Some characteristics of new tissue-binding proteins for metabolites of vitamin D other than 1,25-dihydroxyvitamin D.

Protein(s) have been found in a wide range of tissues which have a high affinity for 25-hydroxycholecalciferol. Of the tissues examined only erythrocytes do not have this protein. The properties of the protein have been examined and it has been found that the association constatns range from 2 - 10(9) to 5 - 10(9) M-1 and the sedimentation constants between 5.0 and 6.0 S. It was not possible to distinguish the proteins from the different tissues by their S values, mobility on gel electrophoresis or behaviour on ion-exchange chromatography. These techniques were all used, however, to show that the tissue 25-hydroxycholecalciferol binding protein is distinct from the main plasma binding protein for this steroid and from the intestinal 1,25-dihydroxycholecalciferol-binding protein. A protein has been in the plasma of rachitic animals but not of normals, which is apparently indistinguishable from this new tissue 25-hydroxycholecalciferol-binding protein. The steroid specificity of this new binding protein has been shown to be dependent upon a C-25 hydroxyl group, and an intact conjugated double bond system. Possible functions for this protein have been briefly discussed.


N-Acetylgalactosamine 4,6-bissulfate in rat urine. I. Isolation, identification and chemical synthesis.

By starting with 4 1 of rat urine, it was possible to obtain a sulfate ester of hexosamine in crystalline form. A series of identification procedures including chemical analyses, enzymatic digestion, proton magnetic resonance spectroscopy and infrared spectroscopy showed that this substance is 2-acetamido-2-deoxy-D-galactose 4,6-bissulfate. The trivial name for this compound is N-acetylgalactosamine 4,6-bissulfate; Quantitation by isotopic techniques indicated the urine possessed an average concentration of 8 muM N-acetylgalactosamine 4,6-bissulfate. Further extension of these studies necessitated the chemical synthesis of N-acetylgalactosamine 4,6-bissulfate and related compounds to be used for references or as biological substrates. Direct sulfation of N-acetylgalactosamine was attempted first, and strong preference for attack on the primary hydroxyl group (position 6) was found for chlorosulfonic acid. Thus, the reaction with 2.2 molar equivalents of the sulfating agent gave N-acetylgalactosamine 6-sulfate and its derivatives bearing a second sulfate at either position 1 (minor) or position 3 (major). The lack of sulfation at position 4 could be attributed to steric effects of the sulfate group preferentially attached to position 6. Another experiment in which UDP-N-acetylgalactosamine 4-sulfate was used in place of the free sugar led to the formation of a bissulfated sugar-nucleotide which, on subsequent hydrolysis with mild acid, afforded N-acetylgalactosamine 4,6-bissulfate, the same compound as that obtained from rat urine.


N-Acetylgalactosamine 4,6-bissulfate in rat urine. II. Occurrence in rat cartilage and blood.

The intraperitoneal injection of inorganic [35S]sulfate to rat was followed by the rapid appearance in urine of a labeled compound which behaved as N-acetylgalactosamine 4,6-bissulfate on paper chromatography and paper electrophoresis and when treated with two sulfatases with a high degree of specificity toward the sulfate bonds at positions 4 and 6, respectively. Enzymatically-prepared N-acetylgalactosamine 4,6-[6-35S]bissulfate was injected intravenously into rats. Of the injected dose, 90% was excreted unchanged in the urine during the subsequent 12 h, suggesting that the urinary N-acetylgalactosamine 4,6-bissulfate may derive from blood as renal filtrate. Examination of the rats injected with inorganic [35S]sulfate revealed the presence of labeled N-acetylgalactosamine 4,6-bissulfate at significant levels in the blood and cartilage, but at much lower levels in the liver. The cartilage component was highest in its rate of 35S uptake, suggesting that the blood component may derive at least in some part from the cartilage. Exposure of surviving cartilage slices to inorganic [35S]sulfate, followed by extraction of the slices with hot 50% ethanol yielded a number of radioactive compounds, of which three were characterized as UDP-N-acetylgalactosamine-4,6-[35S]bissulfate, N-acetylgalactosamine-1-phosphate 4,6-[35S]bissulfate and N-acetylgalactosamine 4,6-[35S]bissulfate. By subjecting the prelabeled tissue to chase incubation, it was possible to show that the UDP-N-acetylgalactosamine-4,6-bissulfate in the tissue disappeared with an approximate half-life of 10 min with a concomitant appearance in the medium of N-acetylgalactosamine 4,6-bissulfate and its 1-phosphate ester. These results suggest the occurrence in cartilage of an enzymatic system which is responsible for rapid turnover of UDP-N-acetylgalactosamine-4,6-bissulfate and possibly required for the rapid secretion of N-acetylgalactosamine 4,6-bissulfate into extracellular field.


The glutathione thiol-disulfide status in the sea urchin egg during fertilization and the first cell division cycle.

The intracellular levels of GSH, GSSG, and protein-glutathione disulfide (protein-SSG) have been measured in the eggs and developing embryos of the sea urchins Lytechinus pictus and Stronglyocentrotus purpuratus. Total cellular glutathione is maintained in a very highly reduced state during these initial stages of development. Thus for unfertilized eggs of L. pictus the results (mumol/g dry weight) were 11 +/- 1 for GSH, 0.02 +/- 0.01 for GSSG, and 0.07 +/- 0.02 for protein-SSG. No significant change in these values was observed upon fertilization of the eggs or during the first cell division cycle. The values obtained with S. purpuratus were somewhat greater, but were also found to exhibit no significant variations upon fertilization or cell division. These observations indicate that changes in the total cellular glutathione thiol-disulfide status are not involved in the control mechanisms which operate during fertilization of the first cell division cycle in the sea urchin egg.


Branched-chain amino acid oxidation by isolated rat tissue preparations.

Branched-chain amino acid transaminase activity, branced-chain alpha-keto acid dehydrogenase activity, and leucine oxidation were measured in homogenates and slices of several rat tissues. Transaminase activity was highest in heart, while dehydrogenase activity was highest in liver. Leucine oxidation in isolated tissues may be limited by either transaminase or dehydrogenase activity depending upon the relative activities of these two enzymes in the tissue. The results suggest that, as the load of branched-chain amino acids increases, the liver may become an increasingly important site for the degradation of branched-chain alpha-keto acids.


A new agglutinating activity from wheat flour inhibited by tryptophan.

A new compound endowed with agglutinating activity, designated the flour agglutinin, was extracted from wheat flour with water and purified by gel filtration and ion-exchange chromatography. The haptenic inhibitors of the plant agglutinins do not affect flour agglutinin activity which, on the other hand, is inhibited by D- and L-tryptophan. Flour agglutinin has a molecular weight of about 5 - 10(4) as determined by gel filtration. It consists of a neutral heteropolysaccharide constituted of D-xylose and L-arabinose, and is homogeneous as judged by sedimentation analysis. Flour agglutinin activity is destroyed by treatment with Cellulase 2000 and periodate, but is not affected by alpha-amylase and proteolytic enzymes. Compared to germ agglutinin, flour agglutinin exhibits a peculiar range of cell specificity. It agglutinates several normal cell types, but has no effects on some neoplastic cells tested. Tryptic digestion of erythrocytes does not affect their susceptibility to flour agglutinin-induced agglutination.


Comparison of secreted and extracted forms of rat pituitary prolactin.

Prolactin secreted by rat anterior pituitary explants into organ culture medium was purified by salt fractionation and gel filtration. A yield of 22 mg/g was obtained, which clearly represented de novo synthesis and secretion of the hormone. Comparative characterization studies were performed on the secreted prolactin and pituitary extracted rat prolactin obtained from the National Institute of Arthritis, Metabolism and Digestive Disease. The biological and immunological activity estimates of both forms were comparable, although the specific activities of the secreted prolactin were somewhat lower than those of the pituitary prolactin. The secreted and extracted forms of prolactin appeared to be identical in primary structure as evidenced by similar amino acid compositions and identical NH2-terminal sequences. Circular dichroism spectra suggested that there may be differences in tertiary structure, since the positive tryptophan band at 292 nm, which eas observed with extracted hormone, was absent in the secreted prolactin.


Effects of prostaglandins and catecholamines on rat spleen adenylate cyclase in vitro.

The results reported here show some characteristics of adenylate cylase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F- under conditions where cyclic nucleotide degradative pathways are effectively inhibited. Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 X g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E1 and E2, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F1 alpha and F2 alpha. Stimulation by prostaglandin E1 and E2 is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E1 is equal to that caused by optimal fluoride concentrations. Stimulation caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.


Characterization and partial purification of a substance in the pineal gland which inhibits cell multiplication in vitro.

A substance which inhibits the in vitro multiplication of 3 cell strains, 37 RC, KB and NCTC clone 929, was characterized in the sheep pineal gland and partially purified using three successive chromatography techniques, respectively on Sephadex G-25, CM-cellulose and Biogel P 60 columns. The sheep cerebral cortex and liver also contain, but at much lower concentration than in the pineal, substance(s) that behave in different tests like the factor isolated from the pineal. The nature of the antimitotic substance from the pineal is as yet unknown. It is not destroyed by treatment with proteolytic enzymes, nor by boiling with 6 M HC1. It was established that it is different from the known antiblastic drugs such as Daunomycin and Methotrexate and from some active substances known to be present in the pineal, such as melatonin, secotonin and norepinephrine, which, in the same conditions, did not show any antimitotic activity. It was shown that when the concentration of the pineal factor in the culture medium was high enough (10 mug/ml), the inhibition of the KB cells multiplication was complete and irreversible. Microscopic examination of the treated cells showed that the morphological alteration was rapid (3--6 h) and deep, with shrinkage of both cytoplasm and nucleus, while with antiblastic drugs, morphological alteration proceeded slower (1--3 days) and appeared less pronounced.


Diffusion coefficient and molecular weight of type 5 adenovirus by photon-correlation spectroscopy.

The technique of photon-correlation spectroscopy (intensity fluctuation spectroscopy) is applied to light scatter from type 5 adenovirus undergoing Brownian motion in solution and the translation diffusion coefficient (D20,w) measured to be 0,367 +/- 0.003 Fick units. Using Svedberg's equation with previously determined parameters, a molecular weight of 165 - 10(6) +/- 5 - 10(6) is obtained.


A proton magnetic resonance study of water in human stratum corneum.

Proton magnetic resonance spectroscopy has been used to study the nature of water in human stratum corneum. For a single planar sheet of stratum corneum mounted at a specific orientation to the applied magnetic fild, three distinct absorptions may be seen having different chemical shifts and spin-lattice relaxation times (T1). All T1 values for these resonances are smaller than that for normal liquid water. One absorption is unusual in that the resonance position is dependent upon the orientation of the sample within the field.


