Spectroscopic studies of specifically deuterium labeled membrane systems. Nuclear magnetic resonance investigation of the effects of cholesterol in model systems. Deuterium nuclear magnetic resonance spectra of dimyristoylphosphatidylcholines specifically labeled in positions 2', 3', 4', 6', 8', 10', 12', and 14', of the 2 chain, of an N-deuteriomethylphosphatidylcholine, and of cholesterol-3alpha-d1, have been obtained by the Fourier transform method at 5.46 and 3.52 T on two "home-built" widebore superconducting magnet spectrometers, as a function of temperature and composition. Data on the specifically deuterium-labeled cholesterol molecule (in nonsonicated membrane systems) permits an estimate of the most probable angle of tilt of the sterol in the membrane, and evaluation of the order parameter (Salpha) describing rigid body motions in the bilayer. Segmental order parameters derived from the data presented allow calculation of individual chain segment projections onto the director axis and, consequently, estimation of effective chain length. It is shown that mathematical models which include chain tilt as well as those which neglect this type of rigid body motion give essentially identical results when applied to the dimyristoylphosphatidylcholine and dimyristoylphosphatidylcholine-cholesterol bilayer systems (in excess water, between 23 and 60 degrees C). Results of calculations of chain length and membrane thickness of a dimyristoylphosphatidylcholine-30 mol % cholesterol membrane system at 23 degrees C give excellent agreement when compared with recent high-resolution neutron diffraction data obtained on specifically deuterium labeled lecithin-cholesterol systems. No evidence for formation of lecithin-cholesterol complexes having lifetimes of approximately 30 ms has been found. Below the pure-lipid gel-liquid crystal phase transition temperature Tc but in the presence of cholesterol, we have obtained further evidence for 1-chain/2-chain nonequivalence. At 10 degrees C, the 2' segment of the 2 chain, but not the 2' segment of the 1 chain or the 3', 6', or 12' segments of the 2 chain, is broadened almost beyond detection. These results are in agreement with similar effects reported recently for the dipalmitoylphosphatidylcholine-cholesterol system and may indicate a bent configuration for the 2 chain, in the lecithin-cholesterol system. Further cooling below Tc results in loss of the 1-chain 2'-position signal intensity plus 2-chain 3', 6', and 12' signals simultaneously. The increase in length of the 2 chain of dimyristoylphosphatidylcholine upon addition of 30 mol % cholesterol of 23 degrees C is about 2.3 A. Addition of cholesterol to a choline-labeled lecithin results in complex behavior of the head group deuterium quadrupole splitting as a function of temperature, and cholesterol mole fraction. Above approximately 20 mol % cholesterol, the main effect is a decrease in quadrupole splitting as cholesterol content increases, the opposite effect to that observed with hydrocarbon chains.


Separation and sequence of dipeptides using gas chromatography and mass spectrometry of their trimethylsilylated derivatives. Dipeptides containing all 20 common amino acids in the amino and carboxyl positions were trimethylsilylated under a variety of conditions. The dipeptides (about 200 of the possible 400) were synthesized by conventional means or derived from polypeptides digested with dipeptidyl aminopeptidase. Derivatives were prepared in high yield by reaction with N,O-bis(trimethylsilyl)trifluoroacetamide in acetonitrile. Reaction products were identified by mass spectrometry. Numerous stationary phases and supports were compared in the gas chromatographic analysis of the derivatives. Best results were obtained with a 0.2 X 60 cm column packed with Chromosorb 750 coated with a 1% solution of OV-1. All Me3Si dipeptides except those containing arginine eluted in less than 30 min when the column was temperature programmed from 100 to 280 degrees C. The typical mass spectrum of trimethylsilylated dipeptides contains two significant ions used for identification: the ion derived from the amino terminal residue by beta cleavage of the central CH-CO bond and the molecular ion minus a methyl group. Other useful ions also have been noted. A sufficient variety of samples have been examined to establish predictable fragmentations which should allow the identification of all 400 possible dipeptides.


Probes of the mechanism of zymogen catalysis. Trypsinogen and chymotrypsinogen hydrolyze p-nitrophenyl esters of several peptides and tert-butyloxycarbonyl amino acids. The best substrate found for chymotrypsinogen was Boc-Ala-ONp and for trypsinogen Z-Gly-Hyp-Gly-ONp. Comparison of the kinetic parameters indicates that in the zymogens the catalytic site is distorted and reduced in effectiveness by about two orders of magnitude, in addition to a 10 000-fold decrease in catalysis due to a distortion of the primary substrate binding site (Kerr, M.A., Walsh, K.A., & Neurath, H. (1976) Biochemistry 15, 5566). Using Boc-Ala-ONp as substrate and certain aldehydes and borates as inhibitors, the zymogens were tested for the integrity of the "oxyanion hole", but these results were largely inconclusive. Probes for the secondary binding sites indicated their presence in trypsinogen and their absence in chymotrypsinogen.


Structure of mutagen nucleic acid complexes in solution. Proton chemical shifts in 9-aminoacridine complexes with dG-dC, dC-dG, and dA-dT-dG-dC-dA-dT. The influence of self-complementary oligodeoxynucleotides on the chemical shifts of protons of the mutagenic acridine dye 9-aminoacridine has been measured. Upfield shifts indicative of intercalative binding are found in the cases of dG-dC, dC-dG, and dA-dT-dG-dC-dA-dT but not in dA-dT. Geometries for the complexes that are compatibile with the chemical-shift data and the X-ray structure of the complex between ri5C-rG and 9-aminoacridine determined by Sakore et al. [Sakore, T.D., Jain, S.C., Tsai, C., and Sobell, H.M. (1977), Proc. Natl. Acad. Sci. U.S.A. 74, 188--192] can be identified using recent theoretical estimates of shifts induced by nucleotide bases.


Infrared spectroscopy of polynucleotides in the carbonyl region in H2O solution: A.U systems. Strong absorbance of water at 1645 cm-1 has previously prevented dependable observation of infrared spectra of H2O solutions in the carbonyl region. We have obtained reliable spectra in pure water and in alcohol-water mixtures in this region and used them to study A.U polynucleotide interaction. Spectra in pure water were obtained by computer subtraction of solvent from that of solution measured in short path length cells. Mixed solvents, by diluting the water, permit use of longer cells and standard double-beam compensation methods. Poly(adenylic acid) in H20 solution has a strong NH2 deformation band at 1659 cm-1 coupled to an A ring vibration at 1605 cm-1. Poly(uridylic acid) has a broad band at approximately 1695 cm-1, predominantly C2 carbonyl stretch, an unresolved CR carbonyl band (probably near 1660 cm-1), and a ring vibration at approximately 1636 cm-1. The large changes these spectra undergo when helical structures are formed are discussed and compared with changes observed in D2O.


Organization of 5-methylcytosine in chromosomal DNA. The 5-methylcytosine residues of L-cells have been labeled with [methyl-3H]-L-methionine and their chromatin localization studied using deoxyribonucleases. The kinetics of micrococcal nuclease digestion showed that the methylated cytosine residues are concentrated within regions resistant to nuclease digestion and preferentially missing from those regions between nucleosomes which are nuclease sensitive. Using DNA hybridization kinetic analysis, it is shown that 5-methylcytosine is abundant in highly repeated sequences but is also present in middle repetitive and unique sequence DNA.


T4 polynucleotide ligase catalyzed joining on triple-stranded nucleic acids. dT1O will form triple-stranded complexes with dAn and these complexes can serve as substrate for T4 polynucleotide ligase (EC 6.5.1.1). The rate of phosphodiester formation was found to be approximately the same as for the double-stranded complex and, furthermore, the rate appears to be similar on the two strands in the complex. Joining of dT1O also took place in the presence of the double-stranded complexes dAn.dTn and dAn.rUn. Polyamines increase the rate of joining catalyzed by T4 polynucleotide ligase under certain conditions.


Conformational analysis of pyridoxal Schiff's bases. Nuclear magnetic resonance studies of the conformations about the C4-C4', Calpha-Cbeta, and N-Calpha bonds of the pyridoxal Schiff's bases of amino acids. The solution conformations of a series of pyridoxal-amino acid Schiff's bases were analyzed using 13C and 1H nuclear magnetic resonance techniques. The 13C--1H coupling constants were assigned based on model compounds and isotopic labeling. The predominant conformation of the the C4--C4' bond was found to be "cis" based on nuclear Overhauser effect (NOE) measurements and the "simultaneous" upfield shift of both H4' and H5' in the Schiff's bases of aromatic amino acids. Going from the monoanion (pD 8.2) to the dianion (pD 12.3), changes in these two effects suggested an increasing contribution of the "trans" conformer. The conformation of the N--Calpha bond was found to be approximately the same for all the Schiff's bases studied based on the long-range coupling constants 3J(C4'--Halpha) of these compounds, and the NOE studies indicate that there is a close spatial relationship between H4' and Halpha. The conformations of the Calpha--Cbeta bond of the Schiff's bases of aromatic amino acids were determined by stereospecific deuterium labeling at the beta position. A pi--pi interaction between the aromatic ring and the pi system of pyridoxal was observed which disappeared upon saturation of the aromatic ring.


Conformation-reactivity relationship for pyridoxal Schiff's bases. Rates of racemization and alpha-hydrogen exchange of the pyridoxal Schiff's bases of amino acids. The role of stereoelectronic effects in controlling the reaction specificity of biological reactions involving pyridoxal phosphate-amino acid Schiff's bases was tested with nonenzymatic models. The rates of racemization and Halpha exchange of a series of pyridoxal-amino acid Schiff's bases were determined. The order of these rates does not parallel the predictions based solely on electronic or steric effect, but parallels the proportions of the reactive conformers (e.g., conformers with the Calpha--Halpha bond orthogonal to the pi system) estimated by CPK models. The special reactivity of the phenylalanine Schiff's base was consistent with a special conformation in which some type of pi--pi interaction increases the proportion of exchangeable conformers, thus further substantiating the role of conformation in governing the reactivity of the Calpha--Halpha bond. Furthermore, semiempirical calculations of the conformation about the Calpha00N bond were performed using the CAMSEA conformational analysis program. The results of conformational calculations are consistent with the results of conformational analysis by nuclear magnetic resonance. The order of reactivity of the the Calpha--Halpha bond of the SB dianion, pH 12.0, predicted by calculation based on stereoelectronic effects, though not quantatively parallel to the observed rate constants, is qualitatively in agreement with the experimental results.


