Effects of diet, energy restriction and diabetes on hexose transport in the rat.

1. Effects of dietary composition, energy restriction, and diabetes on hexose absorption were examined by feeding male rats isoenergetic, semi-synthetic diets of differing carbohydrate and protein content. Diets were carbohydrate, (g/kg): 890 sucrose; carbohydrate-protien, 500 sucrose, 390 casein; or protein, 890 casein. An additional group was fed on commercial rat chow ad lib. 2. Hexose (3-O-methyl-D-glucose) absorption was measured by luminal perfusion of the entire small intestine in situ. Absorption by the total small intestine, i.e. absorption per rat, and absorption per g dry weight of mucosa (specific absorption) were calculated. 3. When semi-synthetic diets were fed at 210 kJ/d to normal animals absorption depended on composition of diets: carbohydrate enhanced or protein suppressed hexose absorption. Dietary carbohydrate as glucose, dextrimaltose or starch gave the same hexose absorption response as sucrose. 4. When diets of normal rats were restricted to 118 kJ/d, specific absorption was independent of dietary composition and was increased for all dietary groups to the level of the group fed on the carbohydrate diet at 210 kJ/d. 5. When diabetic rats were given 210 kJ/d, hexose specific absorption was the same for all diabetic groups independent of dietary composition and was equal to that of controls given carbohydrate, but greater than that of protein-fed controls. 6. Thus, when two of the three stimuli (i.e. carbohydrate diet plus energy restriction or diabetes) were combined, the effect was not additive, and the response of hexose specific absorption to diabetes and energy restriction was the same: absorption was independent of dietary composition and was stimulated relative to controls fed on diets containing protein. 7. The pattern of response of total small intestinal hexose absorption to the stimuli of dietary composition, energy restriction and diabetes was similar to that of specific absorption. 8. Compared with groups given semi-synthetic diets, rats eating commercial rat chow ad lib. (approximately 286 kJ/d) showed increased mucosal mass and decreased specific absorption, but total absorption was similar to that of the carbohydrate and carbohydrate-protein-fed groups. 9. In a separate study in control rats, specific and total intestinal absorption of L-leucine did not respond to dietary composition, i.e. level of protein fed.


The effect of wheat bran and pectin on paracetamol absorption in the rat.

1. The absorption of an oral dose of paracetamol was measured in rats given either a basal fibre-free diet, or the basal diet with either pectin or bran added. 2. Urinary excretion of the oral dose was significantly greater during the first 8 h in the pectin-fed rats compared with those on basal diet, though cumulative excretion after 72 h was the same. 3. Free paracetamol levels in the plasma were significantly higher in the pectin-fed rats compared with those on basal diet at 30, 60, 90 and 120 min after the oral dose. 4. The plasma half-life of intravenously-injected paracetamol was shorter in the pectin-fed rats than in those on basal diet alone, but the antipyrine half-lives were not significantly different. 5. Pectin feeding had no effect on either the apparent volume of distribution of paracetamol and antipyrine, or on the rate of gastric empyting. 6. Perfusion of the whole length of the small bowel showed a significantly greater capacity for paracetamol absorption in the pectin-fed rats. 7. Bran had no effect on paracetamol absorption. 8. It was concluded that dietary fibre intake affects drug absorption and that the effect varies with the type of fibre. Unexpectedly pectin accelerates rather than retards absorption of paracetamol, though the mechansim of this effect is unknown.


The effect of dietary fats on the composition of the liver endoplasmic reticulum and oxidative drug metabolsim.

1. The dependence of the rate of oxidative demethylation in the liver endoplasmic reticulum on the fatty acid composition of the endoplasmic reticulum has been studied by varying the lipid content of the diet. 2. The rate of oxidative demethylation was markedly dependent on the percentage of linoleic acid (18:2) incorporated into the membrane. Feeding diets containing (g/kg) 100 coconut oil, 100 lard or 100 maize oil caused respectively the incorporation of 7.6, 10.3 and 25.1% linoleic acid (18:2) and a demethylation rate 3.26, 3.15 and 5.03 nmol formaldehyde/min per mg protein. Feeding 100 g herring oil/kg diet caused incorporation of only 5.1% C18:2 but also 27.2% omega 3 unsaturated fatty acids, including 8.7% eicosapentaenoic acid (20:5) and 17.0% docosahexaenoic acid (22.6) and caused a very high rate of oxidative demethylation (6.53 nmol formaldehyde/min per mg protein). 3. Destruction of the polyunsaturated fatty acids in herring oil by irradiation with 400 krad caused incorporation of a smaller quantity of 3 omega unsaturated acids into the endoplasmic reticulum and decreased the rate of oxidative demethylation (4.83 nmol formaldehyde/min per mg protein). 4. The inductive effects of phenobarbitone on oxidative demethylation were partially dependent on changes in the fatty acid composition of the endoplasmic reticulum. Phenobarbitone (100 mg/kg) increased the percentage of C18:2 from 25.1 to 29.4% in rats given a maize-oil diet, increased the percentage of C20:5 from 8.7 to 10.3% in rats given a herring-oil diet and decreased the percentage of arachidonic acid (20:4) and C22.6 in rats given a lard, maize-oil, herring-oil or irradiated-herring-oil diet. 5. Intraperitoneal alpha-tocopherol (50 mg/kg) increased the percentage of C20:4 from 11.1 to 13.1% in rats given a lard diet and from 5.9 to 7.3% in rats given a herring-oil diet. 6. It is concluded that dietary C18:2 is an important factor in the regulation of the rate of oxidative demethylation in the liver endoplasmic reticulum but this may be replaced effectively by dietary C20:5 omega 3 and C22:6 omega 3 acids. Oxidative demethylation is regulated by changes in the fatty acid composition of the membranes of the liver endoplasmic reticulum.


Trace nutrients. 3 Magnesium, copper, zinc, vitamin B6, vitamin B12 and folic acid in the British household food supply.

1. Intakes of magnesium, copper, zinc, vitamin B6, vitamin B12 and folic acid in Britain were calculated by applying the values selected for the 4th edition of McCance and Widdowson's The Composition of Foods (Paul & Southgate, 1978) to the amounts of food recorded in the National Food Survey made during 1976 (Ministry of Agriculture, Fisheries and Food, 1977). 2. National average intakes were (/person per d): Mg 249 mg, Cu 1.51 mg, Zn 9.1 mg, vitamin B6 1.36 mg, vitamin B12 6.6 micrograms free folic acid 105 microgram and total folic acid 190 micrograms. Corresponding intakes (/person) in families with four or more children were 10-20% lower. 3. A comparison of intakes with those recommended in Canada (Department of National Health and Welfare, 1976) and the USA (National Research Council, 1974) or by WHO (1973) indicated that for folic acid average values were particularly low, and only for vitamin B12 were the recommendations significantly exceeded. 4. Contributions from alcoholic drinks and confectionery were also calculated and found, on average, to be significant for cu, vitamin B12 and folic acid. 5. The losses of B-vitamins which might occur on cooking are discussed.


The effects of a supplement of dietary fibre on faecal excretion by human subjects.

1. Four human subjects on strictly controlled diets were given a fibre supplement, 25 g Ispaghula husk (Isogel)/d, for 3 weeks. 2. Replicate diets and faeces were collected during two 5 d balance periods. The first period served as control for the second which occurred after the supplement had been fed for 2 weeks. 3. Diets and faeces were analysed for total solids, gross energy, total nitrogen, fat, available and unavailable carbohydrates. 4. Three of the four subjects showed a considerable increase in faecal bulk; total faecal weight was more than doubled in two subjects. In two subjects the increase was brought about mainly by extra fibre in the faeces accompanied by a higher proportion of faecal water whereas in one subject it was also an increased excretion of nutrients. One subject showed very little change in faecal bulk. 5. High values for the apparent digestibility of fibre were found during the control period; from 0.70 to 0.80. There was more variation during the experimental period when apparent digestibility of total fibre ranged from 0.47 to 0.82. Values arrived at for the minimum apparent digestibility of the fibre supplement were generally high; one subject appeared to digest Isogel completely. Isogel was degraded preferentially to the food-derived fibre; in particular, cellulose in the faeces was increased during the experimental period. 6. Only one subject showed distinct decreases in the apparent digestibility of energy, N and fat in the diet. The results do not therefore agree with the generally held view that increased fibre in the diet decreases the apparent digestibility of the other nutrients.


Guar gum and reduction of post-prandial glycaemia: effect of incorporation into solid food, liquid food, and both.

1. The influence of the dose and the form in which guar gum was given on the degree of "flattening" of blood glucose curves was studied in five subjects using meals of bread and soup containing 5 or 10 g guar gum. 2. When 5 g guar gum was added to bread the peak increase of blood glucose was reduced by 41% (P less than 0.002), with 5 g guar in soup, the reduction was 54% (P less than 0.001) while a reduction of 68% (P less than 0.001) was seen with 10 g guar gum (5 g in bread and 5 g in soup). The corresponding reduction in insulin peak increases were 37% (P less than 0.002), 50% (P less than 0.001) and 65% (P less than 0.001) respectively. 3. The difference between the two 5 g doses was significant with respect to the reduction of the peak increases in blood glucose and serum insulin; however the difference between the 5 g dose in bread and the 10 g dose was significantly different (P less than 0.02 for glucose, P less than 0.01 for insulin). 4. The results indicate that as little as 5 g guar gum may reduce the glycaemia following a 45 g carbohydrate meal, but perhaps due to earlier and more complete mixing, guar gum is most effective when added to the liquid phase of the meal.


The contribution of protozoa to the protein entering the duodenum of sheep.