Detection of stable isotopes of lithium or boron with the help of A (n,alpha) nuclear reaction. Application to the use of 6Li as a tracer for unidirectional flux measurements and to the microlocalization of lithium in animal histologic preparations.

In the particular case of boron and lithium we examine the possibilities of using stable isotopes for experiments of isotopic labelling and microlocalization, as no radioisotopes exist. The detection is made with the help of a specific nuclear reaction, using homogeneous detectors. The first experimental applications are given: transepithelial fluxes of lithium (frog skin) have shown Liefflux values larger than the influx ones. Detailed microlocalization of lithium have been made on histological preparations of mice having received lithium treatment: particularly important contents are found in the hypophysis, the salivary glands, the bladder, the kidney (especially the pelvis), the intestinal system and certain parts of the brain (particularly the hippocampus); the liver, however remains very poor in lithium. Physiological implications are examined.


The proteolytic nature of commercial samples of galactose oxidase. Purification of the enzyme by a simple affinity method.

Several commercially available samples of galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) were found to contain high proteolytic activity on proteins such as fibrinogen, transferrin, albumin and casein. A simple, efficient method was devised for the purification of galactose oxidase which relies on the affinity of the enzyme for agarose (Sepharose 6B). The purified galactose oxidase was recovered in high yield free from proteolytic activity. The enzymic affinity for Sepharose and Sephadex was investigated to clarify the absorption mechanism.


The reaction of sulfhydryl reagents with bovine hepatic monoamine oxidase. Evidence for the presence of two cysteine residues essential for activity.

The bovine liver monoamine oxidase (EC 1.4.3.4) was found to be inactivated by various well-known sulfhydryl reagents like p-mercuribenzoate, methylmercuric iodide and 5,5'-dithiobis-(2-nitro benzoic acid). The present investigation shows that the inactivation of the enzyme results from reactions of these reagents with 2 out of 8 titratable sulfhydryl groups per 10(5) g of the enzyme. The substrate, benzylamine, and competitive inhibitors like benzaldehyde, p-nitrobenzaldehyde, benzyl alcohol protected the enzyme from inactivation by the mercurials or the Ellman reagent. The inactivation experiments with these sulfhydryl reagents, the protection experiments, and the kinetics as well as physicochemical observations suggest that there are only two cysteine residues that are required for activity of the enzyme. It is possible that these two residues may be active-center residues.


Interaction of glutathione-insulin transhydrogenase (disulfide interchange enzyme) with phospholipids.

The effect of the addition of several phospholipids (lysophosphatidylcholine, alpha-lecithin, phosphatidylserine, phosphatidylethanolamine, lysophosphatidyl-ethanolamine, sphingomyelin, and disphosphatidylglycerol and phosphatidic acid) and related compounds (glycerophosphocholine, alpha- and beta-glycerophosphate, choline, serine, glycerol, dipalmitoylglycerol, and stearic acid) on the ability of purified (from beef pancreas) and microsomal (rat liver) glutathione-insulin transhydrogenase (Glutathione:protein-disulphide oxidoreductase, EC 1.8.4.2) to degrade insulin has been examined. With purified enzyme, except for phosphatidic acid and phosphatidylserine, all other phospholipids tested caused a slight activation at low concentration with phosphatidylethanolamine causing the highest activation. Lysophosphatidylcholine and phosphatidic acid are the only agents which cause inhibition of activity. The reaction rate as a function of concentration of inhibitor is hyperbolic for phosphatidic acid ([I]0.5 = 25 muM) and biphasic for lysophosphatidylcholine ([I]0.5 = 270 muM). Kinetic studies show that the two phospholipids are noncompetitive versus both substrates (insulin and GSH). Further, the structures of the phospholipids are quite different from the substrates and products of the reaction catalyzed by the enzyme. These dats, together with the data obtained with microsomes (see below), support the possibility that phospholipids, in particular lysolecithin and phosphatidic acid, might function by an interaction at an allosteric site or sites to bring about a conformational change in the enzyme. With a microsomal fraction, four phospholipids (lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylethanolamine, and phosphatidic acid) caused an increase in GSH-insulin transhydrogenase activity. At low concentration the addition of each of these phospholipids led to a 2.5-fold increase in GSH-insulin transhydrogenase activity. At higher concentration, lysophosphatidylcholine almost totally inhibited the microsomal GSH-insulin transhydrogenase activity, as it did with purified enzyme, while phosphatidic acid showed only a slight inhibition, in contrast to its effect on purified enzyme. With the microsomal fraction in which GSH-insulin transhydrogenase activity had been previously unmasked by Triton X-100 treatment, the addition of small amounts of lysophosphatidylcholine and phosphatidic acid produced, as expected, only slight increase in the transhydrogenase activity for both phospholipids; again, only lysophosphatidylcholine but not phosphatidic acid caused inhibition when higher levels were used. It is concluded that the four phospholipids and Triton X-100 increase the GSH-insulin transhydrogenase activity in the microsomes by unmasking the catalytic site without fully unmasking the allosteric site, the point of reaction with the phosphatidic acid.


The further heterogeneity of creatine kinase. Presence of isoenzymes of cathodic mobility in rat tissues.

Supernatant fractions from rat tissue homogenates were examined for creatine kinase (EC 2.7.3.2) isoenzymes by agarose gel electrophoresis at pH 8.6. The distribution of the various isoenzymes, and their relative contributions to the total tissue creatine kinase, is stated. A previously unreported isoenzyme associated with brain and kidney preparations is described. It is a minor contributor to brain creatine-kinase activity but is the major isoenzyme for kidney. The presence of an additional isoenzyme for cardiac tissue was confirmed; this isoenzyme exhibits similar levels of activity to that of the MB isoenzyme.


An essential tryptophan in the active site of phospholipase A2 from the venom of Bitis gabonica.

The role of tryptophan in phospholipase A2 (EC 3.1.1.4) from the venom of the gaboon viper, Bitis gabonica, has been investigated. Modification of the enzyme with N-bromosuccinimide and 2-nitrophenylsulfenylchloride showed that the two tryptophan residues in the enzyme, viz. Trp-28 and Trp-59, differ in reactivity towards the reagents. Only Trp-28 reacted with N-bromosuccinimide while a preferential reaction occurred between Trp-59 and 2-nitrophenyl-sulfenylchloride. In each case it was found that loss of enzyme activity was specifically correlated with modification of TRP-28. CD spectra indicated that neither the local nor the gross conformation of the enzyme was altered by modification of Trp-28 and it was therefore concluded that Trp-28 is crucial for enzyme activity. The active enzyme was protected against N-bromosuccinimide inactivation by micellar concentrations of substrate or substrate analogue, suggesting that Trp-28 is involved in substrate binding.


Differences in subunit activities in acetylcholinesterase as possible cause for apparent deviation from normal Michaelis-Menten kinetics.

1. Form Gp of acetylcholinesterase (EC 3.1.1.7) from electric eel gave curved Lineweaver-Burk plots with acetylcholine. The Hill coefficients were 0.50-0.55 at low ionic strength and increased to 0.93 with increasing ionic strength. In presence of atropine or hexamethonium normal Michaelis-Menten kinetics were observed. 2. Inhibition of the enzyme by iPr2P-F was biphasic. One half of the total enzyme activity decreased at a faster rate than the other half. With [3H]iPr2P-F, the extent of labelling was determined for the light and heavy subunits. At low [3H]iPr2P-F concentration only the light subunit was phosphorylated. Higher [3H]iPr2P-F concentrations and prolonged treatment increased the amount of label in the heavy subunit. 3. From these data it is concluded that the two subunits are labelled at different rates indicating different reactivity towards iPr2P-F as well as towards acetylcholine. These data might account for the apparent non-Michaelis-Menten type kinetics obtained at low ionic strength.


Further characterization of subunit III of bovine procarboxypeptidase A-S6 as a non activatable zymogen.

The homology of Subunit III of bovine procarboxypeptidase A-S6 (EC 3.4.12.-) with Subunit II (bovine chymotrypsinogen C) of the same complex, already reported in a previous publication (Puigserver, A. and Desnuelle, P. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2442-2445) has been extended to the position of the single methionine of the chains. The sequence linked by 4 disulfide bridges out of 5 are also probably homologous in the 2 proteins. The last bridge is displaced in Subunit III as a consequence of the deletion of the N-terminal half-cystine. Subunit II, which is not activatable by trypsin, due to the loss of essential residues in the N-terminal region, has conserved a weakly functional active site reacting with concentrated diisopropylfluorophosphate at exactly the same rate as that of Subunit II.


A sensitive isotopic assay method for S-adenosylhomocysteine hydrolase. Some properties of the enzyme from rat liver.

A rapid and sensitive isotopic method is presented for the assay of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) activity, based on the formation of radioactive S-adenosylhomocysteine labelled in the adenosine portion. The radioactive product is separated either by low-voltage paper electrophoresis or by using phosphocellulose ion-exchange paper. Some kinetic properties of the enzyme from rat liver have shown to be clearly different from those reported earlier for this enzyme. The use of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent inhibitor  of adenosine deaminase, makes it possible to measure the S-adenosylhomocysteine hydrolase activity in tissues with a high adenosine deaminase activity, e.g. in intestinal mucosa.


Partial purification and properties of N-acetylhistamine deacetylase.

The enzyme catalyzing the deacetylaction of N-acetylhistamine was partially purified about 160-fold from rat liver extract and its properties were investigated. The purification procedure included DEAE-cellulose chromatography, precipitation with ammonium sulfate and DEAE-cellulose rechromatography. The enzyme contains a labile -SH group that is essential for its activity. Mn2+ and Co2+ enhanced the deacetylation reaction at low concentration. The molecular weight of the deacetylase was estimated to be about 70 000 from gel-filtration. Among various acetyl derivatives tested so far, N-acetylhistamine and to a lesser extent N-acetyltyramine served as the substrates. The Km value was 0.3 mM at the optimum pH 8.0 for N-acetylhistamine. Diphenhysramine, an antihistaminergic agent, inhibited the deacetylation remarkably.


The regulation of mouse liver ornithine decarboxylase by metabolites.