Effect of membrane phospholipid compositional changes on adenylate cyclase in LM cells. Adenylate cyclase activities were examined in mouse LM cell membranes which had been supplemented with polar head groups and/or fatty acids. Basal, fluoride-, and PGE1-stimulated activities varied systematically with changes in phospholipid composition, and PGE1-stimulated activities correlated with the average degree of unsaturation of the phospholipid fatty acids or with the primary amino group character of the phospholipid polar head groups. In addition, the Km for ATP of basal adenylate cyclase was systematically changed by both polar head group and fatty acid supplementation. Alteration of the membrane lipid composition also changed the temperature dependence of the enzyme and the lag time between addition of PGE1 and the onset of a change in catalytic rate. However, none of the alterations in the enzyme activity could be correlated with the viscosities of supplemented membranes and, instead, seemed to be characteristic for a specific polar head group or fatty acid composition. The data suggest a specific interaction of the enzyme with phospholipids and indicate that structural features of phospholipids may play a role in regulating adenylate cyclase activity. It is proposed that adenylate cyclase can exist in several different conformations in the membrane depending upon the phospholipid composition.


Further studies on the interaction between polynucleotides and antibodies to poly(inosinic acid). poly(cytidylic acid). Interactions between antibodies to poly(inosinic acid).poly(cytidylic acid) [poly(I).poly(C)] (or Fab fragments) and double-stranded polynucleotides have been studied by several techniques. In quantitative precipitation tests, the amounts of precipitated antibodies depend upon salt concentration. This effect of salt is reversible. From the comparison of the association constants (deduced from fluorescence measurements) for the binding of Fab fragments to several polynucleotides, a direct interaction between atoms or groups of the bases, which can be involved in hydrogen bonds, can be excluded. The complexes between poly(I).poly(C) and Fab fragments interact with ethidium bromide. The association constants for the binding of ethidium bromide to poly(I).poly(C) and to poly(I).poly(C)-Fab fragments complexes are about the same, but Fab fragments reduce the number of base pairs available to ethidium bromide binding. At saturation of poly(I).poly(C) by Fab fragments, the number of binding sites of ethidium bromide is only reduced by a factor of two. It is concluded that all the nucleotide residues covered by the Fab fragment binding site do not interact with the amino acid residues. Circular dichroism studies of the complexes between Fab fragments and polynucleotides are in favor of some conformational change of the polynucleotides.


Characterization of the poly(adenylic acid) sequences in RNA synthesized in vitro by mouse myeloma nuclei. The size and quantity of poly(A)sequences made by mouse myeloma nuclei in vitro are dependent on the concentration of KCI, ATP, other ribonucleoside triphosphates, as well as the nature of the divalent cation in the reaction medium. Reduction of the KC1 concentration from 120 mM to 5 mM, for example, stimulates poly(A) synthesis 10- to 20-fold. These poly (A) sequences are similar in size to cellular nuclear poly(A), but the RNA molecules to which they are attached are much shorter than poly(A) containing RNA molecules made at 120 mM KC1. Presence of Mn2+ in the medium led to a much more heterogenous population of poly(A) sequences. From such observations we have found reaction conditions in which nuclei synthesize molecules that resemble native nuclear poly(A) + RNA. Not only are the lengths and amounts of the poly(A) sequences similar, but they also undergo a terminal turnover like that of the poly(A) in hnRNA. An oligo(A) sequence that resembles the oligo(A) found in non-poly(A) containing hnRNA of mouse myeloma and HeLa cells is also synthesized in vitro. These observations suggest that some processing functions are retained during the in vitro incubation of these nuclei.


Procollagen complementary DNA, a probe for messenger RNA purification and the number of type I collagen genes. Type I procollagen mRNAs were separated from contaminating low-abundance messenger and nuclear RNAs by chromatography over Sepharose 4B in 0.65 M NaCl at room temperature. All of 27S rRNA and four-fifths of procollagen mRNAs bind to Sepharose under these conditions, while 18S rRNA and about three-fourths of other poly(A)-containing RNAs do not bind. AMV reverse transcriptase was used to prepare complementary DNA to procollagen mRNA at each purification step. Hybridization studies, in RNA excess, were carried out to establish the enrichment at each step both with respect to total RNA and to poly(A)-containing RNA. While "purified" procollagen mRNA preparations still consist of about 50% 27S rRNA, over 80% of cDNA prepared from it back hybridizes to its template at a log of cr0t1/2 of -1.9. This type I procollagen cDNA hybridizes in DNA excess to DNA isolated from chicken erythrocytes and from embryonic chick calvaria at a log c0t1/2 of 3.1, demonstrating that procollagen cDNA is complementary to unique gene sequences in both tissues and that procollagen genes are not reiterated.


Developmental regulation in cotton seed germination: polyadenylation of stored messenger RNA. Evidence that RNA preexisting in the cotyledons of mature cotton seed (stored mRNA) is polyadenylated during the first day of germination is presented, based on three different experimental data sets. First, actinomycin D is found to inhibit 32PO4 incorporation into mRNA-poly(A) by 62%, into mRNA by 70%, but into poly(A) only 30%. Second, far more 32PO4 and [2-3H]adenosine are incorporated into the poly(A) portion of mRNA-poly(A) than into the mRNA portion as would be expected from their relative sizes and base composition. This underlabeling of the mRNA moiety is enhanced when cotyledons are germinated in actinomycin D. However, an expected distribution of the isotopes between the mRNA and poly(A) moieties is found in cotyledons labeled later in germination. Third, spectral measurements of the absolute amount of mRNA-poly(A) accumulated during the first day of germination in cotyledons germinated in actinomycin D are larger than would be expected from the 70% inhibition of mRNA labeling caused by the drug. The three sets of data suggest that over 50% of the total mass of mRNA polyadenylated during early germination exists in the mature seed. Its complexity, however, has not been measured. These data may explain the sensitivity of much of germination enzyme synthesis to inhibition by 3'dAdo during early germination and its insensitivity to actinomycin D during this period.


Biochemical and electron microscopic characterization of DNA-RNA complexes from HeLa cell mitochondria. The previous electron microscopic investigations on the occurrence in HeLa cell mitochondria of transcription complexes of mitochondrial DNA [Aloni, Y., and Attardi, G. (1972a), J. Mol. Biol. 70, 363-373] have been extended with the aim of obtaining these complexes in a reasonably pure form for biochemical analysis. By using conditions designed to minimize losses of such structures and any possible contamination by nuclear DNA, it has been shown that a substantial fraction (40 to 50%) of mitochondrial DNA can be isolated from exponentially growing HeLa cells in the form of fastsedimenting complexes with RNA. These complexes have been characterized with respect to density and sedimentation properties, content in newly synthesized RNA, stability of the association of RNA with DNA, presence of different forms of mitochondrial DNA, and electron microscopic appearance. The properties of these complexes, as well as the results of reconstruction experiments, strongly suggest that the majority of such structures represent true transcriptional intermediates. The occurrence in this fraction of replicating or newly replicated mitochondrial DNA molecules has been observed. Although the presence of single-stranded DNA segments makes the replicative intermediates particularly susceptible to aggregation with free RNA, electron microscopic observations point to the possibility that these intermediates may be recruited for transcription.


Covalent structure of collagen: amino acid sequence of five consecutive CNBr peptides from type III collagen of human liver. Type III collagen was solubilized from human liver by limited pepsin digestion and purified by differential salt precipitation and carboxymethylcellulose chromatography. Digestion with cyanogen bromide yielded the nine distinct peptides previously described and an additional tripeptide not recognized in earlier studies. Five of these peptides, alpha1 (III)-CB1, 2, 4, 8, and 10, were further purified by molecular sieve and/or ion exchange chromatography. They contained 12, 40, 149, 125 and 3 amino acid residues, respectively. The amino acid sequence of these peptides was determined by automated Edman degradation of tryptic (before and after maleylation), chymotryptic, thermolytic or hydroxylamine-derived peptide fragments as well as the intact peptides. The alignment of these five peptides within the collagen chain is deduced to be 1-8-10-2-4 by homology with known alpha1 (I) sequences. The known CNBr peptide alignment of the NH2-terminal portion of type III collagen so far would, therefore, be alpha1 (III)-CB3-7-6-1-8-10-2-4 and correspond to the homologous region of alpha1 (I)-CB0-1-2-4-5-8-3 or residues 11-567 of the alpha1 (III) collagen chain.


Structural studies on rabbit transferrin: isolation and characterization of the glycopeptides. The structure of rabbit transferrin was investigated with regard to number, size, and composition of the heteropolysaccharide units and their relative location on the polypeptide chain. The composition and molecular weight of the Pronase glycopeptides revealed that rabbit transferrin contains two heteropolysaccharide units, each composed of 2 sialic acid residues, 2 galactose residues, 3 mannose residues, and 4-N-acetylglucosamine residues. The composition and molecular weight of the tryptic glycopeptides further substantiated the existence of two identical heteropolysaccharide units and revealed that both units have identical amino acid residues in the immediate vicinity of the carbohydrate attachment sites to the polypeptide chain, suggesting a sequence homology surrounding the two glycosylation sites. Characterization of the cyanogen bromide fragments from rabbit transferrin indicated that both heteropolysaccharide units are located within a single polypeptide fragment representing approximately one-third of the molecule.


Use of dyes to estimate the electrical potential of the mitochondrial membrane. A number of cationic or anionic fluorescent dyes were investigated as possible monitors of the membrane potential of rat liver mitochondria, and giant mitochondria isolated from the liver of mice maintained on a diet containing cuprizone. The fluorescence of four dyes (8-anilino-1-naphthalenesulfonic acid, merocyanine 540, 3,3'-dipropyl-thiocarbocyanine, and bis[1,3-dibutylbarbituric acid-(5)]-pentamethine oxonol) was found to respond appropriately to changes in an apparent K+ diffusion potential. Generally, valinomycin-induced K+ diffusion potentials as calculated using the Nernst equation were used to calibrate the dependence of the fluorescence on the membrane potential. The appropriateness of this approach was verified for two dyes using microelectrodes in giant mitochondria. The apparent membrane potential change induced by the addition of succinate was variable but was very low and generally less than 60 mV in magnitude. The results are consistent with the notion that a large membrane potential is not established upon the initiation of metabolism and that the membrane potential does not play a significant role in the observed ADP phosphorylation.


Sulfhydryls of platelet tubulin: their role in polymerization and colchicine binding. Sulfhydryls and disulfides of platelet tubulin have been quantified, their accessibility and reactivity measured, and their role in polymerization and colchicine binding evaluated. Platelet tubulin isolated by two cycles of temperature-dependent polymerization--depolymerization was found to contain 12 free sulfhydryl groups per tubulin monomer all of which reacted rapidly with p-chloromercuribenzoate. One sulfhydryl was inaccessible to dithiobis(nitrobenzoic acid). Under anaerobic conditions of tubulin extraction, one intrachain disulfide bridge was found per tubulin monomer. Polymerization of tubulin reduced the number of sulfhydryls by one which were able to react with p-chloromercuribenzoate or dithiobis(nicotinic acid) but did not affect the disulfide bridge. Polymerizability of platelet tubulin was very sensitive to blocking of free sulfhydryl groups. Complete inhibition of microtubule assembly was obtained when the number of free sulfhydryls per tubulin was reduced by 3 but could be reversed by the addition of dithiothreitol. Colchicine binding, on the other hand, was only minimally influenced by blocking of sulfhydryls.


