylcholine introduced into the outer monolayer of such vesicles by intervesicular transfer of lysophosphatidylcholine remains virtually completely available for hydrolysis by lysophospholipases, corroborating the interpretation that transbilayer movement of lysophosphatidylcholine in these vesicles is an extremely slow process. In handshaken liposomes consisting of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine 15-20% of lysophosphatidylcholine is readily available for exogenous lysophospholipase. This pool may represent lysophosphatidylcholine in the outer monolayer of the liposomes.


A possible role of the phosphorylation of synaptic membrane proteins in the control of calcium ion permeability.

Incubation of synaptosomes under conditions which result in complete phosphorylation of membrane bound accepter proteins does not affect the permeability to Na+ or K+ as measured by a spectrophotometric method. This technique was not, however, sensitive enough to determine permeability to Ca2+ which was thus estimated, using 45Ca2+. It was found that although phosphorylation did not affect the equilibrium binding of 45Ca it did lower the rate of both Ca2+ uptake and efflux. The most likely interpretation of these results is that phosphorylation of proteins in the synaptic membrane lowers the permeability of the membrane to Ca2+. This could have a role in the regulation of synaptic transmission.


Irreversibel inactivation of the membrane-bound enzyme IIlac of the lactose phosphotransferase system of Staphylococcus aureus by triton X-100 and protection by substrates.

Enzyme IIlac, the membrane-bound component of the lactose phosphotransferase system of Staphylococcus aureus, catalyzes the phosphorylation-transport reaction below: (see article).  (The sugar can be lactose or one of its analogs.) The effects of the non-ionic detergents Triton X-100, Brij 35, and Tween 40 on the activity of Enzyme IIlac were studied. Especially striking effects were observed using Triton X-100, a detergent previously used to solubilize and isolate this enzyme. A systematic study of Triton effects over a range of concentrations and temperatures demonstrated three aspects of Triton-membrane interaction. At 0.1% Triton and 25 degrees C Enzyme IIlac is activated, but remains particulate. At 0.5% Triton and 0.5% Triton and 37 degrees C, it is rapidly and irreversibly inactivated. Sugar substrates and inhibitory sugar analogs protect Enzyme IIlac against inactivation; the effect is specific for beta-galactosides. The other substrate of Enzyme IIlac, phospho-Factor IIIlac, does not affect Triton inactivation, and the product analog galactose 6-phosphate slightly enhances the inactivation rate.