1. Four sheep, each fitted with a rumen fistula and a re-entrant cannula at the proximal duodenum were fed a semi-purified diet containing urea as the only nitrogen source. The quantities of total protozoal amino acid-N (TPAN) present in the rumen and entering the duodenum were determined when the mean rumen dilution rate (D) was low (0.034/h) and when D was increased to 0.078/h by the intraruminal infusion of artificial saliva. 2. Increasing the dilution rate had no significant effect upon the proportions of TPAN present in the total microbial amino-acid-N (TMAN) of the rumen fluid and duodenal digesta. With both dilution rates the mean proportion of TPAN in the duodenal TMAN (0.24) was markedly less than the equivalent proportion (0.45) found in the rumen fluid. 3. The daily flow of TPAN, as measured at the duodenal cannula at both dilution rates were equivalent to only 41% of the flow of TPAN as predicted from measurements of rumen outflow, indicating that a substantial proportion of rumen protozoal protein was retained within the rumen.


The influence of vitamin A status on the response of chickens to aflatoxin B1 and changes in liver lipid metabolism associated with aflatoxicosis.

1 A series of experiments were conducted to investigate the effects of dietary retinol status on chickens ingesting aflatoxin B1. The effects of dietary supplementation with biotin and alpha-tocopherol were also examined. 2. Aflatoxin B1 levels greater than 1 mg/kg diet had a detrimental effect on 'liveability', body-weight gain, food intake and food conversion efficiency. When fed for more than 2 weeks aflatoxin increased relative liver weight and liver lipid concentration. These effects were less pronounced with avitaminotic A chickens. 3. A synergistic effect on hydropericardium development was observed between aflatoxin B1 and retinol. This effect was not observed when the dietary level of alpha-tocopherol was increased tenfold. 4. The specific activities of certain hepatic lipogenic and amino acid-metabolizing enzymes were influenced by aflatoxin ingestion. A reduction in lipogenic enzyme activity was observed before a reduction in the activities of amino acid-metabolizing enzymes. 5. Liver fatty acid composition was significantly influenced by aflatoxin B1. The extent of these changes was reduced by the inclusion of additional dietary biotin.


Efficiency of utilization of volatile fatty acids for maintenance and energy retention by sheep.

1. Two experiments were conducted with lambs sustained entirely by intragastric infusion of volatile fatty acids (VFA), protein, minerals and vitamins. 2. In the first experiment to determine the effects of VFA on nitrogen retention four mixtures of VFA (B, C, D and E) were used containing acetic, propionic and butyric acid in the following molar proportions respectively: 45, 45 and 10; 55, 35 and 10; 65, 25 and 10; 75, 15 and 10. The level of infusion was 836 kJ/live weight0.75 per d and the design was a 4 X 4 Latin square with 14 d periods. There were no significant differences in the N balance between the different mixtures of VFA though mixture B tended to give the highest N retention. 3. Thirty-two lambs were used in the second experiment for measurements of heat production in closed-circuit respiration chambers. Six mixtures of VFA were used. These included mixtures B-E from Expt 1 and in addition two mixtures (A and F) containing acetic, propionic and butyric acid in the following molar proportions respectively: 35, 55 and 10; 85, 5 and 10. The heat production was measured both at 450 and 900 kJ/W0.75 per d, except for mixture F, where it was not possible to achieve a rate of infusion in excess of 675 kJ/W0.75 per d. 4. The energy required for maintenance was determined to be 0.45 +/- 0.02 MJ/kg live weight0.75 per d regardless of the mixture used. 5. The efficiency of utilization for fattening (kf) values for the six mixtures were 0.78, 0.64, 0.57, 0.61, 0.61 and 0.59 for mixtures A, B, C, D, E and F respectively. Only mixture A was significantly better utilized than the other mixtures. This mixture also gave the most efficient N utilization. 6. It is concluded from this evidence that differences in kf for diets normally given to ruminants cannot be attributed to differences in utilization of volatile fatty acids.


The sustenance of growing and fattening ruminants by intragastric infusion of volatile fatty acid and protein.

A technique is described for feeding young sheep for long periods entirely by the infusion of volatile fatty acids into the rumen and protein and other essential nutrients into the abomasum. When the energy infused was twice the estimated requirement for maintenance, growth rate and nitrogen retention were essentially the same as in normally-fed sheep.


The effects of pregnancy and lactation on the activities of trypsin and alpha-chymotrypsin in the rat pancreas.

1. The food intake, pancreas weight and trypsin (EC 3.4.21.4) and alpha-chymotrypsin (EC 3.4.21.1) activities in the pancreas were measured in rats during pregnancy and lactation and after the young were weaned. 2. All the quantities measured increased significantly during lactation and had returned to their original values by 4 weeks after weaning. Food intake and pancreas weight were highest after the second week of lactation. Total trypsin and alpha-chymotrypsin activity, and the activity per g tissue, fell during pregnancy and rose during lactation, reaching a maximum 1 week after weaning. 3. From these and other results it is suggested that the hypertrophy and hypersecretion of pregnancy and lactation are initiated by changes insulin secretion and mediated by the trophic effects of gut hormones, and that differences in the nature and timing of the response may be controlled by nutrient availability.


Ulcerative dermatitis in rats with over fifteen generations of protein malnutrition.

1. Male and female rats with histories of up to twenty generations of protein malnutrition were found to be at a higher risk for the development of ulcerative dermatitis than rats maintained on a low-protein diet for one generation or in controls on an adequate-protein intake. 2. In all groups, female rats were more likely to have dermatitis than male rats. 3. Bacteriologic examination was performed in the intergenerationally malnourished and control animals; Staphylococcus aureus was isolated from the skins of animals in both groups, whether or not any lesion was present. In these two groups of animals, experimental inoculation with S. aureus produced dermatitis only in the malnourished animals.


A comparison of the influence of breast-feeding and bottle-feeding on the fatty acid composition of the erythrocytes.

The proportion of long-chain polyunsaturated derivatives of linoleic and linolenic acids in the erythrocyte lipids of infants fed a cow's milk formula was lower than in breast-fed infants. Although th formula provided only 0.55% of the total energy as linoleic acid, essential fatty acid deficiency was not detected; the triene:tetraene ratio was 0.09.


Pyridoxal 5'-phosphate binding site of pig heart alanine aminotransferase.

After borohydride reduction, carboxymethylation, and tryptic digestion of the holoenzyme of pig heart alanine aminotransferase, a single icosapeptide containing the N6-(phosphopyridoxyl)lysine residue was isolated by a combination of gel filtration and ion-exchange chromatogrpahy. Its primary structure was determined as Gln-Glu-Leu-Ala-Ser-Phe-His-Ser-Val-Ser-Lsy(Pxy)-Gly-Phe-Met-Gly-Glu-Cys-Gly-Phe-Arg.


Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino)naphthalene.

Environmentally sensitive fluorescent probes involve two groups, an electron donor and an electron acceptor, attached to an aromatic ring system, and maximal effects may be expected when these groups are as far apart as feasible. The syntheisis, characterization, and spectroscopic properties of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), a compound that fulfills these conditions, are described. The maximum of emission is at 401 nm in cyclohexane solution and at 531 nm in water solution, indicating an increase of dipole moment of approximately 20 D units on excitation to the lowest singlet state. The effect of temperature upon the spectral distribution and the bandwidth of fluorescence of PRODAN in 1:1 complexes with albumin shows the existence of a dynamic relaxation process of the protein surroundings within th 2-4 ns of the fluorescence lifetime.


Heat capacity microcalorimetry of the in vitro reconstitution of calf brain microtubules.

The self-assembly of calf brain tubulin, purified by the modified Weisenberg procedure, was examined in an adiabatic differential heat capacity microcalorimeter. Tubulin solutions at concentrations between 6 and 17 mg/mL were heated from 8 to 40 degrees C at heating rates between 0.1 and 1.0 deg/min in a pH 7.0 phosphate buffer containing 1 X 10(-3) M GTP, 1.6 X 10(-2) M MgCl2, and 3.4 M glycerol. The heat capacity change, deltaCp of the microtubule growth reaction was found to be -1600 +/- 500 cal/(deg mol) per 110 000 molecular weight tubulin dimer incorporated into microtubules, in agreement with the reported van't Hoff deltaCp value of -1500 cal/(deg mol) [Lee, J.C., & Timasheff, S.N. (1977) Biochemistry 16, 1754-1765]. The assembly reaction is characterized by a complex heat uptake pattern comprising both endothermic and exothermic processes.


Measurements of fatty acid synthesis by incorporation of deuterium from deuterated water.

Fatty acid synthesis by perfused livers of rat is measured by using D2O as tracer. The newly synthesized, deuterium-labeled fatty acids are separated from unlabeled fatty acids by gas chromatography using glass capillary columns. The areas of the deuterium-labeled peaks are proportional to the amounts of fatty acids synthesized. The absolute amounts of the individual fatty acids synthesized are obtained by use of an internal standard. The number of deuterium atoms incorporated, as determined by mass spectrometry, is proportional to the D2O concentration of the perfusate, except at very high concentrations of D2O. The relative retention times of the newly synthesized, deuterium-labeled fatty acids are proportional to their deuterium content.


Mechanism of enhancement of polynucleotide binding to cells by mutagens.