Isolation of six novel phosphoinositol-containing sphingolipids from tobacco leaves. A concentrate of phosphosphingolipids from tobacco leaves has been resolved into two groups by chromatography on diethylaminoethylcellulose. The first group eluted contains no acetyl residues, whereas the second group contains one N-acetyl/mol of phosphorus. Three lipids from each group have been purified by chromatography on Porasil columns. The chemical composition of these six novel lipids shows them to be related to the previously characterized phosphosphingolipids PSL-I, N-acetylglucosamine[glucuronic acid, inositol phosphate]ceramide and PSL-II, glucosamine[glucuronic acid, inositol phosphate] ceramide [Kaul, K.,and Lester,R. L. (1975), Plant Physiol. 55, 120]. The novel lipids are: PSL-IA, PSL-I-[Ara2Gal2]; PSL-IB, PSL-I-[Ara3Gal2]; PSL-IC, PSL-I-[Ara4Gal2]; PSL-IIA, PSL-II-[Ara3Gal]; PSL-IIB, PSL-II-[Ara2or3Gal2]; PSL-IIC, PSL-II-[Ara2Gal2Man]. The unusual finding of unacetylated hexosamine in the PSL-II group of lipids appears not to be an artifact, as suggested by experiments involving rapid harvesting and processing of the tobacco leaves. Some of the fractions obtained by chromatography on diethylaminoethylcellulose appear to contain phosphosphingolipids with 16 or more sugar residues.


Fusion of phosphatidylcholine bilayer vesicles: role of free fatty acid. The transformation of DMPC vesicles containing myristic acid to larger structures was examined by nuclear magnetic resonance techniques. The process was found to be characterized by a rate which is approximately first order in vesicle concentration. Rates, however, show a dramatic increase with increasing myristic acid content. The fatty acid is characterized as being active under conditions in which it is neutral and dispersed in the bulk bilayer phase. In addition a 1:1 correlation was noted between leakage of contents and vesicle transformation. On the basis of these observations a mechanism is postulated involving stabilization of leaks in bilayer membranes by myristic acid, and the resultant formation of activated sites for vesicle transformation. The relation to fusion in natural systems is discussed.


Ligand binding sites and subunit interactions of Torpedo californica acetylcholine receptor. A [3H]bisazido derivative of ethidium bromide was synthesized to identify sites of interaction of ethidium with the acetylcholine receptor from Torpedo californica and to aid in localization of ligand binding sites. For purified solubilized acetylcholine receptor it was shown (a) that the photolabel was competitive with ethidium bromide, (b) dodecyl sulfate--polyacrylamide gel electrophoresis revealed that all four polypeptide components were labeled with [3H]ethidium azide, and (c) alpha-bungarotoxin inhibited the labeling of the 40 000-dalton subunit. Photolabeling of acetylcholine--receptor enriched membrane fragments led to the following conclusions: (a) the photochemical reaction was more selective than for purified acetylcholine receptor, since the 40 000-dalton subunit was preferentially labeled; this result demonstrated differences in the topography of receptor subunits depending on whether the molecule was in detergent solution or in a membrane-bound state, (b) alpha-bungarotoxin inhibited labeling of the 40 000-dalton subunit, (c) ligand-induced conformational changes resulted in different subunit labeling patterns. The results imply that conformational changes generated at the 40 000 molecular weight subunit upon cholinergic ligand interaction cause further intermolecular structural changes that involve subunits of higher molecular weight. These higher molecular weight subunits therefore belong to a supramolecular complex of polypeptides associated with the postsynaptic membrane.


Conformation examination of uridine diphosphoglucose using lanthanide-nitrilotriacetate chelates as shift probes. A 13C NMR study utilizing the lanthanide-induced shift (LIS) technique was carried out on uridine diphosphoglucose (UDPG) in order to determine the solution conformation. The neutral, water soluble nitrilotriacetate chelates, Ln(NTA), were used in preference to the bare ions due to the potency of the latter in hydrolyzing the UDPG. In addition to the primary binding site at the pyrophosphate moiety, secondary binding to the uracil carbonyl groups was observed and found to be a function of the size of the lanthanide ion; the largest ions showed the greatest relative affinity for the carbonyl groups. This effect was overcome by extrapolating the observed shift ratios to zero Ln(NTA)/[UDPG]. The extrapolated shift ratios were relatively constant for a series of five lanthanides indicating the solution conformation of UDPG is insensitive to metal ion size. The conformation of the UDPG was found to be extended so that the uracil carbons as well as carbons 1', 2', and 3' of the ribose are outside of the pseudo-contact shift cone with the remaining carbons inside the cone.


Isolation of the ADP, ATP carrier as the carboxyatractylate . protein complex from mitochondria. The procedure for the isolation from mitochondria of the undenatured ADP, ATP carrier is described. The condition of retaining the nativity are elaborated. 1. As indicator for the ADP, ATP carrier (35S)- or (3H) carboxyatractylate were used. By preloading the mitochondria with carboxyatractylate, a stable carboxyatractylate . protein complex could be retained after solubilization with Triton X-100. Among the polyoxyethylene detergents emulphogen is also solubilizing, whereas Brij and Lubrol fail to solubilize. 2. When unloaded mitochondria are solubilized the capacity for binding carboxyatractylate disappears rapidly, particularly at 20 degrees C. 3. When mitochondria are preloaded with atractylate, the binding after solubilization with Triton X-100 is considerably lower than with carboxyatractylate, indicating that the high affinity of carboxyatractylate is required for effectively protecting the protein. 4. For purification hydroxyapatite is most effective. The carboxyatractylate-protein complex appears in the pass-through whereas the bulk of other mitochondrial proteins are retained such that a 7-fold purification is obtained. The nonadsorptivity to hydroxyapatite is dependent on the undenatured state maintained in the carboxyatractylate . protein complex. 5. Subsequent gel filtration on Sepharose results in a 1.5-fold further enrichment of specific carboxyatractylate binding up to 17 mumol/g protein, corresponding to a 10-fold purification from mitochondria. This value cannot be increased with further measures. 6. At the last purification step, in sodium dodecyl sulfate polyacrylamide gel electrophoresis virtually a single band of 30 000 molecular weight is found, confirming the purity at this stage. A molecular weight of 60 000 is calculated from the carboxyatractylate binding, indicating that the carboxyatractylate protein complex consists of two 30 000 subunits. From this the protein share of the ADP, ATP carrier in beef heart mitochondria can be calculated to amount to 9.5%9 7. The intact carboxyatractylate . protein complex is protected against proteolytic degradation. The release of carboxyatractylate ensues a conformational change of protein as assayed by conformation specific antibodies, concomitant with unmasking of proteolytic site as assayed by tryptic digestion. 8. The amino acid composition indicates hydrophobicity (39% polarity) and a high content of basic amino acid such as lysine and arginine. There is 1.5 mol percent cysteine and a blocked N-terminal. 9. From the solubilized complex (35S) carboxyatractylate can be removed by carboxyatractylate, ADP and ATP but not by ITP, etc., indicating the presence of recognizing sites specific fof ADP, ATP and therefore, identity with the ADP, ATP carrier. 10. Other reported procedures for isolating the ADP, ATP carrier are shown to either fail or have lower yield than the present, original procedure.


Glutamate transport in pig heart mitochondria. Binding and structural properties of high-glutamate affinity proteolipid: reconstitution studies. Previously, a proteolipid that can bind glutamate with high affinity has been isolated from pig heart mitochondrial membranes. A final affinity chromatography on gamma-methylglutamate-albumin coreticulated on glass fiber was necessary. This procedure includes long dialysis steps which tend to denature the high-glutamate affinity proteolipid. Here is described a new method of isolation which avoids long dialysis steps and yields greater amounts of the high-glutamate affinity proteolipid. The binding of glutamate or aspartate on high-glutamate affinity proteolipid has been studied by gel filtration, by equilibrium dialysis or by a new procedure of rapid centrifugation based on the insolubility of high-glutamate affinity proteolipid in water. The latter method permits the detection of low and high affinity sites for glutamate with a Kd 60 mM and 55 muM, respectively. Among a series of analogues, aspartate appeared to be the best competitor: Kd = 30 muM and two Ki values, 0.37 mM (at high glutamate concentration) and 3.8 muM (at low glutamate concentration). High-glutamate affinity proteolipid binds 0.4 nmol of glutamate but only 0.1 nmol of aspartate per mg protein. The sites for glutamate and aspartate appear to be different but interdependent. In the presence of high-glutamate affinity proteolipid, externally added glutamate stimulated the efflux of aspartate from preloaded liposomes. High-glutamate affinity proteolipid contains cardiolipin, phosphatidyl choline and phosphatidyl ethanolamine the distribution of which is different from that of the inner membrane. The effects of various phospholipases, trypsin, and thiol reagents were studied on the binding of glutamate. High-glutamate affinity proteolipid binds 9 nmol N-ethylmaleimide per mg protein but only 6.1 nmol in the presence of glutamate. The dissociation of high-glutamate affinity proteolipid caused by thiol reagents yielded a soluble protein fraction with higher affinity for glutamate. Electrophoresis and an immunological approach allowed the detection and titration of the glutamate dehydrogenase and aspartate aminotransferase present in high-glutamate affinity proteolipid in inhibited forms, the latter being 26-fold more concentrated than the former.


Studies on the uptake of porphyrin by isolated rat liver mitochondria. 1. The uptake of deuteroporphyrin by isolated rat liver mitochondria proceeds by two different mechanisms, a passive binding, and a mechanism sensitive to CCCP plus valinomycin, with different pH, temperature and time dependencies. 2. The CCCP plus valinomycin-sensitive uptake of deuteroporphyrin parallels the transmembrane potassium gradient ([K+in]/[K+out]). 3. Only that deuteroporphyrin taken up in parallel to the transmembrane potassium gradient is accessible to ferrochelatase. 4. The uptake of deuteroporphyrin at high concentrations is followed by series of damaging effects on the mitochondria: uncoupling, dissipation of the mitochondrial energy potential, increased ion permeability and leakage of endogenous potassium. 5. The detrimental effects of porphyrins at high concentrations on mitochondrial structure might explain the apparently unrelated metabolic aberrations characteristic of certain porphyric diseases.