The enzyme ornithine decarboxylase (L-Ornithine carboxy-lyase, EC 4.1.1.17), has been partially purified from the livers of mice subjected to partial hepatectomy (6-8 h previously). Mouse liver ornithine decarboxylase requires pyridoxal phosphate, and dithiothreitol for maximal activity. The enzyme has a pH optimum of 7.3, it is inhibited in the presence of 0.3 M phosphate, glycine, Tricine and Tris. It shows no dependence on metal ions and is inhibited by high salt concentrations, particularly ammonium salts. The kinetics of the enzyme have been studied with putrescine (and analogs), spermidine and spermine, in the presence of both high and low levels of pyridoxal phosphate. High concentrations of pyridoxal phosphate inhibit the enzyme. The enzyme is also inhibited by low concentrations of putrescine (1 mM). As the concentration of putrescine increased to 10 mM, non-competitive inhibition was observed, this could be reversed by addition of higher levels of pyridoxal phosphate. Spermidine and spermine inhibit (noncompetitively) only at high concentrations (10 mM). Ornithine inhibits at high concentrations (2 mM). Spectral studies have shown that the observed kinetics of competitive inhibition at low concentrations of polyamine changing to noncompetitive inhibition at high polyamine concentrations are due to competition between enzyme and substrate (or inhibitor) for free (non-enzyme bound) pyridoxal phosphate. Noncompetitive inhibition arises through the formation of transient Schiff base complexes between amines and free pyridoxal phosphate. It also appears that the binding of substrate to the active site takes place through Schiff base formation with enzyme bound pyridoxal phosphate.


The structure of human thrombin in relation to autolytic degradation.

Human thrombin was obtained by activation of human prothrombin with venom of the Australian Taipan (Oxyuranus scutellatus scutellatus). This thrombin was precipitated with ammonium sulphate (75% saturation) and subsequently purified by gel-filtration (Sephadex G-75), ion-exchange (CM-Sephadex C-50) and affinity (aminobenzamidine-CH-Sepharose) chromatography. The final preparation (affinity thrombin) had a specific activity of 2340 Iowa units per absorbance unit (A1cm280). Thrombin proteins focused between 5 and 7, while prothrombin proteins focused to pH values less than 5. SDS-acrylamide gel electrophoresis indicated molecular weights of greater than 70 000 for prothrombin and 39 000, 28 000, 25 000-23 000 and 15 000-13 000 for affinity thrombin proteins. The 39 000-dalton species predominated (greater than 90%) when the enzyme was inhibited with phenylmethanesulphonyl fluoride prior to dialysis for SDS electrophoresis. Lack of such inhibition reduced the amount of the 39 000-dalton species to less than 60% with concomitant increase of the smaller species. Peptide mapping studies indicated that the smaller species were structurally related to the 39 000-dalton species. The amino acid compositions of the histidine and/or tyrosine containing peptides indicated a high degree of homology with bovine thrombin. It has been established that human thrombin can exist in at least two secondary structural forms, of different molecular weights, probably due to autolytic degradation of the largest (39 000-dalton) form.


Urea-extractable protein from human epidermis.

The major protein fraction extracted from human epidermis by urea has the following characteristics: (1) A molecular weight of approximately 600 000. (2) A copper content of 0.03% giving a minimum molecular weight of 212 000. (3) A non-protein moiety indicated by (a) protein/dry weight ratio (b) anomalous ultraviolet absorption spectrum (c) high-voltage electrophoresis after mild alkaline or acid treatment (d) an excess of imidazole groups by Pauly reaction compared with the number of histidine residues by amino acid analysis. The non-protein component is not urocanic acid or RNA as has been reported (Bernstein, I.A. (1970), J. Soc. Cosmet, Chem. 21, 583-594 and Sibrack, L. A., Gray, R. H. and Bernstein, I. A. (1974), J. Invest. Dermatol. 62, 394-405). (4) The presence of interchain disulphide linkages indicated by polyacrylamide gel electrophoresis in sodium dodecyl sulphate with and without beta-mercaptoethanol. (5) Polyacrylamide gel electrophoresis in presence and absence of 6 M urea suggests that aggregates are broken down in urea.


Interaction between proteins and detergents which contain a hydrocarbon chain longer than 16 carbon atoms. III. Competitive inhibition of trypsin by cetyldimethylbenzylammonium chloride.

Tryptic activity was competitively inhibited by cationic detergents which contain a cetyl group or longer hydrocarbon chains. Since the cetyl group is much longer than the side chains of lysine or arginine residues of substrates for trypsin, the nature of inhibition by cetyldimethylbenzylammonium chloride was examined using Na-benzoyl-L-arginine-p-nitroanilide as substrate and compared to that by butylamine. The inhibition by cetyldimethylbenzylammonium chloride occurred instantaneously and was completely reversible. The inhibitory effect of cetyldimethylbenzylammonium chloride was strongly dependent on both pH and salt concentration, contrasting with inhibition by butylamine which was relatively indifferent to these changes. The Ki value of cetyldimethylbenzylammonium chloride at pH 7.5 and 25 degrees C was calculated to be 2.0 +/- 0.3 mM, which is equal to that of butylamine within experimental errors. The standard entropy change of binding of cetyldimethylbenzylammonium chloride (44 +/- 2 cal/mol/degree) was much larger than for butylamine, indicating the formation of an efficient hydrophobic bond between the cetyl group and the enzyme.


The protein composition of bovine myelin-free axons.

The proteins of axons prepared from myelinated axons and isolated as myelin-free entities were separated by sodium dodecyl sulfate polyacrylamide electrophoresis and found to consist of more than 10 different molecular weight species. The molecular weights range from 13 000 to over 200 000 with a prominent protein of molecular weight 47 000. The amino acid composition of the seven major proteins showed that the protein with a molecular weigt of 47 000 is distinct from all the other proteins analyzed. A group of three low molecular weight proteins have amino acid compositions which are similar to each other as do a group of three high molecular weight proteins although the two groups are distinctly different from each other and the major axonal protein. Histones, DNA, myelin basic protein and glycoprotein were absent from the proteins but neurotubule protein was present as indicated by cochicine binding activity in the axonal preparations. The cellular origin of these proteins and their relationship to other central nervous system proteins are discussed.


Structure of a light-adapting hormone from the shrimp, Pandalus borealis.

The structure of a light-adapting hormone of the shrimp, Pandalus borealis, has been determined. The hormone, which had been isolated from Pandalus eyestalks and which adapts the shrimp to brighter light conditions by causing the pigment in the distal retinal pigment cells of the eye to move into a more proximal position, is the peptide: Asn-Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pro-Arg-Val-Met-Thr-Glu-Ala-NH2. The structure was obtained by sequence analysis by the dansyl-Edman method of the intact hormone and of isolated tryptic and thermolytic peptides.


Structural requirements for steroid binding and quenching of albumin fluorescence in bovine plasma albumin.

1. Steroids interact with bovine plasma albumin at a binding region that involves tryptophanyl, tyrosyl, arginyl and lysyl residues. The function of the tryptophanyl residues is demonstrated by: (a) the decrease of albumin binding affinity after modification of one tryptophanyl with 2-nitrophenylsulfenyl chloride; (b) steroid quenching of albumin tryptophanyl fluorescence; and (c) steroid quenching of 1-anilinonaphth-alene-8-sulfonate fluorescence, when it is excited by energy transfer from excited tryptophanyls. The function of tyrosyl residues is demonstrated by the decrease of albumin binding affinity after nitration of 30% tyrosyls with tetranitromethane, or deprotonation of tyrosyls by variation of pH. The function of arginyl and lysyl residues is demonstrated by the decrease of binding affinity after modification of these residues with glyoxal, formaldehyde or acetic anhydride. The presence of both apolar (Trp, Tyr and Lys (deprotonated)) and polar (Arg and Lys(protonated)) residues at the steroid binding site fits in well with the site relative apolarity, when expressed on the Kosower scale (Kosower, E.M. (1958) J. Am. Chem. Soc. 80, 3253-3260). 2. The contribution of specific amino acid residues to steroid binding depends to some extent on the steroid structure, as exemplified by the quantitatively different role of arginyl (or lysyl) residues in albumin interaction with testosterone acetate and epitestosterone, respectively, or that of tyrosyl residues in albumin interaction with 11-deoxycorticosterone and epitestosterone, respectively. 3. The concerted action of polar and apolar amino acid residues is an essential requirement for steroid binding, since unfolding of albumin polypeptide chain by guanidine-HC1, urea, or by reduction of disulfide bridges with 2-mercaptoethanol, strongly decreases steroid binding to albumin while, conversely, reoxidation and refolding of the unfolded polypeptide chain restore albumin affinity for steroids. 4. Parallel determinations of steroid binding constants by equilibrium dialysis and fluorimetric titration, as well as the general pattern of the pH and temperature effects on steroid quenching of albumin fluorescence, confirm the validity of the fluorescence quenching titration as an effective method for measuring albumin-steroid molecular interactions.


Specific fluorescent derivatives of macromolecules. A fluorescence study of some specifically modified derivatives of chymotrypsin, trypsin and subtilisin.

The 5-dimethylaminonaphthalene-1-sulfonyl group was specifically introduced into the active site region of the serine proteinases: alpha-chymotrypsin, trypsin and subtilisin Carlsberg by the method of affinity-labeling. The resulting fluorescent derivatives were studied by a variety of fluorescence techniques and the results were correlated with structural data available on these enzymes from X-ray analysis. As model compounds for the Dns-proteinases, the absorption and fluorescence properties of Dns-amide and Dns-ethyl ester were studied in ethanol/water and p-dioxane/water mixtures. The fluorescence emission transtion energies and quantum yields were related to four commonly employed solvent-polarity scales. Best correlations for different solvents were obatined with the empirical "Z" and "Y" scales. From inspection of the fluorescence emission transition energies of the Dns group in the Dns-proteinases and comparision with the model compound studies it was possible to assign "Z" values for the apparent microenvironment polarities of the Dns group in the Dns-proteinases. The apparent polarities of the microenvironments of the Dns group in Dns-Ser 195-chymotrypsin (Dns-chymotrypsin (I)); (Dns-Phe-CH2)-His 57-chymotrypsin;  (Dns-Lys-CH2)-His 46-trypsin; and Dns-Ser 221--subtilisin Carlsberg (Dns-subtilisin (I)) are in the range of 89.5-92.5 on the "Z" scale. The apparent microenvironment polarity of the Dns group in Dns-Ser 183-trypsin (Dns-trypsin (I)) appears to be below 76.7 on the "Z" scale. The Dns group in Dns-chymotrypsin (I) and (Dns-Phe-CH2)-His 57-chymotrypsin appears to be rigidly bound as evaluated by fluorescence polarization studies. The effect of 2H2O on the fluorescence emission quantum yields of Dns-amide and Dns-ethyl ester was examined. In both cases the ratios of quantum yields in 2H2O:ethanol (8:2) to quantum yields in H2O:ethanol (8:2) was about 1.8. The 2H2O effect upon the fluorescence emission quantum yields of the Dns group has been used to investigate solvent accessibility of this chromophore in the Dns-proteinases. Acessibility studies using 2H2O are very promising and have some definite advantages over other existing methods. Energy transfer between the Trp residues and the bound Dns group was investigated in the Dns-proteinases. The mean transfer distance calculated from the observed transfer efficiencies are 18.1 A, 19.7 A and 18.4 A for (Dns-Phe-CH2)-His 57-chymotrypsin, Dns-chymotrypsin (I) and (Dns-Lys-CH2)-His 46-trypsin, respectivly. From models built using X-ray crystallographic coordinates for the protein atoms, the mean distance of separation between the Trp residues and the bound Dns group for the same set of conjugates ar 18.6 A, 17.5 A and 17.5 A, respectively. Considering the inherent difficulties in energy transfer studies, the results are in excellent agreement with the X-ray data.