The binding of polyuridylate to cells is substantially increased by proflavine. This enhanced binding is saturable with respect to time and to the concentration of both proflavine and polyuridylate. Enhancement is observed only when cells are exposed to both proflavine and polyuridylate together and depends cooperatively on the proflavine concentration. The resulting complex formed between the cell, proflavine, and polyuridylate can be dissociated with salt but not with sucrose solutions. An increase in the binding of polyuridylate to cells similar to that observed with proflavine was also obtained with cationic dyes such as acridine orange, 9-aminoacridine, and Hoechst 33258, while the introduction of a bulky polysaccharide residue, dextran, into the dyes cancels these effects. Similarly, cationic aromatic compounds such as primaquine and quinacrine which carry bulky nonplanar substituents or aliphatic cationic compounds like ethylenediamine do not enhance binding. Proflavine is unable to augment the binding of a basic macromolecule, diethylaminoethylaminoethyldextran, to cells. The model proposed for the enhanced binding of polyuridylate is based on the cooperative formation of stacked complexes of cationic dye located between the cell surface and the bound polyuridylate.


Time-dependent changes in the size distribution of distearoylphosphatidylcholine vesicles.

The results of transmission electron microscopic and ultracentrifugal studies of the size distributions of sonicated distearoylphosphatidylcholine vesicles are reported. Small vesicles (d approximately 300 A) were prepared by sonication of pure 1,2-distearoyl-3-sn-phosphatidylcholine in water and incubated at 4, 21, 40, 53, and 65 degrees C. The vesicle size distributions changed as a function of time at all temperatures below the phase-transition temperature but remained constant at the transition temperature and above. The sizes of structures to which the small vesicles are converted are the same at all temperatures, although the rates of conversion differ. The primary structures formed are identified as larger vesicles. The rate of loss of small vesicles is found to increase with decreasing temperature. At 4 and 21 degrees C small vesicles are converted to amorphous material, possibly irregular fragments of neat phase, in addition to being converted to larger vesicles. Trace amounts of an impurity commonly produced in the synthesis of 1,2-distearoyl-3-sn-phosphatidylcholine, 1,3-distearoyl-2-sn-phosphatidylcholine, are found to dramatically reduce the rate of loss of small vesicles at 21 degrees C.


Folding pathways of immunoglobulin domains. The folding kinetics of the Cgamma3 domain of human IgG1.

The in vitro folding kinetics of a fragment corresponding to an intact dimer of the Cgamma3 domain of human IgG1 (pFc') were monitored via the large changes in tryptophan fluorescence which accompany these processes. In going from the guanidine hydrochloride (Gdn.HCl) induced unfolded state (4.0 M Gdn.HCl) to the native state (0.5 M Gdn.HCl), three well-separated first-order processes were observed having time constants of 5, 50, and 350 s and roughly equal amplitudes. These values were concentration independent, a fact consistent with there being no fluorescence change accompanying dimerization. These time constants are one to two orders of magnitude slower than those observed for proteins of similar size such as ribonuclease or cytochrome c, most probably reflecting the complex processes involved in forming the correct beta-sheet arrangement of immunoglobulin domains. The corresponding unfolding transition is biphasic having time constant values of 50 and 500 s, the latter comprising 80% of the fluorescence change. These data indicate the presence of at least one species with intermediate fluorescence along the unfolding pathway. Gdn.HCl concentration jumps were also performed over various intervals within the transition zone. The results are not consistent with a fully reversible mechanism. In the absence of the intrachain disulfide bond, pFc' exists in an unfolded state even at 0.5 M Gdn.HCl. In a concomitant refolding and reoxidation experiment (at 0.5 M Gdn.HCl and using an optimal disulfide interchange catalytic system), the time constant for disulfide formation was in the range of 80--200 s and the fluorescence change revealed a lag phase analyzable in terms of rate-limiting reoxidation and refolding times consistent with those observed for the initially disulfide bonded species. Under similar conditions but a 4 M Gdn.HCl, reoxidation was more than two orders of magnitude slower, suggesting that reoxidation is directed by a refolding nucleation event.


Contributions by ionic and steric features of ligands to their binding with phosphorylcholine-specific immunoglobulin IgA H-8 as determined by fluorescence spectroscopy.

The murine myeloma IgA H-8 Fab' fragment which exhibits a binding specificity for phosphorylcholine was assayed for its ability to bind with a number of charged ligands. Monitoring of the ligand-induced changes of protein fluorescence provided a fast and accurate method of determining the equilibrium binding constants. The binding data along with fluorescence spectral properties of the protein permitted an assessment of the relative importance of some binding parameters as well as an evaluation of certain ionic and steric contributions made by ligands exhibiting significant binding affinity for the antibody fragment. Among the conclusions reached is that the dielectric of the binding site microenvironment is important in determining the strength of binding and that hydrophobic groups surrounding a quaternary cationic ligand are important in creating an appropriate binding site of low dielectric value.


Organization and heterogeneity of sequences within a repeating unit of human Y chromosome deoxyribonucleic acid.

Fragments of 3.4 kilobases (kb) are released from DNA of human males, but not DNA of human females, by cleavage with restriction endonucleases HaeIII, EcoRI, or EcoRII. Most, if not all, reiterated DNA which is specific for the Y chromosome (it-Y DNA) is present within these male-specific 3.4-kb molecules. Although such 3.4-kb molecules are themselves localized to the Y chromosome, this is not true for all sequences within them. At least two distinguishable types of reiterated sequences are found within each 3.4-kb molecule. One type consists of at least two families which are highly reiterated and are not confined to the Y chromosome. The other type is composed of an estimated minimum of 39 families, each moderately reiterated and localized to the Y chromosome. Y-specific and non-Y-specific sequences are interspersed with one another in the same 3.4-kb molecule. In the average 3.4-kb molecule, three 800 nucleotide lengths of Y-specific sequences alternate with four 250 nucleotide lengths of non-Y-specific sequences. Since the total number of families of Y-specific sequences, calculated on the basis of reiteration frequency and total abundance in a male genome, greatly exceeds the number of Y -specific sequences present in a single 3.4-kb molecule, it necessarily follows that the population of these 3.4-kb molecules is heterogeneous.


Interaction between wheat germ RNA polymerase II and adenovirus 2 DNA. Evidence for two types of stable binary complexes.

Transcription of Adenovirus 2 DNA (Ad 2 DNA) by wheat germ RNA polymerase II in vitro satisfies criteria that have been used to establish that Escherichia coli or coliphage transcription in vitro is initiated at true promoters. (1) Wheat germ RNA polymerase forms highly stable complexes at specific sites on Ad 2 DNA, with a Kassoc of (4--5) X 10(10) M-1. (2) Electron microscopic visualization of enzyme bound to Ad 2 DNA reveals the location of eight strong binding sites, at least five of which appear to correspond to promoters that have been identified in studies of Ad 2 transcription in vivo [Evans, R. M., Fraser, N., Ziff, E., Weber, J., Wilson, M., & Darnell, J.E. (1977) Cell 12, 733--739; Berk, A.J., & Sharp, P.A. (1977) Cell 12, 45--55; Weinmann, R., & Aiello, L. O. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1662--1666]. (3) Transcription of Ad 2 DNA from preformed complexes with wheat germ polymerase is capable of escaping the action of rifamycin AF/013 and is relatively resistant to polyriboinosinic acid. In addition, our results are consistent with a two-state model for the interaction of wheat germ RNA polymerase with Ad 2 DNA, indicating that the mechansisms of transcription initiation and promoter-site selection in eucaryotes may be very similar to mechanisms elucidated in procaryotic systems.


Amino acid sequence and sites of phosphorylation in a highly acidic region of nucleolar nonhistone protein C23.

Novikoff hepatoma nucleolar nonhistone proteins, C23 and B23, contain highly acidic phosphorylated regions (Mamrack, M. D., et al. (1977) Biochem. Biophys. Res. Commun. 76, 150--157). Tryptic peptides from protein C23 containing these regions were purified by DEAE-Sephadex columns and paper electrophoresis at pH 1.8. One of these, peptide C23-Ca, was sequenced by combined automated and conventional methods. The proposed amino acid sequence is shown in eq 1. This peptide was found in three 32P-labeled forms with phosphoryl groups at positions 8 and 25, and probably 28. The highly acidic sequences adjacent to the phosphorylation sites represent a unique class of phosphorylation sites different from those in histones or substrates for cytoplasmic cAMP-dependent kinases. Ala-Ala-Pro-Ala-A5la-Pro-Ala-Ser-Glu-A10sp-Glu-Asp-Glu-Glu-A15sp-Asp-Asp-Asp-Glu-A20sp-Asp-Asp-Asp-Asp-S25er-Gln-Glu-Ser-Glu-G30lu-Glu-Asp-Glu-Glu-V35al-Met-Glu-Ile-Thr-P40ro-Ala-Lys (1).


Subunit structure and peptide mapping of junctional and extrajunctional acetylcholine receptors from rat muscle.

We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated.


Steady-state kinetics of mouse DNA polymerase beta.

DNA polymerase beta from mouse myeloma has been purified to near homogeneity, and its properties have been examined. The enzyme did not catalyze a detectable level of dNTP turnover, pyrophosphate exchange, pyrophosphorolysis, 3'-exonuclease degradation, or 5'-exonuclease degradation. Steady-state kinetic studies point to an ordered bibi mechanism for the polymerization reaction. Metal activation, which is required for polymerization, did not alter the Km for either the dNTP or the template--primer.


Cytochrome function in the cyclic electron transport pathway of chloroplasts.

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the alpha-bands of cytochrome b6 and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b6 and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 microM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH4Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift with formation half-time of about 2 ms. Ths inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isoprophy-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b6 and f by factors of ten or more. These observations indicate that cytochrome b6, plastoquinone and cytochrome f participated in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 mumol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO2 fixation.


Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria.

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained. It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.


The kinetic relationship between the C-550 absorbance change, the reduction of Q(delta A320) and the variable fluorescence yield change in chloroplasts at room temperature.