Derivative absorption spectroscopy from 5--300 K of Prosthecochloris aestuarii. Absorption spectra of the bacteriochlorophyll a-protein from Prosthecochloris aesturaii were measured at temperatures from 2.9 to 300 K. Fourth and eight derivatives of the spectra were calculated from the digital data. From an analysis of 34 scans taken from 750 to 850 nm at 5 K, and 130 scans taken from 822 to 838 nm, we find evidence for nine peaks, six of which are probably 0--0 excitonic and three probably higher vibronic features. The major peaks are resolved in the derivative spectra to 300 K, and all shift with temperature by less than 1 nm compared to their 5 K positions, except for the 825 nm peak which shifts about 2 nm. The most prominent fourth derivative peak at 300 K shifts from 812.9 nm in the standard buffer solution to 814.1 nm in the cryogenic solution in which our low temperature measurements were made. We conclude that the conformation of the protein at 5 K is essentially the same as at 300 K.


Determination and modification of the redox state of the secondary acceptor of photosystem II in the dark. The redox state of the secondary electron acceptor B of Photosystem II was studied using flourescence measurements. Preillumination of algae or chloroplasts with a variable number of short saturating flashes followed rapidly by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea induces oscillations of the initial level of fluorescence. The phase of these oscillations is characteristic of a given B/B-ratio in the dark-adapted samples. We conclude from our results that about 50% of the secondary electron acceptors are singly reduced in the dark in chlorella cells, but that more than 70% are fully oxidized in the dark adapted chloroplasts. Benzoquinone treatment modifies this distribution in Clorella leading to the same situation as in chloroplasts, i.e. more than 70% of the secondary acceptors are oxidized in the dark. The same ratio is observed if these algae are illuminated and then dark-adapted, unless an artificial donor (hydroxylamine) is added before this illumination. In that case about 50% B- is generated and stabilized in the dark.


Interactions between photosystem II components in chloroplast membranes. A correlation between the existence of a low potential species of cytochrome b-559 and low chlorophyll fluorescence in inhibited and developing chloroplasts. 1. Chloroplasts inhibited by incubation with hydroxylamine in the light exhibit a low fluorescence yield upon illumination in the presence of dithionite sufficient to completely reduce the primary acceptor, Q. In the absence of magnesium ions, the fluorescence yield is the same as in control chloroplasts, suggesting that the reason for the low yield is a defect in the mechanism by which Mg2+ enhances the fluorescence. These chloroplasts were previouly shown to contain only low potential (Em7.8 = +80 mV) cytochrome b-559 (Horton, P. and Croze, E (1977) Biochim. Biophys. Acta 462, 86-101). 2. In Photosystem II particles, in heat-treated chloroplasts and in trypsin-digested chloroplasts, high potential cytochrome b-559 is absent and the variable fluorescence yield is again low. 3. Peas grown under intermittent light contain only one-fifth of the content of high potential cytochrome b-559 seen in fully greened plants, yet show high rates of water to methyl viologen electron transport. Aquisition of the high potential cytochrome b-559 accompanies synthesis of chlorophyll b, the onset of Mg-stimulated fluorescence and an increased variable yield of fluorescence. A similar correlation was seen during greening of dark-grown barley. 4. It is proposed that the high potential state of cytochrome b-559 is due to the same membrane properties which allow cation enhanced variable fluorescence, so that the presence of low potential cytochrome b-559 is accompanied by a decrease in variable fluorescence yield.


Phospholipid substitution of the purple membrane. The stoichiometry of light-induced proton release by phospholipid-substituted purple membranes. The method of Warren et al. (1974, Proc. Natl. Acad. Sci. U.S. 71, 622--626) was employed to substitute the polar lipids of the purple membrane of Halobacterium halobium by different phosphatidylcholine species. Substitution at pH 6.5 yields proteolipid complexes in the form of bent open sheets which have a protein to lipid phosphorus ratio similar to the natural membrane, i.e. about 1 : 10 (mol/mol). The extent of substitution increases with the length of the fatty acid chain of the phosphatidylcholine used. The spectral properties of bacteriorhodopsin are only slightly affected by substitution of 95% of the lipid, except that the photocycle is slowed down appreciably. Due to this slow rate the M412 intermediate of the cycle accumulates in the light. Associated with this accumulation is a net light-induced proton release, which proved insensitive to uncoupler. A comparison between the net proton release and the amount of M412 accumulated, studied as a function of pH, shows that no fixed stoichiometry exists between the two processes. Phospholipid substitution by egg phosphatidylcholine at pH 7.5 or by egg phosphatidylethanolamine leads to preparations of purple membrane with 15 or 25 mol of phospholipid per mol of bacteriorhodopsin, respectively. These preparations seem to consist of closed membrane structures. They take up protons in the light in an uncoupler-sensitive way.


Metabolic effects of some electrofluorimetric dyes. The effect of five electrofluorimetric dyes on mitochondrial metabolism was examined to determine their suitability for mitochondrial studies and other biological uses. The dyes merocyanine 540, 8-anilino-1-naphthalene sulfonic acid and bis(1,3-dibutyl barbituric acid-(5))-pentamethane oxonol were found to be inhibitors of the respiratory chain. However, the first two exerted their effect only at high concentrations. 3.3'-Dihexyl-2,2'-oxacarbocyanine was found to act as an uncoupler. 3,3'-Dipropyl-thiocarbocyanine inhibited beta-hydroxybutyrate respiration while dissociating succinate supported respiration from the phosphorylation of ADP. Merocyanine 540 and 8-anilino-1-naphthalene sulfonic acid may be the best suited for studies of membrane potentials in mitochondria since their effect on metabolism is negligible


Mitochondrial phosphate transport and the N-ethylmaleimide binding proteins of the inner membrane. Mitochondria have been prepared from the flight muscles of mature blow-flies (Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome A inhibit the swelling by 90%. When the mitochondria are inhibited by N-[3H]ethylmaleimide, then solubilized in dodecyl sulfate/mercaptoethanol at 100 degrees C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the N-[3H]ethylmaleimide-labeled mitochondria, solubilized, and electrophoresed on dodecyl sulfate-polyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 . 10(3). The nmol N-[3H]ethylmaleimide bound to each of these proteins per nmol cytochrome A are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol N-[3H]ethylmaleimide/mol cytochrome a at 10 micron cytochrome A. Coty and Pedersen (1975) J. Biol. Chem. 250, 3515-3521) sensitized rat liver mitochondria to N-[3H]ethylmaleimide and identified five labeled proteins. Only the labeled 32 . 10(3) dalton and the 45 . 10(3) dalton proteins are common to both systems.


Linear dichroism of electric field oriented bacteriochlorophyll alpha-protein from green photosynthetic bacteria. Bacteriochlorophyll alpha-protein from Prosthecochloris aestuarii strain 2K was oriented in a pulsed electric field. The room temperature linear dichroism spectrum of the oriented protein in the Qy region of the bacteriochlorophyll alpha absorption exhibits a single asymmetrical peak at 813 nm with a shoulder extending to the blue. The approximately equal 12 nm fullwidth of the linear dichroism peak is only about half that of the 300 K absorption spectrum. The linear dichroism at 813 nm was not saturated at field strengths of up to 15 kV/cm. The time dependence of the linear dichroism suggests that the orienting particles are aggregates of at least some tens of bacteriochlorophyll alpha-protein trimers. The linear dichroism peak coincides in wavelength with the 813-nm peak of the 300 K, 4th derivative absorption spectrum of the protein and is therefore attributed to the bacteriochlorophyll a Qy exciton transition observed in absorption at the same wavelength.


Photovoltages in suspensions of magnetically oriented chloroplasts. The photovoltage of suspensions of magnetically oriented chloroplasts using polarized light of 680 nm has been measured. The magnitude of the photo e.m.f. depends on the polarization of the light and on its direction of propagation with respect to the oriented thylakoid planes. This photo e.m.f. is qualitatively attributed to the Dember effect which arises when inhomogeneous light absorption gives rise to a gradient of positive and negative charges along x , where x is the direction defined by the propagation vector of the light and which is also the direction joining the two electrodes. The photovoltage obtained with the planes of the oriented thylakoids parallel to x depends on the plane of polarization of the incident light and shows that (1) the magnitude of the photovoltage depends on the absorption coefficient (which itself is polarization dependent) and thus on the magnitude of the charge gradient produced by the inhomogeneously absorbed light, and (2) a charge gradient within the planes of the thylakoids can give rise to the macroscopic photovoltage. While our experimental observations are basically in agreement with those previously reported (Fowler, C.F. and Kok, B.(1974) Biochim. Biophys. Acta 357, 308-318 and Witt, H.T. and Zickler, A. (1973) FEBS Lett. 37, 307-310) for unoriented chloroplasts, their interpretation of the origin of this effect in terms of a transmembrane potential must be modified in view of our results obtained with oriented chloroplasts. The macroscopically observed photovoltage of oriented chloroplasts is due to the creation of charge gradients either parallel or perpendicular to the thylakoid planes by a flash of light, the diffusion of these charges and to differences in the mobilities of the negative and positive charges. This interpretation in terms of the Dember effect is completely consistent with the existence of a transmembrane electric field as proposed by Fowler and Kok, as well as by Witt and Zickler. However, macroscopic measurements of the photovoltage using either oriented or unoriented chloroplast suspensions cannot prove that a transmembrane voltage exists, as previously claimed.


A molecular basis for an irreversible thermodynamic description on non-electrolyte permeation through lipid bilayers. The non-electrolyte permeability of liposomal membranes has been investigated according to the concepts of irreversible thermodynamics. A strong interaction between the permeation of solute and water was observed. This solute-solvent interaction can be fully described by assuming that a number of water molecules will copermeate with each molecule of solute. This number of copermeating water molecules is independent of the nature of the permeant and of temperature, but depends on the osmotic concentration of impermeants inside the liposomes.


Non-electrolyte permeability as a tool for studying membrane fluidity. 1. The reflection coefficient for the permeation of thiourea through bilayers of phosphatidylcholine is a function of the fatty-acid composition of the lipid molecules. By means of these reflection coefficients an index for membrane fluidity has been given to each of those lipids, relative to that of egg phosphatidylcholine. 2. The maximum number of water molecules that can copermeate with each molecule of solute by means of solute-solvent interaction is a function of the packing of the lipid molecules in the bilayer. This parameter has been used in this paper for characterizing the fluidity of cholesterol-containing membranes and for membranes with their lipids in the gel state.


Pretransitions in the hydrocarbon chains of phosphatidylethanolamines. A wide angle X-ray diffraction study. The hydrocarbon chain packing of fully hydrated phosphatidylethanolamine multilayers is investigated by X-ray diffraction. An analysis of the wide angle reflections (short spacings) as a function of temperature indicates that, apart from the well-known ordered-disordered lipid phase transition, a second transition takes place at lower temperatures. This transition, which is in the present paper referred to as the pretransition, is characterized by a transformation of the hydrocarbon chain packing. A first model for the chain lattice is presented, which gives rise to the expectation that similar pretransitions might be found with other phospholipids.


