The mechanism of assimilation of constituents of chylomicrons, very low density lipoproteins and remnants - a new theory.

Purified remnant lipoproteins produced from chylomicrons $\text{in vivo}$ or $\text{in vitro}$ by the action of lipoprotein lipase (LPL) contain firmly bound LPL. The perfused rat liver removes the particulate bound LPL and triglyceride-labeled remnants at exactly the same rate, while purified chylomicrons are not removed. Once remnants are removed by the liver, they are not rereleased into the perfusate. These observations have led to the theory that the LPL attached to the remnant is the signal that allows the liver to “recognize” remnants from chylomicrons. This is followed by fusion of the particle with the cell surface and may be associated with the splitting off a low density lipoprotein particle. The remaining lipids of the remnant are further metabolized by the liver triglyceridase and the cholesterol esterase.     

Chlorophyll-protein complexes of a Codium species,including a light-harvesting siphonaxanthin-Chlorophylla ab-protein complex,an evolutionary relic of some Chlorophyta

Eight chlorophyll-protein complexes were isolated from thylakoid membranes of a Codium species, a marine green alga, by mild SDS-polyacrylamide gel electrophoresis. CP 1a¹, CP 1a², CP 1a³ and CP 1a⁴ were partially dissociated Photosystem (PS) I complexes, which in addition to the core reaction centre complex, CP 1, possessed PS I light-harvesting complexes containing chlorophyll (Chl) a, Chl b and siphonaxanthin. LHCP¹ and LHCP³ are orange-brown green chlorophyll $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 0.66) that contain siphonaxanthin and its esterified form, siphonein. CP a and CP 1, the core reaction centre complexes of PS II and PS I, respectively, had similar spectral properties to those isolated from other algae or higher plants. These P-680- or P-700-Chl a-proteins are universally distributed among algae and terrestrial plants; they appear to be highly conserved and have undergone little evolutionary adaptation. Siphonaxanthin and siphonein which are present in the Codium light-harvesting complexes of PS II and PS I are responsible for enhanced absorption in the green region (518 and 538 nm). Efficient energy transfer from both xanthophylls and Chl b to only Chl a in Codium light-harvesting complexes, which have identical fluorescence emission spectra at 77 K to those of the lutein-Chl $\text{a}\text{b-}\text{proteins}$ ($\text{Chl}\text{a}\text{Chl}\text{b}$ ratios of 1.2) of most green algae and all higher plants, proved that the molecular arrangement of these light-harvesting pigments was maintained in the isolated Codium complexes. The siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ allow enhanced absorption of blue-green and green light, the predominant light available in deep ocean waters or shaded subtidal marine habitats. Since there is a variable distribution of lutein, siphonaxanthin and siphonein in marine green algae and siphonaxanthin is found in very ancient algae, these novel siphonein-siphonaxanthin-Chl $\text{a}\text{b-}\text{proteins}$ may be ancient light-harvesting complexes which were evolved in deep water algae.     

Growth inhibition of Halobacterium cutirubrum by cerulenin,a potent inhibitor of fatty acid synthesis

The polar lipids of $\text{Halobacterium cutirubrum}$ are known to consist exclusively of diether derivatives of glycerol, and do not contain fatty acids. However, cerulenin, a specific and potent inhibitor of fatty acid synthesis, was shown to inhibit the growth of this organism. Protection from growth inhibition was demonstrated when fatty acids of 18 carbons were added to the growth medium, but not when palmitic or palmitoleic acids were used. Cerulenin appears to affect synthesis of all polar lipids in this organism while relative levels of protein and nucleic acids were not significantly affected. Growth inhibition by cerulenin supports the conclusion that the fatty acid synthetase system present in $\text{H. cutirubrum}$ is necessary for lipid biosynthesis, despite the fact that fatty acids are not structural components of the lipids of this bacterium. A pathway is proposed to account for these observations.     

Role of membrane proteins and lipids in water diffusion across red cell membranes

When human red cells are treated with the mercurial sulfhydryl reagent, $\text{p-}\text{chloromercuribenzene sulfonate}$, osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104–106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton $\text{T}{}_1$ NMR and obtained a value of 4.0 · 10^?3 cm · s^?1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$ by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.     