Studies on pituitary prolactin. 39. Reaction of the ovine hormone with hydrogen peroxide.

Three methionine-modified derivatives of ovine prolactin have been prepared: two by oxidation of the methionines by H2O2 to sulfoxide (partial and complete), and the third by complete alkylation of the metionines with iodoacetic acid to the carboxymethyl sulfonium salts. The derivatives were characterized by exclusion chromatography, amino acid composition, circular dichroism spectra, relative rates of digestion by trypsin, and biological activity. Partially oxidized prolactin, having four of its seven methionines oxidized, was very similar to the native hormone. The unmodified methionines in partially oxidized prolactin were found to be the residues at positions 36, 81 and 132. The prolactin derivatives in which all the methionines had been oxidized, or alkylated, showed major changes in all parameters examined. In addition, circular dichroism spectra indicated that complete modification of all the methionines in prolactin exposes the normally buried tryptophans.


Optical diffraction of tropoelastin and alpha-elastin coacervates.

Optical diffraction applied to micrographs of coacervated tropoelastin and alpha-elastin show an equatorial repeat around 50 A. This confirms a 50 A center-to-center distance of parallel aligned filaments to be a fundamental property of the tropoelastin and alpha-elastin coacervates. This periodicity is similar to that of mature cross-linked elastin. These results allow the conclusion that hydrophobic association is the predominant driving force for formation of filamentous elastin in vitro. It is suggested that the coacervate is a model for relaxed fibrous elastin.


Further studies on a highly purified glycoprotein from the intimal region of procine aorta.

Highly purified glycoprotein from the intimal region of porcine aorta was isolated with minor modifications of the procedure described previously. The molecular weight of the glycoprotein as determined by sedimentation equilibrium method either in presence of 0.1 M NaCl or 6 M guanidine-HCl containing beta-mercaptoethanol was 72 000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native glycoprotein and its S-carboxyamidomethyl derivative at different acrylamide concentrations showed no difference in the molecular weight indicating the absence of subunits. Attempts to determine the identity of the amino-terminal acid by a dansylation technique indicated that the amino group is not free. The carboxy-terminal amino acid was found to be serine after treatment of the glycoprotein with carboxypeptidase A. The glycoprotein did not contain an alkali-labile (O-glycosidic) carbohydrate-protein linkage as tested by the beta-elimination reaction. The release of monosaccharides from the glycoprotein as a function of time was studied employing mild acid hydrolysis (0.5 M HCl, 80 degrees C) and also by the use of neuraminidase, alpha-D-and beta-D-glucosidases and beta-D-N-acetylglucosaminidase. From the observations of the release of monosaccharides and analogy with standard features determined by other investigators on soluble aortic glycoproteins, a prediction has been made as to the general features of the carbohydrate moiety of the glycoprotein.


Investigations on the primary structure of human plasminogen. Further evidence for sequence homology.

NH2-Terminal sequences were determined by the automated Edman method in four fragments which were isolated from a mixture of fragments obtained by CNBr cleavage of human plasminogen. One of the fragments whose sequence was determined over the first 31 residues shows sequence homologies with the fragment that forms the linkage between the plasmin chains and also with the non-thrombin part of prothrombin.


Comparison of the amino acid sequence of pig heart myoglobin with other ungulate myoglobins.

The primary structure of pig heart myoglobin has been established by study of the tryptic peptides of whole globin and by analysis of the fragments obtained by CNBr cleavage. Thermolysin and chymotrypsin digestion were used to determine the sequence of the M fragment (56-131). Automatic Edman degradation of whole globin and of the M fragment completed the sequence of pig myoglobin. Comparison with other ungulates shows that pig myoglobin is far from other artiodactyls previously studied (ox and sheep) and close to the eutherian ancestral chain.


Characterization of the hemoglobin of Biomphalaria glabrata as a glycoprotein.

1. The hemoglobin of Biomphalaria glabrata is a glycoprotein which contains 3% sugars. 2. The sugar moiety is composed of 2 mol of hexose and one mol of hexosamine per mol of monomer. 3. The hexosamine in the molecule is glucosamine. 4. Mannose, galactose and fucose are present in the molecule in a ratio of 2:1:1. 5. No sialic and uronic acids were detected in this hemoglobin.


A complex-forming glycoprotein heterogeneous in charge and present in human plasma, urine, and cerebrospinal fluid.

A glycoprotein from the urine of one healthy individual was purified by ultrafiltration, ion-exchange chromatography, gel chromatography and immunosorption. The protein contained only one polypeptide chain with an approximate molecular weight of 31 000 and was associated with a brown colour which did not disappear even after total reduction and alkylation of the protein followed by dialysis in 6 M guanidine hydrochloride. The protein appeared homogeneous on sodium dodecyl sulphate-polyacrylamide electrophoresis and gel chromatography. It had only one N-terminal amino acid sequence, Gly-Pro, and gave only one precipitate with a polyvalent antiserum but was found to be very heterorgeneous on agarose gel electrophoresis and on isoelectric focusing. Desialylation of the protein failed to alter this heterogeneity. An electroimmunoassay system was designed to measure the amount of the protein in normal human plasma, urine, and cerebrospinal fluid where the mean concentrations were found to be about 100, 10 and 0.3 mg/l, respectively. The protein was found to occur in normal plasma and urine as free monomers and dimers and as complexes with IgA and albumin.


An assessment of protein secondary structure prediction methods based on amino acid sequence.

Five of the several secondary structure prediction methods based on protein amino acid sequence has been computerized, allowing the calculation of joint prediction histograms which have been shown to be superior to any individual prediction. The known structures of about 40 proteins experimentally determined by X-ray crystallography are compared with the predictions resulting from calculated histograms. The accuracy of the predictions for helices is generally much better than for both beta-sheet regions and for turns. The overall agreement between prediction and observation within the amino terminal half of the protein molecules is clearly superior to that for the carboxyl half, suggesting an amino nucleating core. Predictions for smaller proteins and thermally stable proteins are generally good, indicating the sensitivity of the methods to short-range but not long-range interactions. In less than half the cases tested were the predictions useful; there was no way of knowing ahead of time if a favorable prediction would result. Given the lack of dramatic improvement with an increase in data base for the schemes and the generally poor agreement factors, it appears that a perfect predictive algorithm must include a consideration of energy minimization, thermalization, and long-range interactions. Extreme caution is suggested in applying present prediction routines to unknown protein structures.


Formation of dityrosine cross-links in proteins by oxidation of tyrosine residues.

1. Enzymic oxidation of proteins with peroxidase and hydrogenperoxide at a basic pH value leads to an oxidative phenolic coupling of adjacent tyrosine residues forming cross-linked proteins. 2. Dityrosine (3,3'-bityrosine) was identified as the cross-link in oxidised proteins by thin-layer chromatography, amino acid analysis and fluorescence measurements. 3. Gel filtration experiments with oxidised insulin showed that the cross-linkage is predominantly intermolecular. 4. In tetranitromethane treated proteins, dityrosine could be identified after hydrolysis.


Preparation and properties of a neurotoxin purified from the venom of black widow spider (Latrodectus mactans tredecimguttatus).

A neurotoxin of the venom of the spider Latrodectus mactans tredecimguttatus, has been prepared in a homogeneous form and examined by a variety of techniques. The protein has a molecular weight of 130 000 and its toxicity in mice is about 49 000 LD50 mg pure protein/g body weight. The toxin releases norepinephrine from synaptosomes prepared from rat brain and shows most of the toxic effects of the crude venom preparation.


Reinterpretation of luminiscence properties of neurotoxins from the venom of Middle-Asian corba Naja oxiana eichw.

A new interpretation of previous work (Bukolova-Orlova, T. G., Burstein, E.A. and Yukelson, L. Ya (1974) Biochim. Biophys. Acta 342, 272-280) and some new data on the luminescence of neurotoxins I and II from Naja oxiana venom is given, based on the newer data on their complete amino acid sequences. Very effective excitation energy exchange exists between Trp-27 and Trp-33 in neurotoxin I and between Trp-27 and Trp-28 in neurotoxin II, Which results in the tryptophanyl fluorescence spectra of each of the proteins seeming to be monocomponent ones. The lowered fluorescence quantium yield value, the shortened phosphorescence lifetime (80% of the emission has tau p less than 0.5 s, 20% has tau p = 4.8 s, comparing with usual tau p = 5.5-5.9 s) and decreased phosphorescence to fluorescence ratio (0.042, as compared to the usual 0.4-0.7) for neurotoxin I suggest that the indole chromophore of Trp-27 and/or Trp-33 are in contact with heavy sulfur atoms of disulfide, most probably of Cys(28)-Cys(32). Tryptophanyls in neurotoxin II are exposed to the solvent, however their accessibility in relation to that of the free tryptophan to the negatively charged quencher I- (0.455) is much higher than that for the positively charged Cs+ (0.08), which is probably due to the proximity of cationic Lys-25, Lys-26 and His-31. The difference of accessibility to the negative and positive quenchers is even more pronounced in the case of the neurotoxin I (1.04 and 0 +/- 0.02, respectively), though in its chromophore vicinity along the primary structure there is only one cationic group, Lys-25. This fact together with the analysis of the amino acid sequence, suggest that the space folding of this polypeptide results in the close proximity of Trp-27 and/or Trp-33 with the C-terminal peptide segment 67-73, which contains four cationic groups (His-67, Lys-69, Lys-71 and Arg-72).