Surface potential of phosphatidylserine monolayers. I. Divalent ion binding effect. Surface potentials of phosphatidylserine monolayers have been measured in the presence of different divalent ion concentrations in order to determine the way in which divalent ions bind to the membrane surface. The association constants for divalent ions (Mg2+, Ca2+ and Mn2+) with the phosphatidylserine membrane have been obtained from the experimental data and simple ion binding theory. The order of divalent ion binding to the membrane is Mn2+ greater than Ca2+ greater than Mg2+. However, none of the divalent ions used completely neutralized the negative charge of phosphatidylserine even at relatively high concentrations. The amounts of the divalent ions bound depended upon the concentration of the monovalent ions present in the subphase. It is suggested that the amounts of bound ions obtained from the use of radioisotope tracer methods may include a considerable contribution from the excess free ions in the double layer region of the phosphatidylserine membrane.


The interaction of detergents with bilayer lipid membranes. Detergents are widely used for extracting and purifying membrane proteins. Four such detergents have been studied to find the extent to which they alone can alter black lipid film conductances. The slope of the plot of conductivity versus concentration for Triton X-100 is 4.54 in the range 0.025--0.15 mM; dodecyl sulphate 0.82 in the range 0.01--1 mM; sodium deoxycholate 1.03 in the range 0.01--1 mM and sodium cholate 1.37 in the range 0.1--10 mM. These ranges are below the respective critical micelle concentrations; above these concentrations the membranes break. Bilayer lipid membrane conductivity measured at constant detergent concentration increases with the conductivity of the bathing salt solution with a slope greater than 1, indicating an effect on the putative pore structures induced by detergents.


Effect of amphotericin B on cholesterol-containing liposomes of egg phosphatidylcholine and didocosenoyl phosphatidylcholine. A refinement of the model for the formation of pores by amphotericin B in membranes. (1) Binding and K+-permeability measurements were performed on egg and 22 : 1c/22 : 1c-phosphatidylcholine liposomes with or without cholesterol. (2) Amphotericin B binds specifically to cholesterol in both types of liposome despite the difference in bilayer thickness. (3) Addition of amphotericin B to one side of the cholesterol-containing egg phosphatidylcholine bilayers induces a fast K+ efflux from the outermost compartment of the liposomes. In contrast, the total K+ content of sonicated unilamellar cholesterol-containing egg phosphatidylcholine vesicles is released by amphotericin B. (4) Amphotericin B addition to one side of the cholesterol-containing 22 : 1c/22 : 1c-phosphatidylcholine liposomes does not cause a change in K+ permeability. The presence of amphotericin B on both sides of the bilayer, however, induces an increase in K+ permeability. (5) A model is proposed which accounts for the effect of bilayer thickness on the amphotericin B-induced permeability changes in membranes.


The temperature dependence of erythrocyte water diffusion permeability. 1. The activation energy of the diffusion water exchange in red blood cells increases with temperature. 2. Fetal blood has a higher activation energy for diffusion water exchange than adult blood. 3. Treatment of red cells with p-chloromercuribenzoate alters the activation energy and apparently allows a lipid and a protein pore pathway to be resolved. The permeability and activation energy of the treated cells is in the range found for lipid membranes; and the difference between treated and untreated cells, the "protein" pathway, has a diffusion activation energy comparable with that of free water. 4. A resolution of the discrepancies between the NMR methods of measuring diffusion water exchange is suggested.


Binding sites of iron transferrin on rat reticulocytes. Inhibition by specific antibodies. 1. In the process of iron uptake by precursors of the erythrocytes probably more than one membrane component is involved; besides the specific transferrin receptor, another membrane component with a high iron activity after incubation with 59Fe can be isolated. 2. A striking resemblance exists between rat and human reticulocyte components which are involved in the process of iron uptake. 3. Incubation of reticulocytes with Fab fragments of an antibody against the membrane receptor for transferrin causes a concentration-dependent decrease in transferrin binding and iron uptake. 4. The membrane receptor complex isolated is still heterogeneous; analytical ultracentrifugation studies suggest a molecular weight lower than 230,000. 5. Intact immature red cells are necessary for specific binding of transferrin with the receptor followed by iron uptake. This is the only mechanism for iron uptake. 6. Immunofluorescence studies showed that the receptor for transferrin is localized at the outside of the cell membrane.


Adaptation of biological membranes to temperature. The lack of homeoviscous adaptation in the sarcoplasmic reticulum. Temperature adaptation of biological membranes was examined by comparing the fragmented sarcoplasmic reticulum preparation of goldfish acclimated to different temperatures. Membrane fluidity was estimated using the fluorescence polarization technique. There was considerable variation between preparations, but no consistent differences in fluidity were observed between 5- and 25 degrees C-acclimated goldfish, fish species adapted over an evolutionary period to arctic or desert temperatures, and rat. The fatty acid composition of the sarcoplamic reticulum preparations of differently acclimated goldfish showed differences in the proportion of mono- and polyunsaturated fatty acids while the proportion of saturated fatty acids remained relatively constant. However, the fatty acid composition of sarcoplasmic reticulum phosphoglycerides became more unsaturated in the order rat, desert pupfish, arctic sculpin, which correlates with their respective environmental or body temperature. It is concluded that differences in membrane components other than fatty acids are important in determining membrane dynamic structure. The inability to demonstrate homeoviscous adaptation in sarcoplasmic reticulum is supported by other evidence suggesting that functions of the sarcoplasmic reticulum that are measured in vitro are not affected by such modifications of their phosphoglyceride fatty acid composition as occur during thermal acclimation.


Loss of function of biomembranes and solubilization of membrane proteins during freezing. Isolated thylakoid membranes are damaged during freezing in dilute salt solutions, as shown by the inactivation of photochemical thylakoid reactions. After freezing, a number of membrane proteins were found in the particle-free supernatant. Up to 5% of the total membrane protein was solubilized by freezing, and the pattern of released proteins as seen in sodium dodecyl sulfate gel electrophoretograms was influenced by the nature of the solutes present. Membranes protected by sucrose did not release much protein during freezing. Concentrated salt solutions caused protein release also in the absence of freezing. Among the proteins released were ferredoxin--NADP+ reductase, plastocyanin and coupling factor CF1. Subunits of CF1 were found in different proportions in the supernatants of thylakoid suspensions after freezing in the presence of different salts. Cyclic photophosphorylation was largely inactivated before significant protein release could be detected. It is suggested that protein release is the final consequence of the nonspecific suppression of intramembrane ionic interactions by the high ionic strength created in the vicinity of the membranes by the accumulation of salts during slow freezing. Salt effects on water structure and alterations of nonpolar membrane interactions by the incorporation of (protonated) lipophilic anions from organic salts into the membrane phase during freezing may also be involved.


Sugar uptake into brush border vesicles from dog kidney. II. Kinetics. The kinetics of D-glucose transport over the concentration range 0.07--20 mM have been investigated in a vesiculated membrane preparation from dog kidney cortex. 1. A sodium-dependent and a sodium-independent component of D-glucose uptake are observed. The sodium-dependent component is phlorizin sensitive (KI approximately 0.6 micron) and electrogenic. 2. The sodium-dependent component of D-glucose uptake yields non-linear Eadie-Hofstee plots consistent with the presence of high (GH) and low (GL) affinity sites (KH approximately 0.2 mM, KL approximately 4.5 mM, VL/VH approximately 7 at pH 7.4, 25 degrees C, 100 mM NaC1 gradient). Alternative explanations are cooperative effects of non-Michaelis-Menten kinetics. 3. The initial uptake of D-glucose increases as the intravesicular membrane potential become more negative but the numerical values of KH and KL show little, if any, change. 4. alpha-Methyl-D-glucoside transport is also sodium dependent and phlorizin sensitive (KI approximately 1.9 micron). 5. In contrast to the results for D-glucose, the sodium-dependent component of alpha-methyl-D-glucoside uptake exhibits a nearly linear Eadie-Hofstee plot consistent with a single carrier site with Km approximately 1.9 mM and Vmax approximately 27 nmol/min per mg protein at pH 7.4, 25 degrees C, 100 mM NaCl gradient. 6. The kinetics of D-glucose transport in newborn dog kidney are similar to those in the adult except that the low affinity (GL) system appears to be less well developed.


The distribution of molecular classes of phosphatidylglycerol in the membrane of Acholeplasma laidlawii. A double-label technique has been applied to study the distribution of different molecular classes of phosphatidylglycerol in the membrane of Acholeplasma laidlawii. After growth on oleic acid, 16% of the total phosphatidylglycerol contains two oleic acid residues and 84% contains one oleic acid and one saturated fatty acid. The dioleoyl phosphatidylglycerol is present in equal amounts in the outer and the inner layer of the membrane. Phosphatidylglycerol which is associated with membrane proteins consists exclusively of the class containing only one oleic acid.


Amino acid transport properties of erythrocytes from normal newborn lambs and lambs with an inherited defect in amino acid transport. An amino acid transport defect which occurs in the erythrocytes of adult sheep is also present in foetal erythrocytes from newborn lambs which have inherited the lesion. The transport defect in erythrocytes from adult sheep is associated with high intracellular levels of ornithine and lysine and a markedly diminished GSH concentration. Although the lesion in foetal cells also results in the accumulation of ornithine and lysine, the intracellular GSH concentration is only moderately diminished.


Molecular and structural characteristics of house fly brain acetylcholinesterase. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the brains of house flies (Musca domestica L., tetrachlorvinphos-resistant strain) was examined for molecular and structural features, including molecular weight, Stokes radii, partial specific volumes, sedimentation coefficients and frictional ratios. Acetylcholinesterase purified by affininity chromatography was examined in the electron microscope by negative staining and three molecular forms were clearly observed (monomers, dimers and tetramers). Several tetrameric configurations were observed as well as structures of similar size showing tails. In the preparations of acetylcholinesterase so far examined, no globular structures having more than four monomeric units were observed.


Leaving group effects in butyrylcholinesterase reaction with organophosphorus inhibitors. The kinetic constants k2, KQ and the second-order rate constant ki of butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) inhibition by organophosphorus compounds (C2H5O)2P(O)SX, with both ionic and non-ionic substituents X, were determined at 25 degrees C and pH 7.5 in 0.15 M KCl. The data were analysed in terms of structure-activity relationships and the roles of the leaving group inductive effect and hydrophobicity in the enzyme specificity were established. This made possible calculations of the actual contribution of the substituent ionic charge in the effectivenes of butyrylcholinesterase action. On the basis of the structure-activity relationships for butyrylcholinesterase and acetylcholinesterase the specificities of the enzymes are compared and some common features are discussed.