The light minus dark difference spectrum and the kinetics of the indicator pigment C-550 have been measured at room temperature in isolate, envelope-free chloroplasts in the presence of 3-(3' ,4'-dichlorophenyl)-1,1-dimethylurea (DCMU). The C-550 spectrum indicates a band shift with peaks at 540 and 550 nm and has an isobestic point at 545 nm. On the assumption of 400 chlorophyll molecules per electron transfer chain the differentaial extinction coefficient delta epsilon (540-550) is calculated to be approximately 5 mM-1 . CM-1. The kinetics of the C-550 absorbance change, occurring upin the onset of continuous illumination, are shown to be biphasic and strictly correlated with the kinetics of the complementary area measured from the fluorescence induction curve under identical cinditions and with those of the absorbance increase at 320 nm due to photoreduction of Q. The lighted-induced change in these three parameters can be described as a function of the variable fluorescence yield change occurring under the same conditions. Such functions are non-linear and reveal a heterogeneous dependence of the variable fluorescence yield on the fraction of closed System II reaction centers. It is concluded that for every molecule of the primary electron acceptor Q of Photosystem II that is photochemically reduced there corresponds an equivalent change in the absorbance of the indicator pigment C-550 and in the size of the complementary area. Ths, C-550 and area are two valid parameters for monitoring the primary photochemical activity of System II at the room temperature.


The terminal oxidase of Photobacterium phosphoreum. A novel cytochrome.

The terminal oxidase of Photobacterium phosphoreum has been purified to the electrophoretically homogeneous state and some of its properties have been studied. The enzyme catalyses oxidation of ascorbate in the presence of phenazine methosulphate or N,N,N',N'-tetramethyl-p-phenylenediamine. The reaction is inhibited by cyanide. Nitrite at comparatively high concentrations inhibits the enzyme, but the enzyme does not catalyse nitrite reduction with ascorbate plus the electron mediator as the electron donor. The enzyme shows the absorption peaks at 632, 565, 534 and 436 nm in the reduced form. It has two kinds of haems: protohaem and haem d. Namely, the enzyme is a 'cytochrome bd'-type oxidase; a novel cytochrome.


Effects of dibromothymoquinone on mung bean mitochondrial electron transfer and membrane fluidity.

The effects of the quinone analog dibromothymoquinone on electron transfer in isolated mung bean mitochondria are described. Both the main, cyanide-sensitive and the alternate, cyanide-insensitive pathways are inhibited by dibromothymoquinone but in markedly different fashions. Half-maximal inhibition appeared at 40 microM and 20 microM dibromothymoquinone for the cyanide-sensitive and alternate pathways, respectively. With succinate as the electron donor, dibromothymoquinone inhibited the alternate pathway at a single site; showing a mixed, non-competitive type inhibition. On the succinate, cyanide-sensitive pathway dibromothymoquinone showed two sites of inhibition and neither coincides with the site of inhibition associated with the alternate pathway. With malate as the electron donor, two sites of inhibition by dibromothymoquinone were observed regardless of the pathway measured. Dibromothymoquinone also inhibited the rate of valinomycin-induced swelling of isolated mung bean mitochondria. Steady-state kinetics showed the inhibition to be non-competitive with respect to valinomycin. Additionally dibromothymoquinone was observed to increase the fluorescence polarization associated with the hydrophobic probe 1,6-diphenylhexatriene. The results indicated that dibromothymoquinone decreased the fluidity of the inner mitochondrial membrane and suggested that the inhibition of mitochondrial electron transfer by dibromothymoquinone may be associated with this decrease in membrane fluidity. The relationship of the multisite nature of the inhibition of electron transfer by dibromothymoquinone and the possible role of mobile electron carriers such as ubiquinone on the main and alternate respiratory pathways of higher plants is discussed.


Photosystem II energy coupling in chloroplasts with H2O2 as electron donor.

NH2OH-treated, non-water-splitting chloroplasts can oxidize H2O2 to O2 through Photosystem II at substantial rates (100--250 muequiv . h-1 . mg-1 chlorophyll with 5 mM H2O2) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H2O2 leads to Photosystem II leads to dimethylquinone reaction supports phosphorylation with a P/e2 ratio of 0.25--0.35 and proton uptake with H+/e values of 0.67 (pH 8)--0.85 (pH 6). These are close to the P/e2 value of 0.3--0.38 and the H+/e values of 0.7--0.93 found in parallel experiments for the H2O leads to Photosystem II leads to dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor leads to Photosystem II leads to dibromothymoquinone (leads to O2) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I-, ferrocyanide).


Dependence of mammalian DNA replication on DNA supercoiling. I. Effects of ethidium bromide on DNA synthesis in permeable Chinese hamster ovary cells.

Chinese hamster ovary cells labelled with [14C]thymidine were made permeable, incubated with various concentrations of the intercalating dye ethidium bromide, and centrifuged through neutral sucrose gradients. The gradient profiles of these cells were qualitatively similar to those obtained by centrifuging DNA from untreated, lysed permeable cells through gradients containing ethidium bromide. The sedimentation distance of DNA had a biphasic dependence on the concentration of ethidium bromide, suggesting that the dye altered the amount of DNA supercoiling in situ. The effect of ethidium bromide intercalation on incorporation of [3H]dTMP into acid-precipitable material in an in vitro DNA synthesis mixture was measured. The incorporation of [3H]dTMP was unaffected by less than 1 microgram/ml of ethidium bromide, enhanced up to two-fold by 1--10 microgram/ml, and inhibited by concentrations greater than 10 micrograms/ml. Alkaline sucrose gradient analysis revealed a higher percentage of small DNA fragments (6--20 S) in the cells treated with 2 micrograms/ml ethidium bromide than in control cells. These fragments attained parental size within the same time as the fragments in control cells. In cells treated with 2 micrograms/ml ethidium bromide, a significant fraction of newly synthesized DNA resulted from new starts, whereas in untreated cells practically none of the newly synthesized DNA resulted from new starts. These results suggest that relaxation of DNA supercoiled structures ahead of the replication fork generates spurious initiations of DNA synthesis and that in intact cells the rate of chain elongation is limited by supercoiled regions ahead of the growing point.


Dependence of mammalian DNA replication on DNA supercoiling. II. Effects of novobiocin on DNA synthesis in Chinese hamster ovary cells.

Novobiocin, an inhibitor of gyrase-induced DNA supercoiling and DNA replication in prokaryotes, inhibited the incorporation of DNA precursors into DNA in both intact and permeable Chinese hamster ovary cells; much higher concentrations were required for permeable cells, in which no new replicons were initiated. Nucleoids were prepared from cells that were incubated for 60 min with 200 micrograms/ml novobiocin, made permeable, and incubated with 0--50 micrograms/ml ethidium bromide. Sedimentation of the nucleoids in neutral sucrose gradients suggested that the number of supercoils in the average nucleoid had been reduced by prior incubation with novobiocin. In intact cells, novobiocin is required inside the cell for continued inhibition of DNA synthesis, suggesting that it does not act directly on the DNA. Alkaline sucrose gradient profiles of DNA synthesized in the presence of novobiocin in intact cells indicated that the drug inhibited replicon initiation while having little if any effect on chain elongation. These data are consistent with the idea that an activity similar to the bacterial gyrase generates supercoils in mammalian DNA and produces the proper conformation for the initiation of DNA replication.


A rapid method for the purification of supercoiled PM2 DNA by affinity chromatography on H1 histone covalently coupled to agarose.

A simple and rapid method is described for the purification of supercoiled PM2 DNA by affinity chromatography on columns of H1 histone covalently coupled to agarose. The method does not require the use of intercalating agents or ultracentrifugation procedures. Under the conditions most appropriate for purification, elution is carried out in a single step with buffered 0.7 M NaCl after the sample has been loaded onto the column in buffered 0.2 M NaCl. The DNA eluted at the higher salt concentration consists of supercoiled closed circular DNA at greater than 90% purity independently of the ratio of supercoiled to nicked circular DNA in the input mixture.


DNA-binding antibiotics. X-ray structure of the distamycin A analog.

The crystal structure of the antibiotic distamycin A analog containing two pyrrol carboxamide fragments has been determined. The space group of the crystals is P21/b; the unit-cell dimensions are a = 11.169, b = 21.535, c = 7.863 A, alpha = beta = 90 degrees, gamma = 122.45 degrees, Z = 4. The structure is solved by direct methods, and refined with the full-matrix least-squares procedure. The data on the structure of the pyrrol carboxamide backbone allow the following conclusions to be made about the molecular structure of the distamycin type antibiotics: (i) the amide groups have normal trans-configuration with slightly shortened C-C and C-N bonds adjacent to the pyrrol rings, (ii) the N-methyl groups of the pyrrol rings and the oxygen atoms of the amide groups have the same orientation with respect to the backbone. In the distamycin A analog molecule the pyrrol rings and amide group between them are approximately coplanar.


Actions of human DNA glycosylases on uracil-containing DNA, methylated DNA and their reconstituted chromatins.

Extracts of human lymphoblastoid cells catalyzed complete release of uracil (Ura) from PBS1 DNA, which contains Ura instead of thymine as a normal component (Ura-DNA), and 3-methyladenine (3-MeAde) from DNA methylated with methyl methanesulfonate (Me-DNA). These two activities, Ura-DNA glycosylase and 3-MeAde-DNA glycosylase, differed in heat stability. Cell extracts released Ura more rapidly and 3-MeAde more slowly from alkali-denatured preparations of Ura- and Me-DNA, respectively, than from native DNA's. On incubation with reconstituted chromatins, prepared from Ura-DNA and Me-DNA, respectively, with calf thymus chromosomal protein by salt gradient dialysis, cell extracts released all the Ura but only about half of the 3-MeAde residues, although both these chromatins were degraded by micrococcal nuclease until about half of the nucleotides became acid soluble. The activities of Ura-DNA and 3-MeAde-DNA glycosylase of xeroderma pigmentosum cells were similar to those of normal cells.