[Photosynthetic activity in the absence of CPl and CP2 pigmentary complexes (author's transl)].

Various photochemical activities were tested on chloroplasts of Zea mays that received 4 s of light every 4 h during the culture period. Photosystem I and Photosystem II were functioning, as well as the photosynthetic electron transport. These chloroplasts exhibited upon sodium dodecyl sulphate gel electrophoresis neither Complex 1 (Mr 70 000) generally associated with Photosystem I nor Complex 2 Mr 25 000) generally associated with Photosystem II. Chlorophyll is indeed attached to polypeptides of molecular weight 21 000 and 29 000. These results lead us to question the functional role of chloroplast protein-pigment complexes observed by sodium dodecyl sulphate gel electrophoresis.


Studies on the nature of the inhibitory effect of trypsin on the photosynthetic electron transport of system II in spinach chloroplasts.

The effect of trypsin on the photosynthetic electron transport of spinach chloroplasts has been investigated by measurements of the flash-induced absorption changes, indicating chlorophyll a1 at 703 nm, chlorophyll aII at 690 nm and at 515 nm via electrochromism the electrical potential gradient across the thylakoid membrane, respectively, and of the fluorescence induction caused by moderate actinic light. It was found: (1) In the presence of benzyl viologen as electron acceptor and with water as natural electron donor trypsin, incubation leads to a complete suppression of the absorption changes of the electrochromic effect and of chlorophyll aI and chlorophyll aII. (2) Addition of System I electron donors (N-methylphenazonium sulfate plus ascorbate or 2,6-dichlorophenolindphenol plus ascorbate) fully restores the chlorophyll aI photoreaction, whereas the initial amplitude of the electrochromic absorption change at 515 nm amounts about 50% of the control value without trypsin. The chlorophyll aII inhibition remains uneffected by System I electron donors. (3) System II electron donors (benzohydroquinone plus ascorbate or TPB) are unable to overcome the inhibition of electron transport by trypsin. (4) The fluorescence induction curve in 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea-blocked chloroplasts is modified by trypsin. The level of maximal fluorescence is remarkably decreased, whereas the initial fluorescence remains constant. The rise in kinetics is slightly decelerated. From these results, it is concluded that in the linear electron transport from water to benzyl viologen, mild trypsin treatment specifically attacks System II at a site very close to the reaction center, either on the oxidizing or on the reducing side. The reaction center of System II itself is relatively stable against trypsin. Arguments are presented which argue in favor of the trypsin attack being primarily directed at the reducing side of System II.


Studies on the structural and functional organization of system II of photosynthesis. The use of trypsin as a structurally selective inhibitor at the outer surface of the thylakoid membrane.

The effect of trypsin on the photosynthetic electron transport has been investigated in the presence of various electron acceptors (benzyl viologen, p-benzo-quinone, K3[Fe(CN)6]) by measurements of flash-induced oxygen evolution and of the absorption changes at 334 nm, indicating the primary electron acceptor of System II, X 320, and at 515 nm, indicating via electrochromism the electrical potential gradient across the thylakoid membrane. It was found that the effect of trypsin is strongly dependent on the nature of the electron acceptor: (1) Oxygen evolution is completely inhibited in the presence of p-benzo-quinone, but remains nearly unaffected by K3[Fe(CN)]6. (2) The initial amplitude deltaAO of the 334 nm absorption change is insensitive to trypsin in the presence of K3[Fe(CN)6], but the absorption change is abolished if benzyl viologen is used as acceptor. (3) The initial amplitude deltaAO of the 515 nm absorption change decreases by trypsin down to 50% with K3[Fe(CN)6] and is completely suppressed with benzyl viologen. (4) In trypsinated chloroplasts, the above-mentioned activities appear to be rather insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, in contrast to normal chloroplasts. On the basis of these results it is inferred that the primary electron acceptor of System II, X 320, is covered by a proteinaceous component susceptible to tryptic digestion. In addition, it is postulated that this component acts as well as an allosteric protein responsible for the regulation of the electronic interaction between X 320 and the plastoquinone pool, as for the inhibitory effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Various other possible effects caused by the proteinaceous shield and its modification by trypsin are discussed. The present results are in complete agreement with asymmetric membrane models postulating a zig-zag arrangement of the electron transport chain with the reducing side located towards the outer phase and the oxidizing side near the inner phase of the thylakoids.


The effect of magnesium ions on action spectra for reactions mediated  by photosystems I and II in spinach chloroplasts.

Action spectra were measured for positive changes in variable fluorescence (emission greater than 665 nm) excited by a beam of 485 nm chopped at 75 HZ. The action of two further beams were compared, one being variable, the other (reference) constant with respect to wavelength and intensity. Comparison was achieved by alternating the reference and the variable wavelength beams at 0.3 HZ and adjusting the intensity of the latter such as to cancel out any 0.3 HZ component in the 75 HZ fluorescence signal. The relative action then was obtained as the reciprocal of the intensity of the variable wavelength beam. Similarly, action spectra were measured for O2 evolution with ferricyanide/p-phenylenediamine as electron acceptor, and for O2 uptake mediated by methyl viologen with ascorbate 3-(p-chlorophenyl)-1,1-dimethylurea as electron donor in the presence of 2,6-dichlorophenolindophenol. Addition of 5 mM MgCl2 increases the relative action around 480 nm for the change in variable fluorescence and p-phenylenediamine-dependent O2 evolution, and decreases it for methyl viologen-mediated O2 uptake with 2,6-dichlorophenolindo-phenol/ascorbate as electron donor in the presence of 3-(p-chlorophenyl)-1,1-dimethylurea. The change in variable fluorescence and O2 evolution are stimulated by MgCl2, whereas O2 uptake is inhibited by it. The results are discussed in terms of a model assuming a tripartite organization of the photosynthetic pigments (Thornber, J. P. and Highkin, H. R. (1974) Eur. J. Biochem. 41, 109-116; Butler, W. L. and Kitajima, M. (1975) Biochim. Biophys. Acta 396, 72-85). MgCl2 is thought to promote energy transfer to Photosystem II from a light-harvesting pigment complex serving both photosystems.


A demonstration of energy transfer from photosystem II to photosystem I in chloroplasts.

Photosystem I activity of Tris-washed chloroplasts was measured at room temperature as the rate of photoreduction of NADP and as the rate of oxygen uptake mediated  by methyl viologen in both cases using dichlorophenolindophenol plus ascorbate as the source of electrons for Photosystem I. With both assay systems the rate of electron transport by Photosystem  I was stimulated approx. 20% by the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea which caused the Photosystem II reaction centers to close. Photosystem I activity of chloroplasts was measured at low temperature as the rate of photooxidation of P-700. Chloroplasts suspended in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1,1-dimethylurea were frozen to -196 degrees C after adaptation to darkness or after a preillumination at room temperature. The Photosystem II reaction centers of the frozen dark-adapted sample were all open; those of the preilluminated sample were all closed. The rate of photooxidation of P-700 at -196 degrees C with the preilluminated sample was approx. 25% faster than with the dark-adapted sample. We conclude from both the room temperature and the low temperature experiments that there is greater energy transfer from Photosystem II to Photosystem I when the Photosystem II reaction centers are closed and that these results are a direct demonstration of spillover.


Picosecond kinetics of chlorophyll and chlorophyll/quinone solutions in ethanol.

The mechanism of quenching by quinones of the lowest excited singlet state of chlorophyll has been investigated using picosecond laser spectroscopy. With chlorophyll alone, laser excitation resulted in immediate (less than 10 ps) bleaching of the 665 nm band and production of new absorption bands in the regions 460-550 and 800-830 nm. The lifetimes of these changes were greater than 500 ps. Addition of 2,6-dimethylbenzoquinone caused quenching of these absorbance changes. No indication of chlorophyll cation radical formation was obtained. Thus, the interaction between quinone and the chlorophyll excited singlet state results in energy dissipation without measurable formation of radical species having lifetimes longer than 10 ps. This is in marked contrast to the quenching of the chlorophyll lowest triplet state by quinones, during which easily detectable stable radical formation has been observed.


Control of oxidative phosphorylation by the extra-mitochondrial ATP/ADP ratio.

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate+rotenone and glutamate+malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized. The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.


Cooperative binding of calcium to glycerinated skeletal muscle fibers.

The binding of 45Ca2+ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg2+ (no ATP) binding was half-maximal at 1.4-10(-6) M Ca2+ and the maximal amount bound was 1.6 mumol/g protein. At less than 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K' = 0.8 - (10(6) M-1) and the Hill coefficient was 1.0. In the absence of Mg2+, binding was half-maximal at 3 - 10(-7) M Ca2+ and the maximal amount bound was 2.9 mumol/g protein. The Scatchard plot indicated two classes of sites with K' values of about 2 - 10(7) and 2 - 10(6) M-1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of the Mg2+ binding to about half of the total Ca2+ binding sites is suppressed and there is a strong positive cooperativity involving half of the ramaining sites.


Positional and fatty acid specificity of monoacyl- and diacylglycerol 3-phosphate formation by rabbit heart microsomes.

Fatty acid selectivity of acyl-CoA:glycerol-3-phosphate acyltransferase (EC 2.3.1.15) and acyl-CoA:monoacylglycerol-3-phosphate acyltransferase (EC 2.3.1.52) of the microsomal fraction prepared from rabbit heart was studied. 1. The rate of acylation of glycerol 3-phosphate was increased proportionally with the concentration of acyl-CoA. The maximum rate was reached at 0.3 mumol acyl-CoA per ml. Palmitoyl-, oleoyl- and linoleoyl-CoA all served equally well as acyl donors, and produced approximately equal amounts of mono- and diacylglycerol 3-phosphate. The rate of reaction measured in the presence of two acyl-CoA esters was similar to that in the presence of a single acyl-CoA. 2. Treatment of synthesized monoacylglycerol 3-phosphate with Crotalus adamanteus venom phospholipase A2 (EC 3.1.1.4) and that with phosphatidate phosphatase (EC 3.1.3.4) indicated that the heart enzyme synthesized almost exclusively 1-acylglycerol 3-phosphate regardless of the kind of acyl donor. 3. Hydrolysis of diacylglycerol 3-phosphate with phospholipase A2 revealed that there was a slight preference for linoleoyl-CoA at position 2 and a slight discrimination against palmitoyl- and stearoyl-CoA at position 2, when diacylglycerol 3-phosphate was synthesized in the presence of a mixture of acyl-CoA esters. When diacylglycerol 3-phosphate was formed in the presence of a single acyl-CoA, the fatty acid distribution was random. 4. The rate of acylation of 1-palmitoylglycerol 3-phosphate by rabbit heart microsomal fraction was increased proportionally to the increasing concentrations of 1-palmitoylglycerol 3-phosphate up to 50 nmol per ml; higher concentrations were inhibitory. Differences in the activities measured with palmitoyl-, oleoyl- and linoleoyl-CoA as acyl donors were negligible. When stearoyl-, arachidonoyl- and erucoyl-CoA acted as acyl donors, the rates of reaction were low. 5. The acyl-CoA:1-palmitoylglycerol-3-phosphate acyltransferase activity increased proportionally to the increasing concentrations of acyl-CoA up to 10 nmol per ml; acyl donor specificity was similar to that found above [4]. The acyltransferase showed


The lipid intermediates arising during glycoprotein biosynthesis in liver microsomes.