Amount and properties of uptake forms in preparations of alpha-mannosidase from pig kidney. alpha-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) from pig kidney has been shown to exist in multiple forms differing in their capability to be endocytosed by alpha-mannosidase deficient cultured cells. A method is presented to evaluate the amount of "uptake" forms in different preparations of the enzyme. Preparations with different rates of uptake were shown to contain different amount of "uptake" forms and "non-uptake" forms. The content of "uptake" forms in a preparation was identical with that of enzyme molecules bearing a phosphorylated carbohydrate group necessary for the recognition by cell surface receptors.


Purification of NADPH-ferredoxin reductase from rat liver mitochondria. NADPH-ferredoxin reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) has been identified in rat liver mitochondria and purified to homogeneity as judged by sodium dodecyl sulfate (SDS) gel electrophoresis. The protein was detected by its ability to reconstitute NADPH-cytochrome c reductase in the presence of adrenal ferredoxin. The purified protein had properties very similar to adrenal NADPH-ferredoxin reductase. The molecular weight was 52 000, as estimated by gel filtration. On SDS-polyacrylamide gels, mobility was identical to that of adrenal NADPH-ferredoxin reductase (Mr = 52 000). The enzyme exhibited a typical oxidized flavoprotein absorbance spectrum with maxima at 269, 377 and 450 nm and gave an absorbance ratio A450nm/A269nm of 0.138. The fluorescence excitation spectrum was identical to that of FAD. In the presence of NADPH and a ferredoxin, the reductase was found to be active in a reconstituted cytochrome P-450-dependent steroid 26-hydroxylase, which was recently isolated from rat liver mitochondria (Pedersen, J.I. (1978) FEBS Lett. 85, 35-39).


Purification and properties of L-lysine-alpha-ketoglutarate reductase from rat liver mitochondria. L-Lysine-alpha-ketoglutarate reductase (N5-(1,3-dicarboxypropyl)-L-lysine: NADP+ oxidoreductase (L-lysine-forming, EC 1.5.1.8) was purified from rat liver mitochondria to a homogeneous state judged by SDS polyacrylamide gel electrophoresis, and its molecular weight was estimated as 52000. On Sepharose 4B filtration it has a molecular weight of 230 000 and it is suggested that the active enzyme is a tetramer of subunits of similar size. The purified enzyme was clearly separated from saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine:NAD+ oxidoreductase (L-glutamate-forming, EC 1.5.1.9). The reactions of purified L-lysine-alpha-ketoglutarate reductase favored the forward reaction (saccharopine formation) and the rate of the reverse reaction (lysine formation) was only 3--5% that of the forward reaction. The forward reaction was specific for L-lysine, alpha-ketoglutarate and NADPH and followed Michaelis-Menten kinetics, whereas the dose vs. response curve of the reverse reaction was sigmoidal with saccharopine. Among the amino acids examined, ornithine, leucine and tryptophan inhibited the forward reaction competitively. These results are different from earlier reports on human and yeast enzymes. The fact that rats fed on lysine-deficient diet do not lose weight much is discussed in relation to the properties of this enzyme.


Purification and properties of an inhibitory protein of chitin synthetase from Mucor rouxii. A soluble protein inhibitor of chitin synthetase (UDP-2-acetamindo-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase, EC 2.4.1.16) was isolated from the cytoplasm of Mucor rouxii. By gel filtration, the molecular weight of the inhibitor was estimated to be 17 500. The inhibitor was effective against crude or purified (chitosome) preparations of chitin synthetase. Unlike the chitin synthesis inhibitor from Saccharomyces spp., the inhibitor from M. rouxii does not operate by blocking the proteolytic activation of chitin synthetase zymogen but by inhibiting the operation of activated enzyme. Presumably, the inhibitor forms part of the regulatory mechanism of chitin synthesis in the cell.


IV. Synthesis and assay of analogs of adenosine 3',5'-diphosphate as inhibitors of bovine adrenal estrogen sulfotranferase. Analogs of adenosine 3',5'-diphosphate are described which aid in the characterization of the inhibition of estrone sulfurylation by 3'-phosphoadenosine 5'-phosphosulfate as mediated by bovine adrenal estrogen sulfotransferase (3'-5'-phosphosulfate:estrone 3-sulphotransferase, EC 2.8.2.4). The facile conversion of ribonucleosides to 2',3'-cyclic phosphate 5'-phosphate in neat pyrophosphoryl chloride is utilized to provide a reliable route to the requisite intermediates for enzymatic regiospecific conversion to ribonucleoside 3',5'- and 2',5'-diphosphates. The importance of the 3'-phosphate ester to inhibition of estrone sulfurylation is confirmed by Ki measurements. Replacement of the 6-amino group by hydrogen or oxygen leads to considerable loss in affinity for the enzyme as does also dimethylation of the exocylic amino group. Alterations in the pyrimidine ring are not well tolerated by the sulfotransferase but modifications in the imidazole ring as in tubercidin (7 -deazaadenosine) and 8-bromoadenosine 3',5'-diphosphate lead to an enhanced affinity. The latter findings are discussed in terms of an hypothesis of stacking of the aromatic ring of the estrogen substrate and the purine moiety and its analogs.


An improved large scale procedure for the purification of porcine pancreatic lipase. A modified procedure for purifying porcine pancreatic lipase (triacyglycerol acyl-hydrodrolase, EC 3.1.1.3) is described. In comparison to the previous procedure reported by Verger, R., de Hass, G.H., Sarda, L and Desnuelle, P. (1969) Biochim. Biophys. Acta 188, 272--282) it is more rapid, more reproducible and results in a purer enzyme preparation. No colipase could be detected in the mixture of isoenzymes and, naturally, in the different separated lipases. In this process, butanol treatment is omitted. After pancreas powder extraction, a batch procedure was used for adsorption of DEAE-cellulose. Sephadex filtration (pH 8.0) was made in a largeer size column. Finally the isoenzymes were separated on CM-cellulose as in the Verger procedure, but under slightly modified conditions. Lipase LB was fully homogeneous as judged by end group determination, gel electrophoresis (in the presence of absence of sodium dodecyl sulfate) and sedimentation equilibrium.


Successive cleavage of N-terminal Arg1--Pro2 and Lys3-Pro4 from substance P but no release of Arg1-Pro2 from bradykinin, by X-Pro dipeptidyl-aminopeptidase. X-Pro dipeptidyl-aminopeptidase (EC 3.4.14.1) purified homogeneously from the human submaxillary gland was proved to hydrolyze N-terminal dipeptide Arg1-Pro2 and subsequent dipeptide Lys3-Pro4 from substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-gly-Leu-Met-NH2). Km and V values of hydrolysis of substance P were 2.0 mM and 3.6 mumol/min per mg protein, respectively. In contrast, the N-terminal Arg-Pro of bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was not cleaved by the enzyme.


Substituent effect on the elementary processes of the interaction between several benzeneboronic acids and substilisin BPN. Benzeneboronic acid, a transition-state analog for serine proteases, binds to the catalytic center of subtilisin BPN'. The binding mechanism is so-called two-step mechanism; the initial fast association followed by a slow unimolecular process (Nakatani, H., Uehara, Y. and Hiromi, K. (1975) J. Biochem. (Tokyo) 77, 615--616), E + S fast equilibrium ES slow equilibrium ES (E = subtilisin, S = benzenebroonic acid). The structure of the transient complex (ES) at the initial association process was manifested by the substituent effect of benzeneboronic acid on the rate parameters in the elementary processes. The study by the temperature-junp and stopped-flow methods showed that the boron atom in benzeneboronic acid strongly interacts with a nucleophilic site, probably, O gamma of Ser-221 or imidazole of His-64 at the catalytic center, already at the initial fast association.


Cyanate modification of essential lysyl residues in the catalytic subunit of tobacco ribulosebisphosphate carboxylase. Crystalline ribulose-1,5-bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is irreversibly inactivated by incubation with potassium cyanate at pH 7.4. The rate of inactivation is pseudo first-order and linearly dependent on reagent concentration. In the presence of ribulosebisphosphate or high levels of CO2 and Mg2+ the rate constant for inactivation is reduced, suggesting that chemical modification occurs in the active site region of the enzyme. In contrast, neither the effector NADPH nor the activator Mg2+ alone significantly affect the rate of inactivation by cyanate; however, NADPH markedly enhances the protective effect of CO2 and Mg2+. Incubation of the carboxylase with potassium [14C] cyanate in the absence or presence of ribulosebisphosphate revealed that the substrate specifically reduces cyanate incorporation into the large catalytic subunits of the enzyme. Analysis of acid hydrolysates of the radioactive carboxylase indicated that the reagent carbamylates both NH2-terminal groups and lysyl residues in the large and small subunits. Comparison of the substrate-protected enzyme with the inactivated carboxylase revealed that ribulosebisphosphate preferentially reduces lysyl modification within the large subunit. The data here presented indicate that inactivation of ribulosebisphosphate carboxylase by cyanate or its reactive tautomer, isocyanic acid, results from the modification of lysyl residues within the catalytic subunit, presumably at the activator and substrate CO2 binding sites on the enzyme.


Aging of soman-inhibited acetylcholinesterase: inhibitors and accelerators. The influence of 27 possible effectors, mostly bispyridinium salts, upon the dealkylation (aging) of soman-inhibited acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was examined at pH 7.6 and 25 degrees C. In the absence of effectors, the rate constant of the aging process was 4.0. 10(-2) min-1. At 2 mM, the strongest inhibitor reduced the rate to 0.8. 10(-2) min-1, whereas it was raised to 8.2. 10(-2) min-1 by the most potent accelerator.


Unusual substrates for adenosine deaminase from calf intestinal mucosa. A number of adenine nucleosides with exocyclic double bonds either at the 4',5' position in pentofuranosyl nucleosides or at the 5',6' position of hexofuranosyl nucleosides have been found to act as substrates for adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from calf intestinal mucosa. Most of the results obtained are contrary to the accepted minimal structural requirements for substrate activity. These nucleosides had either incorrect anomeric configurations or no hydroxyl group at C-5' or C-3' in the proper configuration; some compounds incorporated both structural changes. There is a possiblity that the unsaturated group has a special role in binding to the enzyme.


Degradation of phenylalanine:pyruvate transaminase after glucagon treatment. Using a single-isotope and immune precipitation technique the half-life (t 1/2) of hepatic phenylalanine:pyruvate transaminase (aminotransferase, EC 2.6.1.--, Number not yet assigned) from glucagon-treated rats was determined to be 2.8 days, similar to that of the control rats (t 1/2 = 3.3 days). The half-life of rat liver total soluble proteins also remained unchanged after glucagon treatment (t 1/2 = 2.7 days in glucagon-treated rats; t 1/2 = 2.8 days in normal). Thus, glucagon has no effect on the degradation of phenylalanine:pyruvate transaminase. Furthermore, the degradation rates are similar for both the holoenzyme and the apoenzyme of phenylalanine:pyruvate transaminase.