Dansylation of bacteriorhodopsin near the retinal attachment site

The purple membrane of $\text{Halobacterium}\text{halobium}$ was reacted with 5-dimethylaminonaphthalene-l-sulfonyl chloride (dansyl chloride) at pH 8.0. Chromophoric and functional properties of the product appear unaltered. Approximately 2 moles of dansyl group were incorporated per mole of bacteriorhodopsin, part bound to bacteriorhodopsin and part bound to lipids. Purification and fragmentation of the protein showed most of the dansyl modification in a fragment containing residues 33 to 56. Amino acid analysis indicates that the major dansylated site is lysine 40. We conclude that, contrary to published models, 1) bacteriorhodopsin folds in a way that exposes lysine 40 at the membrane surface, and 2) this side chain is not involved in the proton pump mechanism.     

Glutathione peroxidase activity of glutathione-s-transferases purified from rat liver.

$\text{Rhizobium}$ hemeproteins P-450$\text{a, b}$, and $\text{c}$ cross react with antibodies to P-450_CAM and P-450_LM-2. Anti P-450_CAM IgG and phenobarbital, each bound to Sepharose 4B, were effective in purification of $\text{Rhizobium}$ P-450$\text{c}$; the latter was more convenient. The amino acid composition of highly purified $\text{Rhizobium}$ P-450$\text{c}$ resembles the compositions of P-450_CAM and P-450_LM-2. These results suggest that P-450 heme proteins of unrelated substrate specificities may nevertheless contain similar structural features.     

The effect of phosphatidylcholine depletion on biochemical and physical properties of a Neurospora crassa membrane mutant

By using the choline starvation process it is possible to deplete the membranes of Neurospora crassa choline auxotroph $\text{chol-1}$ of phosphatidylcholine, without affecting the viability of germinated spores or whole mycelium. Spin label probes were used to examine the possible dependence of the physical state of cellular lipids on the presence of phosphatidylcholine in the membranes.Increased freedom of rotational motion of lipid soluble probes was regularly detected in choline-starved mycelium. The accumulation of neutral lipids (mostly triglycerides) in bulk form was also observed during the choline starvation process. The experiments with isolated and separated lipid classes indicated that the observed increase in fluidity of lipids in choline-starved mycelium is partly due to the difference in physical properties between bulk lipids and membrane lipids. Spin label probe 2N4 ($\text{2-}\text{propyl}\text{-2,5,5-}\text{trimethyl-oxazolidine}\text{-N-}\text{oxyl}$), which can partition at the membrane-water interface, exhibited easier partitioning among membrane lipids of choline-starved mycelium.     

Ribonucleotide reductase activity in green algae

A ribonucleoside diphosphate reductase is demonstrated in the algae, $\text{Scenedesmus}\text{obliquus}$ and $\text{Chlorella}\text{pyrenoidosa}$. In synchronized cultures an activity maximum at the 12th hour of the cell cycle coincides with maximum DNA production. Induction of reductase activity is prevented by cycloheximide. The enzyme requires dithiols for reduction of CDP $\text{in}\text{vitro}$; it is not significantly stimulated by iron or magnesium ions nor dependent upon deoxyadenosylcobalamin. ATP stimulates the reaction but dATP or dTTP act as inhibitors. The ribonucleotide reductase of green algae differs from the B₁₂-requiring enzyme characterized in $\text{Euglena}\text{gracilis}$.     

Occurrence in mammalian liver of a protein which replaces the B protein of E. coli quinolinate synthetase.

$\text{Escherichia coli}$ contains two proteins (A and B) which together convert dihydroxyacetone phosphate and aspartate to quinolinic acid, a precursor of NAD. Although mammalian liver homogenate does not catalyze this reaction it contains a protein which will replace the B protein of the $\text{E. coli}$ system. The behavior of the liver protein on Sephadex G-75 suggests it is much smaller than the $\text{E. coli}$ B protein. Liver B protein also appears to contain tightly bound FAD while FAD is easily removed from the $\text{E. coli}$ B protein. The pH optimum for the hybrid system $\text{E. coli}$ A protein-liver B protein is 9.0 while in the pure $\text{E. coli}$ system the optimum is pH 8.0. The hybrid system is inhibited by NAD to the same extent as the pure $\text{E. coli}$ system.     