Incubation of liver microsomes with GDP [14C] mannose leads to the formation of lipid-linked derivatives of [14C] mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these two types of compounds from each other were found to be deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. This paper presents a column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into two components; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated molecular weight of 1100. However, when this released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into two peaks suggesting that there may be 2 oligosaccharide of approximately the same size but different structures. After brief periods of labeling with GDP [14C]mannose (5 s) an additional oligosaccharide of 3 to 4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP [14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7--8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to three different components, including a lipid bound oligosaccharide containing 3- 5 sugar residues.


Rat intestinal glycolipids. II. Distribution and biosynthesis of glycolipids and ceramide in villus and crypt cells.

Intestinal epithelial cells were isolated from rat intestine and grouped into villus and crypt cell fractions. Glycolipids were purified from each cell fraction and quantitated by fluorimetric determination of glycolipid sphingosine. Significant quantities of ceramide were found in all cell fractions and accounted for approximately 15% of total glycolipid sphingosine. While villus and crypt cell fractions quantitatively contained differing amounts of sphingosine, all cell fractions contained proportionally similar quantities of sphingosine when compared to cellular cholesterol or phospholipid. Individual glycolipids, however, showed significant differences in distribution between villus and crypt cells. Hematoside and glucosylceramide were proportionally increased in villus cells, while crypt cells showed an increase in trihexosylceramide and ceramide content. The rate of UDPglucose : hydroxy fatty acid ceramide glucosyltransferase was higher in villus cells while the rate of UDPgalactose : lactosylceramide galactosyltransferase was 3--4 times increased in crypt cells. These studies demonstrate that significant differences in both the distribution and biosynthesis of individual glycolipids occur in crypt and villus cells of rat intestine and are of possible importance in the process of intestinal cell differentiation.


A comparative study of the glycolipids of human, bird and fish testes and of human sperm.

The glycolipids of human testis and sperm have been compared. Both adult testis and the sperm exhibited remarkably complex, but generally similar, patterns of glycolipids. In particular, both contained appreciable amounts of the sulfogalactosylmonoalkylmonoacylglycerol, recently shown to be the principal glycolipid of the testis and sperm of a number of animals. In contrast, immature (prebuteral) human testis did not contain this compound. To extend knowledge on the possible distribution of sulfogalactosylmonoalkylmonoacylglycerol in the testes of other chordates, we have also analysed the glycolipids of the testes of a number of birds and fish. None of the testes from these species contained the above compound. Instead, sulfogalactosylceramide was found to be a major glycolipid of the testis of mature fowl, duck and skate-fish and sulfogalactosylglucosylceramide of the testis of mature salmon and trout. Immature duck testis contained only a trace of sulfogalactosylceramide. These studies reveal intriguing differences between the sulfatides of various chordates, lend support to the concept that sulfatides increase markedly in testis at a specific stage of spermatogenesis and suggest an important role for sulfatides in testicular and spermatozoal function.


In vitro incorporation of isomeric cis-octadecenoic acids by rat liver mitochondria.

The metabolic fate of positional isomers of cis-octadecenoic acids was compared to that of oleic acid, elaidic acid and stearic acid in rat liver mitochondria. The positional isomers as well as elaidic acid and stearic acid were labelled with 3H and they were incubated in pairs with [1-14C]oleic acid. 3H/14C ratios were determined for the administered mixtures and for the isolated lipid classes. The results suggested that all isomers were readily incorporated into the membraneous structure of mitochondria. Those with the double bond in the middle of the acyl chain resembled oleic acid and they were preferentially incorporated in cholesterol esters, triacylglycerols and in the 2-position of triacylglycerols and of phosphatidylcholines. Those with the double bond away from the middle of the chain were metabolically distinct from oleic acid and behaved like trans fatty acids. They were rapidly taken up by mitochondria. They were preferentially incorporated in phospholipids and they occupied the 1-position in phosphatidylcholines and the 1- and 3-positions in triacylglycerols.


Effects of the thyroid state on cholesterol metabolism in the rat.

An isotopic equilibrium method which permits the in vivo measurements of cholesterol turnover processes was applied to different groups of rats: (1) radiothyroidectomized, (2) low-iodine fed, and (3) L-thyroxin fed. Plasma cholesterol concentrations were enhanced after thyroidectomy and reduced by large dose of L-thyroxin. A low-iodine diet decreased plasma thyroxin level but did not affect plasma cholesterol. Thyroid levels in plasma modified the coefficient of intestinal absorption of cholesterol. After thyroidectomy or under conditions of reduced thyroxin formation this absorption coefficient was enhanced. The absorption coefficient of cholesterol was decreased in rats receiving 31 or 61.5 mug/day of L-thyroxin but was not changed in rats fed 110 mug/day L-thyroxin. The proportion of de novo biosynthetisized cholesterol eliminated into the feces (external secretion) was reduced while thyroxin levels were low. The fecal excretion of cholesterol and the in vitro exchange of cholesterol between erythrocytes and plasma were increased by L-thyroxin ingestion. The rate of cholesterol biosynthesis was decreased after thyroidectomy and enhanced by L-thyroxin feeding. In fact, changes in thyroid state modified indirectly the biosynthesis of cholesterol by its effects on metabolism and on the coefficient of intestinal absorption of cholesterol.


Linoleic acid desaturation activity of liver microsomes of essential fatty acid deficient and sufficient rats.

Studies were carried out to relate the changes of the fatty acid and lipid composition of rat microsomes with the modification of the activity of the linoleic acid desaturation evoked by an essential fatty acid deficient diet. Two steps were shown in the progression of the essential fatty acid deficienty. In a first step shown at three days of essential fatty acid deficiency the fatty acid composition was changed by decreasing linoleic and arachidonic acids and increasing oleic and eicosatrienoic (-9) acids. No change was found in the lipid distribution and approximate V and Km of the linoleic acid desaturation. In this first step the unsaturated/saturated fatty acids ratio fell in spite of the synthesis of eicosatrienoic (n-9) acid that was produced without any change of enzyme activity. In a second step shown at 15 days of essential fatty acid deficiency the change of the fatty acid composition was greater but the unsaturated/saturated acid ratio was restored. An increase of triacylglycerols and a decrease of phospholipids was also detected together with an enhanced activity of linoleic acid desaturation (higher approximate V) and a higher approximate Km. The increase of the V of linoleic acid desaturation is considered to be evoked by an increased level of active delta-6 desaturase. The increased activity of the delta-6 desaturase in this second period is a secondary and important response of the cell to maintain the unsaturated : saturated acid ratio and fluidity of the membrane.


Concentration changes of bile acids in sequential segments of pigeon intestine and their relation to bile acid absorption.

Changes in the concentration of bile acids in sequential segments of pigeon intestine were measured. It was found that the jejunum has the highest concentration of bile acids. When the ratio of bile acids to beta-sitosterol (a non-absorbable dietary marker) was examined in contents of various segments, it was found that the ratio showed a marked decrease (86.6%) from the upper to lower jejunum while the subsequent changes in other segments were not as striking. This indicates that the jejunum is the major site of bile acid absorption in the pigeon, unlike in mammals, where the ileum has been shown to be the major site of bile acid absorption. This notion was confirmed by studies of the effect of ileal bypass in these pigeons. This surgical procedure did not significantly change the fecal excretion of bile acids or neutral sterols indicating that the jejunum is capable of maintaining a normal enterohepatic circulation of bile acids in the absence of the ileum in the pigeons.


Effects of local anaesthetics on phospholipases.

1. The effects of six local anaesthetics have been studied on the activities of soluble phospholipases A2 (EC 3.1.1.4) and lysophospholipase (EC 3.1.1.5). 2. Phospholipase A2 activity in human seminal plasma towards sonicated radioactively-labelled phosphatidylethanolamine was slightly stimulated a low and inhibited at high concentrations of all anaesthetic compounds employed. The order of decreasing potency was chlorpromazine, dibucaine, tetracaine, lidocaine, cocaine and procaine. In line with previous findings, the mode of inhibition was seen to be competitive with respect to Ca2+. 3. Phospholipase A2 activity in crude venom of Crotalus adamanteus was not affected or slightly stimulated by local anaesthetics up to 10(-2) M concentrations, when egg yolk was used as substrate. However, with sonicated radioactively-labelled phosphatidylcholine or phosphatidylethanolamine as substrate, stimulation of phospholipase activity was seen with all local anaesthetics up to 10(-2) M, the order of decreasing potency again being chlorpromazine, dibucaine, tetracaine, lidocaine, cocaine and procaine. The mode of stimulation was seen to be un-competitive with respect to substrate and probably independent of any involvement of Ca2+. 4. As in seminal plasma phospholipase A2, the activity in crude Naja naja venom towards sonicated radioactively labelled phosphatidylcholine was stimulated at low and inhibited at high concentrations of dibucaine and chloropromazine, for example. The mode of inhibition was seen to be competitive with respect to Ca2+, whereas stimulation by the anaesthetic drugs was independent of Ca2+. Binding between drug and enzyme was demonstrated by equilibration filtration of purified phospholipase A2 of Naja naja venom through a Sephadex G 25-fine column, previously equilibrated with 0.5 mM radioactively labelled chlorpromazine. 5. Lysophospholipase activity in rat liver cytosol towards radioactively labelled lysophosphatidylcholine was  inhibited by all local anaesthetics used; the order of decreasing potency was chlorpromazine, dibucaine, tetracaine, cocaine, lidocaine and procaine. The inhibition was un-competitive with respect to substrate. 6. The inhibitory and stimulatory potencies of the local anaesthetics employed closely parallel their lipid solubilities and anaesthetic potencies.


The relationship between chain elongation of palmitoyl-CoA and phospholipid content in rat liver microsomes.