Hybrid isozymes of rat pyruvate kinase. Their subunit structure and developmental changes in the liver. Thin-layer polyacrylamide gel electrophoresis of various rat tissues revealed three major isozymes (types L, M1 and M2) and various intermediate forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40). In vitro dissociation and reassociation of purified enzymes showed that the three major isozymes had homotetrameric structures. L.M2 hybrids and M1.M2 hybrids closely resembled some naturally occurring intermediates; the subunit structure of intermediates isolated from the small intestine (form 3 or form 4) were estimated to be (L)2(M2)2 and (L)(M2)3, respectively. Pyruvate kinase activity after electrophoresis could be estimated quantitatively from densitometric measurements of the electrophoretic pattern. Type L activity in fetal liver was separated from type R activity derived from intrahepatic erythropoietic cells. It changes in three distinct steps during development: it increased during the late fetal period, remained steady during the neonatal period and increased again after weaning. Some of the intermediates found in extracts of early fetal iver were shown to cross-react with both anti-L and anti-M1 serum, suggesting that they might be L.M2 or R.M2 hybrids. These hybrid enzymes were shown to appear only during early fetal and neonatal periods.


Substrate preferences of wild and a mutant house fly acetylcholinesterase and a comparison with the bovine erythrocyte enzyme. Comparisons were made of purified acetylcholinesterase from the heads of wild type house flies with a mutant form (which bound organophosphates and carbamates less tightly). Using 12 substrates and 6 quaternary inhibitors, the only substantial difference was that the Km for butyrylcholine was 25 times greater for the mutant enzyme, suggesting that butyrylcholine and the organophosphates and carbamates shared a common binding site. The pure enzyme from the wild type house fly was also compared with bovine erythrocyte acetylcholinesterase. The major difference was again with butyrylcholine as substrate: the ability to acylate or deacylate was 30 times greater in the fly enzyme (the Km values differed by a factor of 4).


The activation of proacrosin in spermatozoa from ram bull and boar. Acrosin activity was estimated in fractions from washed ram, bull and boar spermatozoa that had been disrupted using a Stansted Cell Disruptor. When p-aminobenzamidine was included in the medium during disruption, all the acrosin (acrosomal proteinase, EC 3.4.21.10) was recovered as its inactive zymogen form, proacrosin. But if spermatozoa were damaged before disruption, of were disrupted in the absence of p-aminobenzamidine, considerable amounts of active acrosin were detectable. It was concluded that conversion of proacrosin to acrosin takes place in spermatozoa only after the acrosome has been rutured. In a sucrose medium, all the proacrosin was bound to the sperm heads. Conversion to acrosin took place readily with all components in a bound state. Using arm sperm heads, the conversion was found to be relatively insensitive to pH, proceeding rapidly above pH 6.5; the rate of conversion was not affected by physiological levels of Ca2+, Mg2+, or Zn2+, although elevated ionic strength caused a solubilization of the acrosin activity and some slowing of the rate. Electrophoretic analysis revealed that several active forms of acrosin were involved, but the final product was a single stable form. Final levels of the active acrosin (expressed as mu mol N-alpha-benzoyl-L-arginine ethyl ester utilised/min per 10(9) heads) were: ram 26.2; bull, 15.9; boar, 133.8. But active site titration revealed that these different levels were not reflected in the numbers of active enzyme molecules on the sperm head; boar acrosin appears to be about three times more active towards benzoyl-arginine ethyl ester than do the acrosins from the other species.


Purification and characterization of 3-dehydroquinate hydrolase and shikmate oxidoreductase. Evidence for a bifunctional enzyme. The basis for the physical association of 3-dehydroquinate dehydratase (3-dehydroquinate hydrolyase, EC 4.2.1.10) and shikimate dehydrogenase (shikimate: NADP+ 3-oxidoreductase, EC 1.1.1.25) in higher plants was investigated. The enzymes were extracted from the moss Physcomitrella patens and were purified to homogeneity. Determinations of subunit sizes were made by sodium dodecyl sulfate gel electrophoresis and gel exclusion chromatography in 6 M guanidinium chloride. Results from these studies demonstrate that both enzyme activities are carried out by a single polypeptide.


Studies of fluoride-preactivated rabbit liver adenylyl cyclase. Rabbit liver adenylyl (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) cyclase was stimulated by preincubation with F- and Mg2+ and the stimulation persisted despite extensive washing and/or detergent solubilization. Optimum preactivation conditions were found to be 4 mM NaF and 2 mM MgCl2; higher or lower concentrations produced submaximum stimulation regardless of preincub ation time. In addition to an enhanced catalytic acticity, the activated enzyme also exhibited different responses to Ca2+ and Cu2+ when compared to the basal enzyme. ATP caused a time-dependent inhibition that could be partially prevented or reversed by F-, but was not completely reversed by washing. This inhibition was not observed when 5'-adenosine(beta, gamma-imide) triphosphate blocks inhibition by ATP. The results support, but do not prove, the proposed molecular basis of F- activation which entails a phosphorylation-dephosphorylation mechanism.


Effect of Fe2+ and Mn2+ on 3-mercaptopicolinate inhibition of cytosolic and mitochondrial phosphoenolpyruvate carboxykinase of five species. Liver cytosolic or mitochondrial fractions of five species were incubated with 30 micrometer Fe2+ or with 100 micrometer Mn2+ prior to assaying for phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) acticity in the presence of 3-mercaptopicolinate. Only the cytosolic carboxykinases were activated 3--4-fold by Fe2+ or Mn2+. Fe2+ enhanced the inhibitory potency of 3-mercaptopicolinate 10--50-fold against the cytosolic and the mitochondrial carboxykinases, but Mn2+ was ineffective. Mn2+ interfered with Fe2+ -enhancement of inhibition by 3-mercaptopicolinate in a manner competitive with Fe2+. It is hypothesized that Fe2+ and 3-mercaptopicolinate form a coordination complex that inhibits the carboxylkinases and that 3-mercaptopicolinate does not blind to a carboxykinase containing Mn2+.


Inhibition of liver prenyltransferase by alkyl phosphonates and phosphonophosphates. n-Pentyl and n-decyl phosphonate and the corresponding phosphonophosphates were found to inhibit cholesterol synthesis from mevalonate in the 10000 X g supernatants of liver homogenates and the synthesis of farnesyl pyrophosphate from geranyl and isopentenyl pyrophosphate by purified liver prenyltransferase. Kinetic analysis of the inhibition of prenyltransferase showed that the phosphonates and the phosphonophosphates interacted with two forms, or two sites, of the enzyme. The order of increasing potency was C5-phosphonate less than C10-phosphonate less than C5-phosphonophosphate less than C10-phosphonophosphate. The phosphonophosphates were at least ten times stronger inhibitors than the phosphonates.


Distribution and metabolism of synthetic alkyl analogs of lysophosphatidylcholine in mice. Stability, distribution and metabolic fate of alkyl analogs of lysophosphatidylcholine have been studied in mice. Analogs employed were 1-hexadecyl- and 1-dodecyl-propanediol-3-phosphorylcholine (2-deoxy-lysophosphatidylcholine) and rac-1-octadecyl-2-methyl-glycero-3-phosphorylcholine (2-methoxy-lysophosphatidylcholine). It is demonstrated that (1) the half-life of these compounds in the organism was increased to the order of days as compared to minutes or hours for natural lysophosphatidylcholine, (2) the analogs were slowly degraded in vivo with partial incorporation of the hydrophobic part into neutral lipids and phosphatidylcholine. Deoxy analogs were apparently degraded faster than methoxy derivatives, and (3) due to this metabolism, accumulation of these compounds, even after repeated injections, did not occur in mice. Intravenous and intraperitoneal injections led to rapid and complete distribution of the lysolipids with slight enrichment in liver, kidneys and intestine. Subcutaneous or oral application resulted in a similar distribution, however, remarkable depot effects at the sites of administration were observed. Experiments with tumor-bearing mice indicated a reduced metabolic turnover and thus a slight enrichment of ether-lysophosphatidylcholine analogs in tumor tissue.


Partial purification and properties of monoacylglycerol lipase and two esterases from isolated rat small intestinal epithelial cells. In intestinal epithelial cells, three enzymes possessing monoacylglycerol hydrolase activity were found and partially purified. Two of these enzymes have properties that justify their classification as an esterase and one as a monoacylglycerol lipase. The three enzymes show similar Km values for monooleoylglycerol and each shows similar activity towards 1- and 2-monopalmitoylglycerol. Antiserum raised in rabbits against rat liver monoacylglycerol lipase inhibits the intestinal lipase completely, suggesting that the enzymes are at least partially similar. The esterases of small intestinal villus cells were not inhibited by the antiserum against liver monoacylglycerol lipase. It was calculated that the esterases account for approx. 2/3 of the monooleoylglycerol hydrolase activity in epithelial cells. Monoacylglycerol lipase also hydrolyzed palmitoyl-CoA, while the esterases did not. The enzymes were inhibited by micellar palmitoyl-CoA. The hypothesis that palmitoyl-CoA is an important regulator for monoacylglycerol acylation is discussed in the light of these new findings.


Monoacylglycerol metabolism in rat small intestinal epithelial cells. 1. In various preparations of rat small intestinal epithelial cells the acylation and deacylation of monoacylglycerol were studied. In the in vitro vascularly perfused intestine, of which the lumen was loaded with monoacylglycerol with or without fatty acids, acylation exceeded deacylation. In contrast, deacylation was much faster in isolated microsomes and in isolated whole cells. 2. In vascularly perfused intestine, without long-chain fatty acids present in the lumen, the amount of di- and triacylglycerol formed was found to be half of that formed in perfusions with long-chain fatty acids in the lumen, while the glycerol formation was increased 1.4-fold. 3. The concentration of monoacylglycerol is an important factor in determining the relative rates of monoacylglycerol acylation and deacylation in microsomes: the ratio acylation/deacylation decreases with increasing monoacylglycerol concentrations. 4. The function of the monoacylglycerol lipase in fat resorption is discussed.


On the mechanism for the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, of cholesterol 7alpha-hydroxylase and of acyl-coenzyme A:cholesterol acyltransferase by free cholesterol. The administration of mevalonic acid to rats by intravenous injection resulted in a dose- and time-dependent increase in the activity of cholesterol 7alpha-hydroxylase in the liver microsomal fraction, a decrease in the microsomal activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and no significant change in the activity of acyl-coenzyme A:cholesterol acyltransferase or in the concentration of free and of esterified cholesterol in the liver microsomal fraction. However, the increased hepatic cholesterogenesis that follows the injection of mevalonic acid resulted in an increase of the size of the intracellular pool of cholesterol that is in the environment of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acts as substrate for cholesterol 7alpha-hydroxylase. The administration of mevalonic acid to rats by stomach tube resulted in an increase in the activity of cholesterol 7alpha-hydroxylase and of acyl-coenzyme A:cholesterol acyltransferase and in the concentration of cholesterol esters in the liver microsomal fraction, while there was a considerable decrease in the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.