Inhibition of DNA repair in ultraviolet-irradiated human cells by hydroxyurea.

The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair:the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonuclease-sensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65--70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well.


Rate-limiting factors for lymphocyte protein synthesis. Ribosome commitment and the capacity of lymphocyte cell-free systems to translate exogenous mRNAs.

The properties of the protein synthesising systems of different lymphocyte preparations have been compared with those of non-lymphoid tissues. Polysome profiles from rat thymocytes, sheep mesenteric lymphocytes, rat liver and mouse ascites tumours showed that the commitment of ribosomes to protein synthesis in lymphocytes was relatively low. Initiation factor activities, assessed on the abilities of post-mitochondrial fractions to support exogenous mRNA translation, were limited or undetectable in lymphoid tissues. While the thymocyte system translated globin mRNA, the response was enhanced by ascites extracts rich in initiation factors. The mesenteric lymphocyte system responded only marginally to globin mRNA and poly(U) but the responses were not enhanced by ascites extracts. The activity of isolated  mesenteric ribosomes was comparable with ribosomes from other tissues, indicating that extraribosomal factors were responsible for the poor overall activity of the mesenteric system. Finally, the effects of cycloheximide on the recruitment of polysomes in lymphocytes indicated that the commitment of ribosomes to protein synthesis might be restricted by both limited mRNA availability and limited capacity for initiation of mRNA translation.


Isolation of ribonuclease-free intact polyribosomes from rat kidney.

High levels of RNAase present in rat kidney have prevented isolation of intact polyribosomes from this tissue. This problem has been circumvented by a thorough in situ arterial perfusion of rat kidney, coupled with homogenization of the perfused rat kidney in heparin and detergents-fortified high-speed supernatant prepared from rat liver. This procedure reduced RNAase activity in the homogenate by as much as 70%. Sedimentation of the polyribosomes from this homogenate through a layer of 2.0 M sucrose resulted in a 78--80% yield of polyribosomes from the rat kidney. The resulting polyribosomal pellet contained less than 8% of the RNAase activity present in polyribosomes from non-perfused rat kidney. The remaining RNAase activity was separated from the larger polyribomes by sucrose density gradient centrifugation. The majority of the polyribosomes were larger than tetramers. This procedure also incrased both the yield and size of polyribosomes from rat and mouse liver.


Peptidyl-puromycin synthesis by free and membrane-bound ribosomes.

The peptidyl transferase reaction, as measured by the formation of peptidyl-puromycin, was compared for free ribosomes and ribosomes bound to two types of membrane, the endoplasmic reticulum and the outer nuclear membrane. In most respects the reaction catalyzed by the three types of ribosome was similar, demonstrating that interaction of the 60 S ribosomal subunit with the membrane has little effect on the functioning of peptidyl transferase, a 60 S protein. However, both the rate and extent of synthesis of peptidyl puromycin were lower for ribosomes bound to the nuclear membrane than for free or microsome-bound ribosomes. This difference appears to be a direct consequence of the ribosome-membrane interaction, since ribosomes stripped from the nuclear membrane could not be distinguished from the other classes of ribosome.


Factors affecting the rate of purine ribonucleotide dephosphorylation in human erythrocytes.

Purine ribonucleotide dephosphorylation was measured in intact human erythrocytes in vitro to evaluate those factors which might regulate this process in vivo. It was found that purine nucleotides which exist predominantly in the triphosphate form (e.g. ATP and GTP) are protected from dephosphorylation while those nucleotides normally present as the monophosphate (e.g. IMP) are susceptible to dephosphorylation. This point was emphasised by studying an individual whose erythrocytes accumulated ITP rather than IMP; erythrocytes from this individual has a more stable pool of inosine phosphates than did erythrocytes from normal individuals. The concentration of intracellular phosphate was also shown to affect the rate of dephosphorylation. The dephosphorylation of IMP was inhibited at intracellular phosphate concentrations above approx. 3 mM. AMP dephosphorylation (in cells whose AMP concentration was increased by incubating them in the presence of 2-deoxyglucose) was inhibited by phosphate more strongly than was found for IMP. In contrast, the dephosphorylation of GMP did not appear to be affected by phosphate concentration. High oxygen tension was a powerful stimulator of IMP dephosphorylation while low oxygen tension protected IMP from dephosphorylation. This finding shows that human erythrocytes are similar to those of other mammals in this regard and points to a possible physiological determinant of purine turnover in these cells.


The cross-resistance of mouse blasticidin S-resistant cell lines to puromycin and sparsomycin, inhibitors of ribosome function.

The antibiotic blasticidin S inhibits peptide-chain elongation in extracts of bacteria and mammalian cells. After spontaneous or nitrosoguanidine mutagenesis, we have isolated 46 blasticidin S-resistant (Blar) cell lines independently from mouse mammary carcinoma cells (FM3a). Among those Blar clones, we studied two clones, a spontaneously induced one (S501) and a nitrosoguanidine mutagenized one (N742) in more detail. The resistant phenotype of these Blar cells is retained without change for at least four months in the absence of the antibiotic. These Blar cells are 10- to 20-fold more resistant to the cytotoxic action of the antibiotic than their parental cells in vivo. Polyuridylate dependent polyphenylalanine synthesis in vitro with S-30 extracts either from N742 or S501 is 10- to 50-fold more resistant to the inhibitory action of blasticidin S compared to the parental FM3a cells. Ribosomes from FM3a and N742 are fractionated into 40-S and 60-S subunits, and polyphenylalanine synthesis by mixing them in various combinations with S-100 fraction from mouse leukemia L5178Y cells indicating that the resistant phenotype of Blar cells is due to the alteration of 60 S ribosomal subunit. We also found that these two Blar cell lines (N742 and S501) show cross-resistance to gougerotin, puromycin and sparsomycin, but not to emetine or cycloheximide. The polyribosomal pattern of FM3a (Blas) and N742 was compared when the cells were incubated with 3 microgram/ml puromycin for 6 h. Puromycin treatment of Blas cells induced accumulation of monosomes and ribosomal subunits, while little if any transition of polyribosomes into monosome and ribosomal subunits appeared in its counterpart N742 treated with the same dose of puromycin.


Evidence for a single species of DNA ligase localized in nuclei of rat liver.

DNA ligase activity was distributed mainly in the extract of nuclei rapidly isolated from rat liver and about 20% of the nuclear activity in the cytoplasmic fraction. The DNA ligases of nuclear extract and cytoplasmic fraction showed similar gel-filtration patterns and the same sedimentation coefficient of 5.5 S. The appearance of 4-S enzyme from nuclei or nuclear extract was partly inhibited by phenylmethylsulfonyl fluoride. These results suggest that the major activity of DNA ligase in rat liver is due to a single species of the enzyme localized in nuclei and that 4-S nuclear DNA ligase reported previously is an artifact arising from 5.5-S enzyme during the isolation procedure.


The crystal and molecular structure of 9-alpha-D-arabinofuranosyladenine.

The glycosyl torsional angles in two crystallographically-independent molecules of alpha-araA are -73 and -64 degrees, both of which are in the "anti" region. The sugar conformations are C(3')-endo and C(2')-exo-C(3')-endo.


Nucleoside phosphotransferase from yellow lupin seedling cotyledons.

Nucleoside phosphotransferase (nucleotide:3'-deoxynucleoside 5'-phosphotransferase, EC 2.7.1.77) from yellow lupin seedling cotyledons was purified and the active enzyme consists of a single polypeptide chain, Mr = 72 000 +/- 3000. In transphosphorylation, purine and pyrimidine nucleosides are good phosphate acceptors and 5'-nucleotides are effective phosphate donors. Among 2'- and 3'-nucleotides, only 3'-AMP and 3'-psi MP acted as phosphate donors, and p-nitrophenylphosphate appeared less active in this regard. The purine and pyrimidine bases inhibit transphosphorylation. The Km values determined for the inosine:5'-AMP pair were 400 micrometers for both the compounds. The enzyme showed optimum activity at pH 8.0 in mM Tris-HCl buffer. Antisulfhydryl reagents and EDTA did not affect enzyme activity.


Aliphatic ketones are acetylcholinesterase inhibitors but not transition state analogs.

A number of C4--C9 aliphatic ketones are acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitors, with Ki values in the 0.7--5 mM range. Comparison to analogous substrates would suggest that these ketones are transition state analogs; e.g. 2-pentanone binds to the enzyme approx. 550 times more tightly than ethylacetate. However, a number of other criteria contradict this conclusions: (1) the binding is insensitive to ketone structure: isomeric ketones, cycloalkanones, and sterically hindered ketones have similar inhibitory potencies. (2) Analogous alcohols are also good inhibitors even though they cannot form hemiketals with the enzyme. (3) Representative ketones are relatively ineffective at blocking inactivation of the enzyme by methylsulfonyl fluoride, indicating that ketones do not bind principally at the hydrolytic site. (4) A competition experiment shows that binding of tetramethylammonium chloride excludes binding of 2-pentanone, suggesting that ketones bind to the anionic rather than the hydrolytic site. Thus, observation of tight binding relative to a substrate is not a sufficient criterion to establish that an inhibitor is a transition state analog.


The susceptibility of urokinase to affinity labeling by peptides of arginine chloromethyl ketone.