The relationship between the chain elongation of palmitoyl-CoA and phospholipid content in rat liver microsomes was studied. When liver microsomes were incubated with phospholipase C, microsomal phospholipids were linearly hydrolyzed during 10 min of incubation under the present experimental conditions. Coincident with the decrease in microsomal phospholipid content by phospholipase C treatment, the chain elongation activity also decreased linearly. The decreased chain elongation activity in phospholipase C-treated microsomes was completely or partially recovered by the addition of a sonicated dispersion of phosphatidylcholine, microsomal phospholipids or phosphatidylcholine/phosphatidylethanolamine mixtures. The extent of recovery of decreased activity by a sonicated dispersion of phosphatidylcholine/phosphatidylethanolamine mixture was gradually reduced by increasing amounts of phosphatidylethanolamine in the dispersion. In addition, the chain elongation activity in native nicrosomes was more stimulated by the addition of a sonicated dispersion of phosphatidylcholine alone than by that of phosphatidylcholine/phosphatidylethanolamine mixtures. The chain elongation activity of palmitoyl-CoA was inhibited by the addition of stearoyl-CoA which is the end-product of this reaction. The inhibitory effect of stearoyl-CoA was partially eliminated by the addition of a sonicated dispersion of phosphatidylcholine. The increase of the chain elongation activity in native and phospholipase C-treated microsomes by the addition of a sonicated dispersion of phosphatidylcholine was not related to the activity of fatty acyl-CoA hydrolase.


Metabolism of 8,11,14-eicosatrienoic acid in human platelets.

The following labeled compounds were isolated and identified after incubation of 8,11,14-eicosatrien [1-14C] oic acid with human platelets: 12-L-hydroxy-8,10,14-eicosatrienoic acid, 8,11,12-trihydroxy-9,14-eicosadienoic acid, 8,9,12-trihydroxy-10,14-eicosadienoic acid, 12-L-hydroxy-8,10-heptadecadienoic acid, prostaglandin E1, prostaglandin D1, and 8-(1-hydroxy-3-oxopropyl)-9,12-dihydroxy-10-heptadecenoic acid (thromboxane B1).


Triacylglycerol contents and in vivo lipogenesis of ob/ob, db/db and Avy/a mice.

The triacylglycerol content and the in vivo lipogenesis rates of the liver and the carcass of 12 groups of mice were studied. They were mice of three strains and affected by mutations at three loci: C57BL/6J-ob/ob and normal mice; C57BL/KsJ-db/db and normal mice; and VY/WfL-Avy/a and normal mice. Each type of mice was studied at two body weight levels, before and after the mutants became grossly obese. It was found that the C57BL/6J-ob/ob and the C57BL/KsJ-db/db mice had the characteristics of juvenile type obesity. They had higher lipogenesis rates and accumulated more triacylglycerol when they were young. They gained weight rapidly mainly due to the accumulation of more triacylglycerol as they matured. Their total triacylglycerol content could reach 50% of their body weight. At maturity, their lipogenesis rates had decreased to normal. In contrast, the VY/WfL-Avy/a mice had the characteristics of maturity-onset type obesity. When they were young, they did not have higher lipogenesis rates and had only a moderate amount of triacylglycerol stored. They did not gain weight rapidly as they matured. However, when they reached maturity, their lipogenesis rate did not decrease. Their body triacylglycerol content was about 25% of their weight.


Studies on lysophospholipases. VIII. Immunochemical differences between two lysophospholipases from beef liver.

1. Two distinct lysophospholipases have previously been obtained in homogeneous form from beef liver. In this paper, we demonstrate that ageing of a beef liver homogenate does not result in a change in the ratio of the two enzymatic activities, indicating that no interconversion of the lysophospholipases took place. 2. Possible partial structural relationships between the two enzymes were explored by immunochemical techniques. Rabbit antisera raised against each individual lysophospholipase showed no cross-reactivity with the other enzyme. This was concluded from immuno double-diffusion experiments and from the results of immunoprecipitation of enzymatic activities in solution. 3. Lysophospholipase and esterase activity in the purified preparation of lysophospholipase II from beef liver were concomitantly precipitated by anti-serum against lysophospholipase II. This is further proof that both enzymatic activities reside in a single polypeptide chain, in agreement with previous results of isoelectric focusing experiments.


Cholesterol sulfate in rat tissues. Tissue distribution, developmental change and brain subcellular localization.

1. A reliable micromethod for the determination of the tissue level of cholesterol sulfate has been developed. Cholesterol sulfate was separated from the bulk of the free cholesterol by silica gel column chromatography, and the cholesterol sulfate fraction subjected to benzoylation. A small amount of contaminating free cholesterol and other lipids remaining in this fraction were converted to benzoyl esters while the cholesterol sulfate remained unreacted. The cholesterol sulfate was then separated from the benzoylated contaminants by a second silica gel chromatography column and subjected to solvolysis. The liberated cholesterol was determined by gas-liquid chromatography. 2. The cholesterol sulfate contents of the visceral organs of 43-day-old rats were determined. Every tissue examined contained small amounts of this sulfate. Kidney contained the highest concentration of cholesterol sulfate (250-300 mug/g dry tissue weight) followed by spleen (77 mug/g), adrenal gland (50-70 mug/g) and lung (50-57 mug/g). 3. In brain, cholesterol sulfate level rises sharply from 17 mug/g dry weight in 7-day-old rats to more than 50 mug/g in 15-day-olds, then it declines rapidly to 15 mug/g in the 40-day-olds and this level is maintained to adulthood. The developmental pattern in the liver resembles that in the brain, except that the peak is somewhat flatter with the highest value (60 mug/g dry weight) occurring in the 21-day-old animal. In contrast to the above two tissues, the level of kidney cholesterol sulfate increases steadily from 15 mug/g in 7-day-olds and reaches the adult level of approx. 200 mug/g in 50-day-olds. 4. The highest level of cholesterol sulfate in subcellular fractions of rat brain occurred in a fraction rich in nerve endings. The level here was 10 times higher than that in the mitochondrial fraction, which contained the lowest levels of this steroid sulfate.


The effect of portacaval anastomosis on plasma lipoprotein metabolism in rats.

1. The effect of portacaval anastomosis on the metabolism of plasma lipoproteins was investigated in rats. 2. When compared with sham-operated pair-fed controls, plasma high density lipoprotein cholesterol concentration was decreased, plasma low density lipoprotein cholesterol concentration was increased and plasma total cholesterol concentration was unchanged in the portacaval anastomosis rats. Maximal incorporation of [14C]leucine into the total circulating mass of protein was decreased in the very low density lipoprotein and high density lipoprotein fractions and, possibly, in the low density lipoprotein fraction, but there was no change in maximal incorporation into albumin. It is concluded that portacaval anastomosis diminishes the rate of synthesis of high density lipoprotein and very low density lipoprotein proteins and, possibly, of low density lipoprotein proteins.


The glycosphingolipids of rat sublingual and submaxillary glands.

1. Glycosphingolipids have been isolated from rat sublingual and submaxillary glands by the procedure involving lipid extraction, column fractionation and thin-layer chromatography. 2. The major neutral glycosphingolipids in rat sublingual and submaxillary glands were monohexosylceramide, dihexosylceramide, tetrahexosylceramide and pentahexosylceramide. Both types of glands exhibited a low content of trihexosylceramide. The fucose-containing glycosphingolipids were not found. 3. The acidic glycosphingolipids in rat sublingual and submaxillary glands were composed of monohexose sulfatide, dihexose sulfatide and monosialo-and disialogangliosides of hematoside series. In addition, small quantities of gangliosides containing hexosamines were also present. 4. The distribution of acidic and neutral glycosphingolipids was similar in the sublingual and submaxillary glands, except for the tetrahexosylceramide and sultatides. Sublingual glands contained 1.5 and 3.0 times as much tetrahexosylceramide and sulfatides, respectively, as did submaxillary glands. 5. The glycosphingolipids of submaxillary and sublingual glands showed large similarity in fatty acid composition. The fatty acid composition of gangliosides resembled each other, but differed remarkably from those of sulfatides and neutral glycosphingolipids in the docosanoate content.


[Characterisation of D(-)glyceric acid from Nocardia caviae (author's transl)].

A glycolipid was found in a strain of Nocardia caviae. It consists of glucose, myristic, palmitic and stearic acids and a polyhydroxylated acid. The structure of this hydroxyacid was demonstrated by the identification of the product glycerol after LiA1H4 reduction of the glycolipid methyl ester and subsequent hydrolysis, by comparison of the infrared spectra of the hydroxyacid and glyceric acid, by gas chromatography of acetylated methyl and ethyl esters of the polyhydroxylated acid and of standard glyceric acid and by mass spectrometry of the diacetylated methyl ester. The hydroxyacid from the glycolipid is D(-)glyceric acid, a compound rarely found amongst natural products.


Abnormal activation of sterol synthesis in lymphocytes of atherosclerotic-susceptible pigeons and heterozygous familial hypercholesterolemic patients.

Delipidated human sera enhances the incorporation of [2-14C]acetate, but not mevalonate, into digitonin-precipitable sterols of pigeon lymphocytes. Show Racer and White Carneau pigeons exhibit inherited differences in induction of sterol synthesis dissociated from inheritance of hypercholesterolemia. Moreover, lymphocytes of three familial hypercholesterolemic individuals, having serum cholesterol level in the normal range by drug therapy, exhibited a higher activation of sterol synthesis by delipidated sera when compared to cells of normal individuals. It is suggested that genetic abnormality in lymphocytes of familial hypercholesterolemic can be dissociated from hypercholesterolemia.


Metabolism of delta7- and delta5,7-sterols by Phytophthora cactorum.

delta7- and delta5,7-Sterols are converted to the corresponding delta5-sterols by Phytophthora cactorum. When delta7-sterols were supplied to the fungus, delta5- and delta7-sterols were recovered in an acetone extract of the mycelium, the proportion of delta5-sterol being higher in the free sterol fraction than in the esters. When delta5,7-sterols were supplied, delta5-sterols were recovered. No delta5,7-sterols were found in the mycelium. The initial rate of uptake of the sterols was in the order delta5 greater than delta7 greater than delta5,7.


Isolation and characterization of insect vitellogenin. Its identity with hemolymph lipoprotein II.

Several years ago, we isolated two major lipoproteins from the pupal hemolymph of the Cynthia silkworm, both of which contained diacylglycerol as a major lipid, and named them lipoproteins I and II. In this paper, we present definite evidence that the vitellogenin (female protein) of this insect is indeed identical with lipoprotein II.