Photosynthetic electron transport,ATP synthesis and nitrogenase activity in isolated heterocysts of Anabaena cylindrica

Isolated heterocysts of the N₂-fixing blue-green alga Anabaena cylindrica contain the Photosystem I components P-700, bound and soluble ferredoxins and ferredoxin-NADP reductase. They also show Photosystem I activity being able to photoreduce both methylviologen and NADP when ascorbate+dichlorophenol-indophenol acts as reductant. They photophosphorylate (64 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$) and carry out oxidative phosphorylation (8.7 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$). Ninety per cent of the total cell-free extract nitrogenase activity is located in the heterocyst fraction of aerobic cultures.     

Bound nucleotides and phosphorylation in Rhodospirillum rubrum.

Chromatophores from $\text{Rhodospirillum rubrum}$ contain 12 × 10^?3 mol ATP and 8.3 × 10^?3 mol ADP per mol chlorophyll, tightly bound to the coupling ATPase. Under energised conditions, these exchange slowly with added nucleotide. Using single turnover light flashes, it is demonstrated that the release of bound ATP is too slow to be on the direct pathway of photophosphorylation.     

Molybdenum and chlorate resistant mutants in Escherichiacoli K12

Radioactive molybdenum is used to detect the existence of molybdo compounds in $\text{E.}\text{coli}$ K12. Three membrane bound Mo-proteins are found, using sodium dodecyl sulfate. One of them is the nitrate reductase. The nature of the other two is discussed. The soluble fraction of the cellular extract contains a small Mo binding molecule which could be peptidic in nature (MW is about 1,500). Different chlorate resistant mutants are analyzed on the basis of these molybdo-compounds. None of the mutants is found to contain radioactivity bound to nitrate reductase protein. Defects in the biosynthesis of a molybdenum coenzyme is deduced for chlorate resistant pleiotropic mutants.     

Cytochrome P-450 and drug metabolism in intestinal villous and crypt cells of rats: effect of dietary iron.

The amino acid sequence of the $\text{Spirulina maxima}$ ferredoxin has been determined. $\text{Spirulina maxima}$ is a blue green algae and is a procaryote. The ferredoxins of the plant-algal type sequenced to date have all been isolated from eucaryotes. The $\text{S. maxima}$ ferredoxin was composed of 98 amino acids arranged in a single polypeptide chain.The sequences of the various procaryote-eucaryote ferredoxins are compared and the differences discussed.     

Non-sterol metabolism of mevalonate in vitro: artifacts and realities.

Commercial [5-¹⁴C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-¹⁴C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated $\text{R}\text{-5-phospho-[5-}{}^{14}\text{C]mevalonate}$ by ion-exchange chromatography. The artifactual ${}^{14}\text{CO}{}_2$ results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the ${}^{14}\text{CO}{}_2$ and [${}^{14}\text{C}$]cholesterol produced by rat renal cortex slices incubated with purified [5-¹⁴C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that $\text{R}\text{[5-}{}^{14}\text{C]mevalonate}$ is genuinely oxidized to ${}^{14}\text{CO}{}_2\text{in}\text{vitro}$, and that purification of substrate before its use is necessary. Production of ${}^{14}\text{CO}{}_2$ and various [${}^{14}\text{C}$]lipids from purified [5-¹⁴C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