Pro-Gly-ArgCH2Cl, a reagent corresponding to the C-terminal sequence generated in plasminogen on activation by urokinase (EC 3.4.99.26) and probably by other plasminogen activators, was prepared. Pro-Gly-ArgCH2Cl was effective in the inactivation of urokinase at the 10(-6) M level (Ki 68 micrometers and k2 0.47 min-1). In contrast, only a slow inactivation was obtained by 10(-2) M N-tosyllysine chloromethyl ketone. Glu-Gly-ArgCH2Cl, N,N-dimethylaminonaphthalene-5-sulfonyl-Glu-Gly-ArgCH2Cl, and Ac-Gly-Gly-ArgCH2Cl were more reactive than Pro-Gly-ArgCH2Cl against urokinase by factors of 25, 6, and 3, respectively. The effectiveness of arginine chloromethyl ketones as affinity labels is highly dependent on binding in the S2 and S3 sites, thus sequence variations in the reagents exhibited differences in reactivity of up to four orders of magnitude. The most effective reagents had Gly in P2. Ac-Gly-Gly-ArgCH2Cl inactivates urokinase 50 times more rapidly than it does plasmin, thus providing a means of distinguishing the activity of plasmin from its activating protease whereas urokinase is almost inert to Ala-Phe-LysCH2Cl, a reagent which inactivates plasmin at the 10(-7) M level.


Chemical modification of histidyl and lysyl residues in yeast enolase.

Modification of yeast enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11) by diethyl pyrocarbonate at either pH 6.1 or 6.6 caused a biphasic inactivation of the enzyme. In the presence of excess Mg2+, either an equilibrium mixture of substrates or 3-phosphoglycerate, a competitive inhibitor, prevented the second slower phase of inactivation, but had no effect on the first rapid phase. Complete inactivation by diethyl pyrocarbonate correlates with the modification of six histidyl residues/subunit, while 3-phosphoglycerate protects two histidyl residues/subunit from modification. Modification of enolase by two lysine-specific reagents, 2,4,6-trinitrobenzenesulfonate and pyridoxal 5'-phosphate, at pH 8.3 caused a slow loss of enzyme activity. However, substrates did not significantly protect against inactivation by either reagent, and inactivation with 2,4,6-trinitrobenzenesulfonate correlates with the modification of 18 lysyl residues/enzyme subunit.


Associative properties of butyryl-coenzyme A synthetase from ox liver mitochondria.

Butyryl-coenzyme A synthetase (butyrate:CoA ligase (AMP-forming), EC 6.2.1.2) from an acetone-dried powder of ox liver mitochondria was found to have a molecular weight of approx. 40 000. The sedimentation equilibrium analysis suggested the presence in solution of higher molecular weight forms of the enzyme and these could also be obtained by extracting the enzyme from the mitochondrial powder in non-reducing conditions. The enzyme was inhibited by sulphydryl reagents, and was found to have at least one available thiol group/molecule. The relationship between enzymic activity and concentration was non-linear, and suggested that an inactive monomer-active dimer equilibrium was present. The 5--6-fold activation by bovine serum albumin required the presence of free thiol groups in the albumin and involved association of albumin with the enzyme.


Subcellular localisation of di- and tripeptidases in guinea pig and rat enterocytes.

Enterocytes were isolated from rat and guinea pig jejunum and subcellular fractions were prepared by density gradient centrifugation. Gradient fractions were assayed for principal organelle marker enzymes and for di- and tripeptidases. The hydrolases showed a dual localisation with both brush border and cytosol components. In the rat, approximately equal portions of dipeptidase activities were found in the two fractions but, in the guinea pig, three times more activity were found in the two fractions but, in the guinea pig, three times more activity was found in the soluble than in the brush border fractions. Cytosol components in the rat were markedly inhibited by p-hydroxymercuribenzoate. In both species tripeptidase, leucyl-2-naphthylamidases and gamma-glutamyltransferase activities were found predominantly in the brush border fractions.


Studies on the formation of N6-hydroxylysine in cell-free extracts of Aerobacter aerogenes 62-1.

We have investigated conditions optimal for the conversion of L-lysine to its N6-hydroxy derivative by partially purified cell-free extracts of Aerobacter aerogenes 62-1. The enzyme system was highly specific to L-lysine: the D-isomer and, the N2- or N6-derivatives of lysine, and alpha-amino acids were not hydroxylated. Most of the latter compounds had little effect onthe hydroxylation of L-lysine. However, -l-glutamic acid and L-glutamine enhanced the hydroxylation, with half-maximal activation achieved at 100 micrometers concentration of the effector. The Km values for pyruvate and L-(+)-lactate (compounds known to stimulate N-hydroxylysine formation) were found to be approx. 100 micrometers. The data show that N-hydroxylation of the amino acid precedes acylation in the biosynthesis of hydroxamic acid in A. aerogenes 62-1.


The stimulation of erucate metabolism in isolated rat hepatocytes by rapeseed oil and hydrogenated marine oil-containing diets.

1. The metabolism of palmitate and especially of erucate was studied in hepatocytes isolated from rats fed for 3 weeks a diet containing peanut oil (diet, 1), rapeseed oil (diet 2) and partially hydrogenated marine oil (diet 3). 2. The metabolism of palmitate was not significantly influenced by the diet. The rapeseed oil diet caused 1.4 fold and 1.3 fold increase and marine oil diet 3 fold and 2.2 fold increase in the oxidation and chain-shortening respectively of [14-14C]erucic acid in isolated hepatocytes. 3. Cyanide and antimycin A did not inhibit the chain-shortening of erucate in liver cells of rats fed rapeseed oil and peanut oil. The high capacity of the chain-shortening system in hepatocytes of marine oil-fed rats was partially inhibited. 4. Inhibition of the transfer of fatty acids into the mitochondria by lowering the intracellular carnitine concentration and/or by addition of (+)-decanoyl-carnitine resulted in a very pronounced apparent stimulation of the chain-shortening of erucic acid. It is suggested that the chain-shortening system may be virtually independent of the mitochondria, unless the availability of the extramitochondria NAD+ and/or NADP+ is rate-limiting under conditions of extremely low redox potential of the mitochondria. 5. Feeding marine oil or rapeseed oil to the rats induced a 30% increase in catalase activity, a 25--30% increase in urate oxidase activity and a 50% increase in the total CoA in the liver compared to rats fed peanut oil. 6. It is suggested that the increased metabolism of erucate in hepatocytes of marine oil and rapeseed oil-fed rats may be due to the increase in ther peroxisomal beta-oxidation.


Formation of cholic acid via 3 alpha, 7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid in the dog.

The proposed cholic precursor, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-[3H]cholestan-26-oic acid, and [14C]cholesterol were infused intravenously at a constant rate into two dogs for 25 days. If the specific activities of trihydroxy[3H]cholestanoic acid and [3H]cholic acid will be equal after an isotopic steady-state is achieved. The specific activities of [14C]deoxycholic acid (formed from [14C]cholic acid) isolated in the stool of these two dogs were equal the last four days of the infusion indicating that labeled deoxycholic acid (and presumably labeled cholic acid) was in an isotopic steady-state. However, the specific activities of trihydroxy[3H]cholestanoic acid were 3.3 and 5.7 times greater than the specific activities of [3H]cholic acid, respectively. These data suggest that either an alternate route of cholic acid synthesis exists exclusive of trihydroxycholestanoic acid or that an isotopic steady state of trihydroxycholestanoic acid cannot be reached during an infusion of labeled trihydroxycholestanoic acid.


Selective measurement of triacylglycerol lipase activities in pig post-heparin plasma.

Immunochemical methods for the selective measurement of pig post-heparin plasma lipoprotein lipase and hepatic lipase are described and validated. A simple two step purification method for porcine hepatic lipase from hepatic perfusate based on affinity chromatography and gel filtration is reported. The activity of the post-heparin plasma lipoprotein lipase and hepatic lipase in swine is reported. It is demonstrated that fasting decreases the activity of post-heparin plasma lipoprotein lipase activity more than two-fold while it does not affect the hepatic lipase activity significantly.


Lipid biosynthesis in the marine flatworm Convoluta roscoffensis and its algal symbiont Platymonas convoluta.

As a part of an investigations on the lipid metabolism in Platyhelminthes, the acoel Convoluta roscoffensis, which harbors the green alga Platymonas convoluta as a symbiont, was studied. Isotopic tracer experiments established that the acoel lacks the ability to synthesize de novo long-chain saturated and unsaturated fatty acids and depends on its algal symbiont for these compounds. The acoel's fatty acid composition closely resembles that of the alga but differs from those of other animals; the acoel's polyunsaturated fatty acids are of the plant type (omega 3 family) rather than of the animal type (omega 6 family). The acoel also lacks the ability to synthesize sterols de novo. It contains 24-methylenecholesterol synthesized by the algae and, in addition, significant amounts of cholesterol, which is probably a host modification product of the algal sterol. With fatty acids provided by the symbiont, the acoel has the ability to synthesize its own complex lipids. The acoel contains relatively large amounts of triglyceride, phosphatidylcholine, and ethanolamine plasmalogen. These compounds are either not present at all or present only in minute amounts in the symbiotic alga. Since acoels belong to the most primitive forms of the present-day flatworms, the observed metabolic defects in this organism suggest that mechanisms for the biosynthesis of fatty acids and sterols were lost early during the evolution of the Platyhelminthes, and that this phenomenon is widespread within the phylum.


Effect of sphingosine and other amphiphilic amines on the biosynthesis of phosphatidylethanolamine and other glycerolipids in isolated rat hepatocytes.