Lesions in post-ribosomal supernatant fractions associated with loss of viability in pea (Pisum arvense) seed.

RNA synthesis and protein synthesis in viable pea embryonic axis tissue commences during the first hour of water imbibition whilst DNA synthesis commences after 8 h of imbibition. Neither DNA synthesis nor protein synthesis could be detected in non-viable axis tissue during the first 24 h of imbibition but some RNA synthesis is detectable during this period. Both post-ribosomal supernatant and ribosomal fractions from imbibed non-viable embryonic axis tissue were impaired in their ability to support polyphenylalanine synthesis in a cell-free protein-synthesising system, yet the same fractions isolated from unimbibed non-viable axis tissue were as efficient as equivalent fractions from unimbibed viable axis tissue in the support of polyphenylalanine synthesis in the cell-free system. A major lesion in elongation factor 1 activity and additional lesions in elongation factor 2 and phenylalanyl-tRNA synthetase activities were detected in the post-ribosomal supernatants isolated from non-viable embryonic axis tissue.


Ion binding and chromatin condensation.

1. The binding of 45Ca2+ to hen erythrocyte chromatin has been studied to help elucidate how cations induce a reversible condensation of this chromatin. 2. As the unbound Ca2+ of the medium rises from 0.5 to 4 mM, Ca2+ is bound to the chromatin with a stability constant of approx. 3.1 and a saturation value of 0.25 Ca2+ per DNA phosphate, or one-half the value for pure DNA. Condensation of the chromatin is half complete when this binding of calcium is roughly half complete. Hence the transition from the uncondensed to the condensed state occurs as repulsion between the free DNA phosphates of erythrocyte chromatin is neutralised by bound cations. Genetically active chromatin may be maintained in an uncondensed state in living cells by the presence of different negative groups that remain unneutralised at the unbound cation concentrations of the cell. 3. That only one-half of the calcium binding sites of DNA are masked in erythrocyte chromatin supports recent models of chromatin structure in which the DNA double helix is wound round a core of histones. 4. Competition for calcium binding sites in the chromatin by other cations was also studied.


DNA uptake by Streptomyces species.

Uptake of homologous and heterologous radioactively labelled DNA was studied in ten Streptomyces species. Among these, S. kasugaensis and S. virginiae were shown to take up 0.5-2.0% of the supplied donor DNA at a well-defined point in their growth cycle. The heterologous donor DNA taken up retained its original buoyant density in CsCl gradients. Other strains either extensively degraded the donor DNA or showed poor uptake efficiency.


Lesions in the ribosomes of non-viable pea (Pisum arvense) embryonic axis tissue.

Ribosomes isolated from either dry viable or non-viable pea embryonic axis tissue were equally effective in the support of polyphenylalanine synthesis in a poly(U)-directed cell-free protein-synthesising system. Ribosomes isolated from imbibed non-viable axis tissue were impaired in their ability to support polyphenylalanine synthesis in the cell-free system. RNA isolated from ribosomes and 40-S ribosomal subunits of dry or imbibed viable axis tissue was found not to be degraded, whereas the equivalent RNA species isolated from non-viable axis tissue showed an increased degree of breakdown as imbibition proceeded. Even though rRNA of imbibed non-viable axis tissue was degraded, the ribosomes and ribosomal subunits of these embryos appeared intact. In viable embryonic axis tissue the percentage of ribosomes present in the cell in the form of polysomes increased during imbibition whereas no polysomes could be detected in ribosomal preparations from dry or imbibed non-viable axis tissue. The breakdown of rRNA in ribosomal particles from non-viable axis tissue may be a contributory factor to senescence and loss of viability in Pisum arvense.


Aminoacylation of Phaseolus vulgaris cytoplasmic, chloroplastic and mitochondrial tRNAsPro and tRNAsLys by homologous and heterologous enzymes.

The cytoplasmic prolyl-tRNA synthetase can be separated by hydroxyapatite chromatography, from the enzyme present in the chloroplasts and in the mitochondria (organellar enzyme). The cytoplasmic lysyl-tRNA synthetase can also be separated from the organellar enzyme. There are two tRNAsPro in the cytoplasm; they can be charged by the cytoplasmic enzyme, but not by the organellar enzyme or the Escherichia coli enzyme. Chloroplasts contain, in addition to the two cytoplasmic tRNAsPro, one chloroplast-specific tRNAPro, which is not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. Mitochondria contain, in addition to the two cytoplasmic tRNAsPro, two mitochondria-specific tRNAsPro, which are not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. There are two tRNAsLys in the cytoplasm. Both can be charged by the cytoplasmic enzyme, but one can be charged by the organellar or E. coli enzyme. Chloroplasts contain in addition to one cytoplasmic tRNALys, one chloroplast-specific tRNALys which can only be charged by the organellar or E. coli enzyme. Mitochondria contain, in addition to one cytoplasmic tRNALys, one mitochondria-specific tRNALys which can only be charged by the organellar or E. coli enzyme.


The buoyant density of three double-stranded RNAs in cesium sulfate.

The buoyant densities in Cs2SO4 of the double-stranded RNA from the bacteriophage phi 6 and the virus-like particles of Helminthosporium maydis and Penicillium chrysogenum were determined by a Taylor series expansion and by the position and slope of the gradient relative to the isopycnic position (hinge point method). Buoyant densities for the three types of double-stranded RNA calculated by the two methods were, respectively, 1.6089 and 1.6083, 1.6065 and 1.6059 and 1.6057 and 1.6050.


Newly synthesized mammalian cell DNA. Separation of Okazaki pieces by thermal chromatography on hydroxyapatite.

The optical melting profile of a fraction of nascent DNA which can be isolated from DNA of logarithmically growing Ehrlich ascites cells by nitrocellullose column chromatography was recorded. In relation to the bulk DNA a slightly lowered Tm and an increased width of the transition was observed. Thermal hydroxyapatite chromatography revealed that melting of the nascent DNA begins at relatively low temperatures in helix regions containing small daughter strand pieces. The pieces were characterized as Okazaki pieces by determining their size and their labeling kinetics during [3H] thymidine pulses. The combination of nitrocellulose chromatography and thermal hydroxyapatite chromatography seems to be suitable for isolating Okazaki pieces.


NMR studies of the structure and stability of the 1 : 2 actinomycin D-d-pG-C complex in aqueous solution.

We describe NMR studies at superconducting fields which characterize aspects of the structure and stability of the 1 : 2 actinomycin-d-pG-C complex in solution as monitored at the Watson-Crick base pairs and backbone phosphate groups. Two guanine N1H resonances (12.17 and 11.66 ppm) are observed in the 360 MHz proton NMR spectra of the complex in water at -4 degrees C. These slowly exchangeable resonances, which demonstrate the presence of two Watson-Crick G + C base pairs in the complex, broaden in a sequential manner with increasing temperature. The terminal and internucleotide phosphates of both d-pG-C molecules are observable in the 145.7 MHz 31P spectra of the 1 : 2 actinomycin-d-pG-C complex at 0 degrees C. The internucleotide phosphate resonance at 1.905 ppm broadens prior to that at 2.385 ppm with increasing temperature, consistent with a sequential breakage of the G + C base pairs in the complex. The lifetime of the complex (4.5 +/- 0.5 X 10(-4) s, 33 degrees C) was deduced from the variation of the d-pG-D internucleotide 31P resonance line width on gradual addition of the antibiotic in solution.


A sedimentation velocity method for the separation of complementary strands of DNA.

A method is described for the rapid separation of the complementary strands of homogeneous DNA's. The method takes advantage of a difference in sedimentation coefficients between the complementary strands when these have been dissociated and complexed with poly(U-G). The separation procedure is readily carried out for preparative purposes by zone sedimentation in linear 5--20% (w/v) sucrose gradients containing 2 M NaCl. In analytical band sedimentation experiments, concentrated solutions of CsCl have been used to effect the separation of the complexed complementary strands. Sedimentation coefficients corresponding to corrected values at infinite dilution for the cesium salt of the nucleic acid complexes at 25 degrees C (s 25, w) have been obtained for the complexed complementary strands of coliphage lambdab2b5c DNA and for the uncomplexed single strands of this DNA. The relative values of the parameters affecting s 25, w have been determined. Under these conditions, for which preferential hydration of the sedimenting species is not involved, it was found that the more highly complexed strand has sustained a 24% increase in mass and a 28% decrease in frictional coefficient relative to uncomplexed DNA, while the strand binding the lesser amount of RNA has increased in mass by 11% and changed in frictional coefficient by an insignificant amount relative to the uncomplexed species from that of the uncomplexed DNA do not contribute significantly to the differences in s 25, w between the complexed complementary strands.


Differentiation between chelated and non-chelated DNA-Pt complexes by atomic absorption spectrophotometry.

As a continuation of our study of the properties of the cis-trans platinum series we have investigated the DNA-Pt complexes of [Pt(dien)Cl]Cl, cis-Pt (en)Cl2, cis-Pt (NH3)2Cl2 and K2[PtCl4] by atomic absorption spectrophotometry. The DNA-Pt complexes correspond to the saturation of the N7-(Guanine) sites. It has been found that the chelate complexes obtained with cis-Pt(en)Cl2, cis-Pt(NH3)2Cl1 and K2[PtCl4] show the same absorbance. The [Pt(dien)Cl]Cl and trans-Pt (NH3)2Cl2 which are bound to the N7  (tuanine) site only, show an absorbance greater than the chelate complexes by a factor of two. In addition, it has been possible in the case of the trans-Pt (NH3)CCl1 complex to follow the fixation of platinum to DNA by atomic absorption spectrophotometry. The result is similar to the ionic chlorine liberation procedure reported previously.


Colony-forming ability of ultraviolet-irradiated xeroderma pigmentosum fibroblasts from different DNA repair complementation groups.

Patients with xeroderma pigmentosum develop severe sunlight-induced damage, including malignant neoplasms, on sun-exposed skin. Some patients also have neurological abnormalities. Xeroderma pigmentosum cells are known to have impaired ability to repair ultraviolet light- or chemical mutagen-induced damage to their DNA, and cell-fusion studies have shown five complementation groups among the DNA excision repair-deficient strains. All xeroderma pigmentosum fibroblast strains we tested had lower colony-forming abilities after ultraviolet irradiation than normal strains. Furthermore, we have found that strains from different complementation groups can have different post-ultraviolet colony-forming abilities and that strains from patients with neurological abnormalities are the most sensitive to ultraviolet light. These results suggest that extremely ineffective repair of damaged DNA in central nervous system neurons may be the cause of the neurological abnormalities.