A selenite-induced decrease in the lipid content of a red alga

Two unicellular marine algae (Dunaliella primolecta and Porphyridium cruentum) have been found to contain a selenium-inducible, non-enzymatic glutathione peroxidase activity when cultured in the presence of selenite. To test the possibility that selenium functions in vivo as an antioxidant in these algae, a detailed examination of the lipid content of algae cultured in the presence or absence of selenite was conducted. If selenium augments the antioxidant defenses of algal cells, an increase in the content of oxidation-sensitive lipids would be expected. The fatty acid, chlorophyll, phospholipid and glycolipid content of the green alga D. primolecta was not affected by growth in selenite. At low light intensity there was a moderate decrease in the chlorophyll and polyunsaturated fatty acid content of the red alga P. cruentum when cultured in selenite. At higher light intensity the content of all fatty acids, phospholipid, glycolipid, chlorophyll, carotenoid and phycoerythrin decreased in P. cruentum grown in selenite. Since growth in selenite did not increase the quantity of oxidation-sensitive lipids in either alga, there is no evidence for an in vivo functioning of selenium as an antioxidant. Instead, the observed decrease in lipids of the red alga P. cruentum can best be explained as a selenite-induced oxidative effect.     

Differential scanning calorimetry and enzymic activity of rat liver microsomes in the presence and absence of Δ1-tetrahydrocannabinol

The thermal transitions of rat liver microsomes and isolated lipids were investigated by using differential scanning calorimetry. Endothermic transitions at ≈?5°C and between ≈18° and 40°C were detected in the membranes and at ≈?10°C and between ≈10° and 20°C in the extracted lipids.Interaction with $\text{Δ}{}^1\text{-}\text{tetrahydrocannabinol}$ of microsomal membranes and of extracted lipids influences the thermotrophic behaviour as revealed by differential scanning calorimetry and eliminates the break in the Arrhenius plot of the enzymic activity of $\text{O-}\text{demethylase}$.     

Lipid-protein interactions in model membranes from bovine brain white matter. An ESR spin label and electron microscopy study

Lipid-protein model membranes, prepared from bovine brain white matter and containing all the lipids and Folch-Lees proteolipids, have been studied in macroscopically oriented multibilayers. To examine the lipid environment the membranes were spin labeled with the cholestane spin label ($\text{3-spiro(2′-(N-}\text{oxyl-4′,4′-dimethyl-oxazolidine))5α-cholestane}$) and a fatty acid spin label ($\text{4′-,4′-dimethyloxazolidine-N-}\text{oxyl}$ derivative of 5-ketostearic acid). The ESR spectra exhibit two components arising from fairly well oriented and completely unoriented lipids. Up to a temperature of 55°C the amount of oriented lipids is almost constant, being about 35%. At higher temperatures this percentage drops rapidly to zero. It is shown that the presence of unoriented lipids arises mainly from disrupted areas in the lipid bilayer structure. This is confirmed by electron microscopy and from an analysis of the temperature dependence of the order parameters of the spin labels. The presence of locally disrupted lipid parts in the bilayer is discussed in relation to the interaction of the brain white matter lipids with Folch-Lees protein.     

Constraint on the substrate cytochrome P-450 binding reaction in bovine adrenocortical microsomes at physiological temperature

The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant ($\text{K′}{}_{\mathrm{<mtext>s</mtext>}}$) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$ and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome $\text{P-450}$ is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome $\text{P-450}$ binding reaction and in the temperature dependency of the $\text{K}{}_{\mathrm{<mtext>s</mtext>}}$.     

Determination of vanilmandelic acid in pig urine and chicken feces by gas-liquid chromatography

A gas chromatography method for the determination of free and bound vanilmandelic acid (VMA) in pig urine and chicken feces has been developed. The method consisted of extraction of the free or bound acids by ethyl acetate under acidic conditions. The ethyl acetate extracts were dried under nitrogen, followed by complete silylation of the phenolic and carboxylic acid groups with BSA (N,O)-bis (trimethylsilyl) acetamid. The solution was distilled at 180°C in a sealed glass tube after which the sample was injected on a stainless steel column (6 ft × .125 in. o.d.) containing 4% SE-30 on $\text{80}\text{100}$ mesh chromosorb GHP. The recovery of the urinary VMA was 82%, and the fecal VMA was 84% through the outlined procedures. Pigs ranging in age from 8 to 12 weeks were found to excrete $\text{2–8 mg urinary VMA}\text{24 hr}$ with no significant difference between the free and bound. Commercial laying hens excreted bound VMA in a range of $\text{1–5 mg}\text{24 hr}$ with a $\text{F}\text{B}$ ratio of 1:3.