The importance of ethanolamine and sphingosine as precursors of phosphoethanolamine was investigated by incubating them with [3H]glycerol and isolated rat hepatocytes. Sphingosine (0.1--0.5 mM) stimulated the synthesis of phosphatidylethanolamine from [3H]glycerol, but the stimulation by ethanolamine was more pronounced. Furthermore, more phosphoethanolamine accumulated in the heptatocytes after incubation with ethanolamine than after incubation with sphingosine. It is concluded that ethanolamine is the most important phosphoethanolamine precursor in rat liver. Higher concentrations of sphingosine caused accumulation of [3H]phosphatidate and inhibition of total glycerolipid synthesis in isolated hepatocytes, when incubated in the presence of [3H]glycerol. These effects were very similar to those of fenfluramine and norfenfluramine described previously. Simpler cationic amphiphilic amines, like oleoylamine and octadecyltrimethylammonium bromide, also caused these effects. Variation of alkyl chain length and amphiphile charge showed that both a positive charge and a certain alkyl chain length were necessary for interference with phosphatidate metabolism. A much wider range of compounds inhibited total glycerolipid synthesis from [3H]glycerol.


Mixed micelles of sphingomyelin and phosphatidylcholine with nonionic surfactants. Effect of temperature and surfactant polydispersity.

Mixed micelle formation of the polydisperse nonionic surfactant Triton X-100 as well as its homogeneous analogue, p-(1,1,3,3-tetramethylbutyl)-phenoxynonaoxyethylene glycol (OPE-9), with bovine brain sphingomyelin or dipalmitoyl phosphatidylcholine has been characterized by column chromatography on 6% agarose. At 40 degrees C, mixtures of OPE-9 and either sphingomyelin or dipalmitoyl phosphatidylcholine give a narrow size distribution for mixed micelles. A this temperature the size distribution of Triton X-100-containing mixed micelles is complicated because of the polydispersity of the oxyethylene chains. At 20 degrees C narrow size distributions are observed for mixed micelles of sphingomyelin/Triton X-100 and sphingomyelin/OPE-9 up to at least 0.06 mol fraction of lipid. For dipalmitoyl phosphatidylcholine this is observed only with OPE-9. At intermediate mol fractions of lipid (around 0.25), two populations of mixed micelles exist for sphingomyelin/Trition X-100, sphingomyelin/OPE-9, and dipalmitoyl phosphatidylcholine/OPE-9. At high mol fractions of lipid only one population of mixed micelles again exists. At 20 degrees C, sphingoymelin forms a clear solution with Triton X-100 and OPE-9 to a lipid mol fraction of at least 0.46 and 0.67, respectively. Dipalmitoyl phosphatidylcholine forms a clear solution with OPE-9 to a lipid mol fraction of at least 0.57 at the same temperature. Triton X-100 and dipalmitoyl phosphatidylcholine do not form stable, clear solutions at 20 degrees C unless the lipid mol fraction is extremely low. These results show that surfactant polydispersity and temperature are important determinants in the solubilization of lipids by nonionic surfactants. It is also shown that pure surfactant micelles and lipid/surfactant mixed micelles do not co-exist in the same solution.


Activation of lipoprotein lipase by native and acylated peptides of apolipoprotein C-II.

Apolipoprotein C-II, a protein found associated with all major classes of plasma lipoproteins, is a potent activator of the enzyme lipoprotein lipase. We have prepared the maleyl, citraconyl and succinyl derivatives of apolipoprotein C-II, and compared the capacities of the intact and tryptically cleaved proteins to activate lipoprotein lipase. The NH2-terminal 50 residue peptide proved virtually inactive, even after removal of the masking groups from the citraconyl derivative. The COOH-terminal 29 residue peptides of maleyl and citraconyl apolipoprotein C-II were more active than the corresponding succinylated peptide. After deacylation of the citraconyl derivative, the COOH-terminal peptide had maximal activity as great as apolipoprotein C-II, although the profile of activation remained dissimilar at low activator concentrations.


Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.


Studies on the biosynthesis of sulfolipids in the Diatom Nitzschia alba.

Labeling of sulfolipids in Nitzschia alba was studied after growth of the cells in media containing L-[35S]cystine, L-[35S], L-[35S]cysteine, L-[35S]-methionine or a mixture of L-[Me-3H]methionine and L-[35S]methionine, [35S]Cysteine or [35S]cystine labeled the deoxyceramide sulfonate and the sulfonium analog, phosphatidylsulfocholine (and its lyso derivative) but not the sterol sulfate nor the sulfoquinovosyl diglyceride; [35S]methionine labeled only the phosphatidylsulfocholine and its lyso derivative. With the [35S]- and [Me-3H]methionine mixture (3H/35S ratio 1.0) the phosphatidylsulfocholine had a 3H/35 S ratio of 1.5 indicating that both sulfonium methyl groups were derived from methionine. Probable biosynthetic pathways for these novel sulfolipids are discussed.


Disulfide-linked peptides in the chloroplast thylakoid membrane.

Two peptides move out from the diagonal when dodecyl sulfate gels of pea leaf chloroplast thylakoid membrane proteins are treated with a reducing agent and electrophoresed in the second dimension. Apparently two of the proteins in the thylakoid membrane are disulfide linked dimers. Additional changes in the electrophoretic mobility of certian peptides occur when thylakoid membranes are denatured in the presence of reducing agents: these peptides probably contain intrapeptide disulfide bonds or prosthetic groups covalently attached to the peptide through disulfide bonds.


Disulfide interaction in situ between two neighbouring proteins in mammalian 60-S ribosomal subunits. Isolation of the contact region of the larger protein.

A disulfide complex is formed in situ under gentle conditions between two neighbouring proteins in the 60-S subunits of mammalian ribosomes. The proteins have been identified as L 4 and L 29. The complex is easily isolated from whole ribosomes, and can be utilized for preparing the two proteins in a very pure state for further characterization. Chymotryptic cleavage of the complex or the isolated larger protein (L 4) in the presence of SDS produces two unequal fragments of this protein in nearly quantitative yield. The smaller fragment (approx. 12 000 daltons) contains the contact sequence. Only this fragment of protein L 4 is labelled when rat liver ribosomes are incuabted with iodo[14C]acetate under conditions of complex formation. Protein L 29 is resistant to chymotrypsin in the presence of sodium dodecyl sulfate.


A theoretical interpretation of the variations of some physical parameters within the [2Fe-2S] ferredoxin group.

A model is proposed to explain the variation of some physical parameters within the reduced [2Fe-2S] ferredoxin group. According to this model, the main effects result from a variable mixing of some d orbitals of the Fe2+ ion owing to rhombic distortion of the active site having the same geometrical character, but different in intensity, for each protein. Some peculiar experimental results such as the axial electron paramagnetic resonance spectra of adrenal ferredoxin and Pseudomonas putida ferredoxin and the electric field gradient tensor of P. putida ferredoxin are explained without assuming properties drastically different from those of the other ferredoxins, as had been suggested in the literature.


Circular dichroism spectra of purified cytochromes P-450 from rabbit liver microsomes.

Circular dichroism (CD) spectra were measured for cytochromes P-450 (P-450) purified from phenobarbital- and 3-methylcholanthrene-induced rabbit liver microsomes. No striking difference in alpha-helix content was seen between phenobarbital-induced P-450 (PB P-450) (50%), phenobarbital-induced P-448 (PB P-448) (40%) and 3-methylcholanthrene-induced P-448 (MC P-448) (45--50%) in terms of ultraviolet CD spectra. Strong negative CD spectra associated with 3-methylcholanthrene transitions for MC P-448 in the near-ultraviolet region (250--310 nm) and weaker negative CD spectra associated with Soret transitions for PBP-448 ([theta] = 50 000) and MCP-448 ([theta] = 160 000), indicated that structures of these preparations are strikingly different from each other. Reduction of P-450 and P-448 led to a remarkable decrease of the Soret CD trough, suggesting that reduction was accompanied by a striking conformational change in the vicinity of the heme. Since CO complexes of reduced P-450 and P-448 showed a CD trough and an S-shaped CD, respectively, associated with the absorption peak at 450 nm, the heme vicinities are remarkably different from each other. The CD spectra in the visible region are also discussed. It was noticed that P-420, the denatured form of P-450, exhibited no CD spectra in the Soret and visible regions.


The binding of calcium to fibrinogen: influence on the clotting process.

The influence of Ca2+ on the basic reaction between thrombin and fibrinogen was investigated. The results demonstrate that: (a) A Ca2+-dependent dimeric intermediate is formed during the early step of the clotting process. This dimeric intermeidate is shown to be formed by the association of an intact fibrinogen molecule and a fibrin monomer devoid in only the peptide A, (b) Ca2+ enhances the proteolytic step as illustrated by the measure of the kinetics of H+ release at pH 8.6. On the basis of these observations it is proposed that Ca2+ catalyses the proteolysis of fibrinogen by thrombin through the formation of a Ca2+-dependent dimer.


Molecular weight and structural studies on cephalopod rhodopsin.

The protein moiety of squid (Watasenia scintillans) rhodopsin has been shown to have a molecular weight of 46 800 by means of amino acid analysis. This value was comparable to the value (51 000) obtained from SDS-polyacrylamide gel electrophoresis. After the squid eyes were incubated at 10 degrees C for 8 days, the rhodopsin showed a molecular weight of 39 000 on electrophoresis. The smaller molecular weight was ascertained by amino acid analysis of the rhodopsin; and may result from autolysis by the lysosomal enzyme. The rhodopsin in rhabdomeric membranes and in detergent solution was treated with chymotrypsin, papain or subtilisin. These enzymes first produced the 39 000 dalton rhodopsin and then cleaved this into the 25 000 and 14 000 dalton peptides without bleaching. The rhodopsin was attacked by proteases and readily lost an approx. 12 000 dalton peptide portion. This portion included the COOH-terminal and was rich in glutamic acid, proline, glycine, alanine and tyrosine residues.