The effects of serum albumin and phospholipid on sterol excretion in tissue culture cells. Serum albumin was as effective as whole serum or alpha-globulins in facilitating sterol release from strain L mouse fibroblasts. Commercial bovine serum albumin preparations, however, had markedly different absolute effects in this regard. These differences were attributable to the variation in phospholipid content of these products. All but one of these albumins enhanced sterol release when supplemented with phospholipid. The exception was fatty acid-poor albumin which contained an adequate amount of phospholipid. Among the phospholipids examined, lecithin proved to be most effective, while phosphatidylethanolamine had little potentiating influence. As the unsaturation of the test lecithins increased, enhancement of sterol release decreased. The potentiating effect of the phospholipid was in turn dependent on the protein used, since the phenomenon was not observed with non-serum proteins like ovalbumin or with non-transport serum proteins such as gamma-globulins. The results of these studies raise the possibility that serum albumin together with phospholipid can play an important role in sterol release in tissue culture cells.


Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) porphyrin compounds. 13C nuclear magnetic resonance spectra have been obtained for a variety of high-spin iron(III) porphyrin compounds and corresponding mu-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric complexes exhibit hyperfine shifts which are downfield with the exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing 13C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of 13C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH2 resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling.


The pentose phosphate pathway in developing chick cornea. Embryonic chick corneas at different stages of development were evaluated for activity of the pentose phosphate pathway. The appearance of activity was concurrent with the onset of corneal transperancy (stage 40). Highest values were found after complete transparency is achieved (stage 45 and after hatching). Phenazine methosulfate, an artificial electron acceptor, increased activity at all stages studied even before endogenous activity was measurable; however, no increase in glucose uptake was observed. Thus, the enzymes for the pathway are present at early stages (i.e., stage 38 and 40) although in latent form. The pathway probably functions in the developing cornea to generate NADPH rather than sugar moieties for macromolecular incorporation.


The 4-amino sugars present in the lipopolysaccharides of vibro cholerae and related vibrios. Brief treatment, with 10 M hydrochloric acid, of the lipopolysaccharides of Vibrio cholerae and related organisms led to the release of 4-amino-4, 6-dideoxy-D-mannose (D-perosamine) and 4-amino-4-deoxyl-L-arabinose. These sugar are highly unstable and undergo complex changes in neutral and basic media. Of the strains examined, the lipopolysaccharides from representatives of the Inaba group contain only D-perosamine, while those of the Ogawa biotype have 4-amino-4-deoxyl-L-arabinose as an additional component. The other Vibrios produce lipopolysaccharides which lack these sugars.


Chemical structure and biodegradability of halogenate aromatic compounds. Substituent effects on 1,2-dioxygenation of benzoic acid. Dioxygenation of substituted benzoic acids by whole cells of 3-chlorobenzoate-utilizing Pseudomonas sp. B 13, benzoate-induced cells of Alcaligenes eutrophus B 9 and toluate-grown cells of Pseudomonas putida mt-2 was examined. Electron-attracting substituents like halogen decreased the reaction rates of benzoate 1,2-dioxygenation. Dioxygenation of substituted benzoic acids by P. putida mt-2 was mostly undisturbed by steric effects of the substituents. Good correlation resulted between the log Vrel values and the Hammett substituent constant sigma. In contrast the reaction rates of dioxygenation by Pseudomonas sp. B 13 and A. eutrophus were decreased predominantly by steric effects of substituents. A non-polar reaction mechanism of benzoate 1,2-dioxygenation is discussed. Results from inhibition studies demonstrate high stereospecificities for the 1,2-dioxygenation by Pseudomonas sp. B 13 of benzoic acids with substituents in ortho- or para-position. In the case of P. putida mt-2 steric handrance by substituents was observed only with orth-substituted benzoic acids. Stereospecificities of the benzoate 1,2-dioxygenation by Pseudomonas sp. B 13 and P. putida mt-2 are illustrated schematically.


Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on dehydrogenation of 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid. The dehydrogenation of substituted 3,5-cyclohexadiene-1,2-diol-1-carboxylic acids by dihydrodihydroxybenzoic acid dehydrogenases from benzoate grown cells of Alcaligenes eutrophus and Pseudomonas sp. B 13 and 3-chlorobenzoate grown cells of the latter organism was examined. No significant differences (Km and Vrel values) were detected for the enzymes from both organisms. The same dihydrodihydroxybenzoic acid dehydrogenase is formed in Pseudomonas sp. B13 during growth on benzoate as well as on 3-chlorobenzoate. The lower turnover rates of 3- and 5-chlorodrodihydroxybenzoic acid compared to dihydrodihydroxybenzoic acid are counterbalanced by an increase in specific activity. With the exception of 4-substituted dihydrodihydroxybenzoic acids exhibiting relative high Km values, only slight sterical and electronic substituent effects are evident. Reaction rates were never reduced to a critical level.


Interaction of phospholipid vesicles with rat hepatocytes in primary monolayer culture. We studied the interaction of positively and negatively charged unilamellar and multilamellar phospholipid vesicles (liposomes) with rat-liver parenchymal cells in primary monolayer culture. Radioactive liposomal phosphatidylcholine was taken up more rapidly and to a larger extent from unilamellar than from multilamellar vesicles. No significant difference in uptake characteristics was observed between vesicles of different charge. The presence of serum greatly reduced uptake of liposomal phosphatidylcholine of both unilamellar and multilamellar vesicles. This serum effect was independent of surface charge of the vesicles. When cells were allowed to take up radioactive liposomal phospholipid and then incubated further in absence of vesicles, part of the radioactivity associated with the cells was released into the medium, most of it as water soluble degradation products. When cells were preincubated with vesicles containing horseradish peroxidase and then, after removal of the vesicles, further incubated, peroxidase activity could be demonstrated in the culture medium, part of it only after addition of Triton X-100. These observations were taken to indicate that part of the phospholipid taken up the cells represented vesicles binding to the cell surface rather than having been internalized. Vesicle-entrapped [125I]albumin was taken up by the cells and rapidly hydrolyzed as indicated by the appearance of radioactivity soluble in trichloroacetic acid within minutes after starting the incubation. No uptake of free albumin could be demonstrated. The kinetics of albumin uptake and release of trichloroacetic acid-soluble radioactivity from the cells suggest that, initially, liposomes are internalized predominantly by endocytosis, while during prolonged incubation fusion of the liposomal membrane with the plasma membrane gradually contributes more substantially to the overall uptake process. The significance of these findings is emphasized with special reference to the use of liposomes as intravenous carriers of enzymes or drugs.


Production of protein HC by human fetal liver explants. Human fetal lever explants were found to secrete protein HC into the medium in molar amounts comparable to those of albumin, alpha 1-antitrypsin and orosomucoid. Incorporation of a radioactive amino acid from the medium into the secreted protein HC demonstrated de novo synthesis. The secreted protein HC had the same size and electrophoretic mobility as protein HC of plasma and urine and gave a reaction of immunochemical identity with the protein in these biological fluids.


Oxidation of cuprous stellacyanin by aminopolycarboxylatocobaltate(III) complexes. Rate parameters are reported for the oxidation of cuprous stellacyanin by Co(PDTA)-(k(25.0 degrees) = 17.9 M(-1)sec(-1), deltaH not equal to = 8.5 kcal/mol, deltaH not equal to = 8.5 kcal/mol, deltaS not equal to = -24 cal/mol-deg; pH 7.0, Mu 0.5 M) and Co(CyDTA)-(k(25.1 degrees) = 17.0 M(-1)sec(-1), deltaH not equal to = 8.7 kcal/mol, deltaS not equal to = -24 cal/mol-deg; pH 7.0 mu 0.5 M). The first order Co(PDTA)- and Co(CyDTA)- dependences observed over wide concentration ranges contrast with the saturation behavior reported previously for Co(EDTA)- as the oxidant. It is concluded that the- CH3 and -(CH2)4-substituents of PDTA and CyDTA, respectively, prevent the alkylated derivatives of Co(EDTA)- from hydrogen bonding with the reduced blue protein, causing precursor complex formation constants to fall far below that of 149M(-1) (25.1 degrees) observed for the EDTA complex. The similarity between deltaH not equal to and deltaS not equal to values for the oxidation of stellacyanin by Co(PDTA)- and Co(CyDTA)- indicates that the size of alkyl substituents linked to the carbon atoms of the EDTA ethylenediamine backbone has little influence on activation requirements for Cu(I) to Co(III) electron transfer. The electron transfer reactivity of aminopolycarboxylatocobalt(III) complexes with cuprous stellacyanin therefore appears to be linked to the accessibility of one or more of the ligated acetate groups to outer-sphere contact with the type 1 Cu(I) center. Saturation in kobsd vs. [oxidant] plots found for the reactions of Co(PDTA)- and Co(CyDTA)- with stellacyanin at pH 6 and at pH 7 in the presence of EDTA is attributed to the formation of "dead-end" oxidant-protein complexes.


Selenium and cancer: effects of selenium and of the diet on the genesis of spontaneous mammary tumors in virgin inbred female C3H/St mice. Inbred female C3H/St mice exhibit the normal incidence of spontaneous mammary adenocarcinoma of 80--100% if they are maintained on a standard commercial laboratory diet containing 0.15 ppm of selenium with meat and dried skimmed milk as major sources of protein. The tumor incidence drops to 42% if animals of the same strain are kept on a diet containing 0.45 ppm of selenium, with fishmeal as the main source of protein. The tumor incidence declines further to 25, 19 and 10% if the animals in addition receive 0.1, 0.5, and 1.0 ppm of selenium in the drinking water. Selenium supplementation at these levels has no noticable adverse effects on weight-grains and survival of the mice. Selenium supplmented groups of animals also remained tumor-free for longer periods than the unsupplemented controls. The results of this study indicate that a diet rich in seafoods and cereals provides more selenium and may in turn lower the probability of cancer development. Reference is made to the average human diet in the U.S.A., which only contains 0.07--0.15 ppm of selenium due to the comparatively low consumption of cereals and seafoods. An equivalent mouse diet would not have any cancer-protecting effect in the C3H/St mice of our study. Australian workers have reported significantly lower tumor incidence in a different strain of C3H mice if it was kept in Australia rather than in the U.S.A. We have found that the Australian feed contained three times more selenium than that employed in the U.S.A. and propose that this difference in selenium content was primarily responsible for these previous observations.