Mouse hepatoma and liver ferritins. Comparative structural studies.

Pure ferritin from male mouse liver produces a single band of monomers (RF = 0.199) with electrophoresis in polyacrylamide gels at pH 9.0. The five sub-bands within this monomeric band appear to represent charge isomers having the same molecular size. Ferritin from BH3 transplantable mouse hepatoma shows two overlapping bands of monomers (RFA = 0.208 and RFB = 0.240); further electrophoretic studies show that these bands represent two subpopulations of molecules differing both in charge and size. Sub-bands are not found in this hepatoma ferritin. The larger tumor ferritin reaches the same end migration position as all liver isoferritins on gradient gels, signifying a very similar or identical molecular size; however, the absence of sub-bands indicates that this hepatoma ferritin differs in charge from the homologous liver proteins. Liver and hepatoma ferritins both produce a single prominent subunit band corresponding to nominal molecular weights of 22 250 and 21 700, with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. With electrophoresis on polyacrylamide gradient slabs containing sodium dodecyl sulfate and dithiothreitol, both liver and hepatoma ferritins now reveal two subunits bands situated at identical positions. The polypeptides of these two closely spaced bands have a nominal molecular weight difference of less than 1000. Neither the hepatoma nor the liver seems to produce the ferritins found in the other tissue. Nevertheless, all these ferritins are composed of the same two types of subunits, albeit in different relative amounts. Observed distinctions in the ferritins from these normal or neoplastic cells must reflect differences in assembly and processing, as well as in the regulated expression of the same ferritin genes.


The prosthetic group of milk lactoperoxidase is protoheme IX.

The prosthetic group of milk lactoperoxidase has been isolated from a Pronase hydrolysate of the enzyme and identified spectroscopically and chromatographically as protoheme IX. Partial degradation of the heme occurred during the proteolysis, possibly as a result of coupled oxidation in the presence of glycine and oxygen. The heme is assumed to be buried in the protein molecule in a crevice, which allows ligands to bind to the iron on one side only. The pyridine hemochrome of lactoperoxidase with alpha-band at 563 nm is interpreted as a mixed ligand complex with pyridine on one side of the heme and a ligand originating from the protein on the other. No experimental evidence supports the view that the heme is covalently bound to the protein through an ester linkage.


Salt and temperature-dependent conformation changes in spectrin from human erythrocyte membranes.

ORD and CD measurements of spectrin, in both the dimer and tetramer association state, indicate a high proportion of alpha-helix in this protein. At temperatures below 27 degrees C and in 0.1 M NaCl, the tetramer has an apparent helix content of 73% and the dimer, 68%. The conformation of both states is dependent on salt concentration and temperature. Low ionic strength solutions of spectrin display lowered sedimentation coefficients and a decreased apparent helix content, indicating perhaps a slight refolding and expansion of the molecule. In addition, spectrin in low ionic strength solutions undergoes a broad temperature-dependent transition spread from 20 to 50 degrees C, while in the presence of salt the transition is sharp and centered on 49 degrees C. The temperature-dependent changes in low ionic strength solutions appear to parallel the dissociation of tetramer to dimer.


Multiple molecular forms of glia maturation factor.

Glia maturation factor from the pig brain can be detected in two molecular forms: the high molecular weight form which is 200 000 dalton in size and the low molecular weight form which is 40 000 dalton in size, as determined by Sephadex gel filtration. The former accounts for 85% of the total biological activity extracted at physiologic pH. The proportion of the low molecular weight form increases following freeze-thawing and ion-exchange chromatography. In addition to the morphological effects, both forms possess mitogenic activity but no esteropeptidase activity. Both forms show similar enzyme susceptibility, being inactivated by papain, ficin and pronase but resistant to subtilisin, thermolysin and trypsin. The high molecular weight form is more resistant to denaturation by low pH, heating and urea than the low molecular weight form. The high molecular weight factor has an isoelectric point of 4.27 whereas the low molecular weight factor has one of 5.04.


Some properties and the complete primary structures of two reduced and S-carboxymethylated polypeptides (S5C1 and S5C10) from Dendroaspis jamesoni kaimosae (Jameson's mamba) venom.

Two polypeptides (protein S5C1 and toxin S5C10) were purified from Dendroaspis jamesoni kaimosae venom. Whereas protein S5C1 comprises 61 amino acid residues, toxin S5C10 contains 58 and they each comprise four disulphide bridges. The complete primary structures of the two polypeptides have been elucidated. The sequences of protein S5C1 and toxin S5C10 are structurally homologous to the short neurotoxins Type I, but they are much less toxic. In toxin S5C10 one of the functionally invariant amino acid residues, lysine 26, of the Type I neurotoxin has been replaced by a serine. In contrast protein S5C1 has the feature that it contains ten or eleven structurally invariant amino acids and apparently only one of the five functionally invariant residues.


Selective extraction of desmosomal proteins by low ionic strength media.

Desmosomes, isolated using an acidic buffer, have been subjected to extraction at low ionic strength. This treatment removes more than 35% of their protein in the form of two polypeptide chains of molecular weight 210 000 and 230 000, but the desmosomes show only subtle changes in ultrastructure. It is concluded that the use of low ionic strength media for desmosome isolation yields residual structures specifically depleted in high molecular weight proteins.


Purification of human transcobalamin II-cyanocobalamin by affinity chromatography using thermolabile immobilization of cyanocobalamin.

Transcobalamin II-cyanocobalamin was isolated from Cohn fraction III of pooled human plasma by affinity chromatography on cyanocobalamin-Sepharose and some conventional separation methods. The affinity ligand cyanocobalamin was coupled to AH-Sepharose by a thermolabile linkage. The unsaturated binding protein was absorbed at 4 degrees C and eluted from the column at 37 degrees C as transcobalamin II-cyanocobalamin complex. The final preparation had a specific cyanocobalamin-binding capacity of 0.98 mol cyanocobalamin/mol transcobalamin II, the yield was 55% and the purification index amounted to 1.1 . 10(6). In dodecyl sulphate polyacrylamide gel electrophoresis one major protein band was observed at a molecular weight of 37 000 and a faint band at a molecular weight of 29 000. In polyacrylamide gel isolectric focusing the pure preparation turned out to be heterogeneous with isoelectric points ranging from pH 6.2 to 6.8, possibly by the occurrence of isoproteins.


The isolectins of Helix pomatia. Separation by isoelectric focusing and preliminary characterization.

Helix pomatia lectin was fractionated into several (at least 12) components by isolectric focusing in PharmalyteTM. Eight of these were recovered from a preparative column experiment and shown to be essentially pure by analytical electrofocusing in polyacrylamide gel slabs. The lectin components had haemagglutinating activity against human group A erythrocytes and similar molecular weights of 80 000 as determined vidual components showed slight variations in composition. The components are considered to be isolectins. Isoelectric focusing of lectin samples prepared from single snails from two different biotopes showed isolectin patterns similar to that of pooled lectin. Gel filtration of the lectin and flat bed electrofocusing of isolated isolectins both in the presence of 6 M ura, indicated that the lectin molecules are formed by different combinations of a range subunits with similar molecular weight but with different isoelectric points.


Separation of the integral membrane glycoproteins E1 and E2 of Semliki Forest virus by affinity chromatography on concanavalin A-Sepharose.

The membrane glycoproteins E1 and E2 of Semliki Forest virus are of about equal size but can be separated from each other by affinity chromatography on a concanavalin A-Sepharose column in the presence of sodium dodecyl sulfate. The E1 protein eluted like glycopeptides containing two peripheral sugar branches composed of N-acetylglucosamine, mannose, galactose and sialic acid. The E2 eluted like glycopeptides containing only N-acetylglucosamine and mannose.


Isolation and partial characterization of the major glycoprotein from the plasma membranes of AH-66 hepatoma cells.

A major glycoprotein of the plasma membranes of AH-66 hepatoma ascites cells was isolated in essentially pure form and in milligram amounts. The plasma membranes were solubilized with a solution containing both 0.3 M lithium diiodosalycylate and 0.2% cetylpyridinium chloride, and further extracted with 50% phenol, followed by gel filtration on Sepharose 6B in the presence of 0.1% Ammonyx-LO at pH 8.0. The apparent molecular weight of the purified glycoprotein was estimated to be 165 000 in 5.6% polyacrylamide gels, of which 54% was carbohydrate and 46% was protein. The chemical composition of the glycoprotein resembles glycophorin A from human erythrocyte membranes in that it has a high content of N-acetylgalactosamine, N-acetylglucosamine, galactose and sialic acid and a particularly large proportion of serine, threonine, aspartic acid and glutamic acid.


Preparation of cyanogen bromide fragments of MM, NN, and MN glycoproteins (glycophorins) from human erythrocyte membranes of single donors.

A procedure is described for the preparation of three cyanogen bromide fragments of the MM, NN, or MN glycoprotein (glycophorin) of the human erythrocyte membranes, from erythrocytes of single donors. The fragments are obtained in pure form and excellent yields by employing procedures which include proteolytic inhibitors during membrane processing, thorough delipidation of the glycoprotein, and CNBr cleavage conditions which lead to quantitative fragmentation without loss of carbohydrates. A phenol-urea extraction resolves the two glycopeptide fragments from the carbohydrate-free fragment. The two glycopeptides are further purified by Bio-Gel P-6 and P-100 chromatography. The three fragments include the amino terminal 8 residue glycopeptide, a large glycopeptide form the middle of the molecule which bears the Asn-linked oligosaccharide and 8--9 O-glycosidically linked units, and a carboxyl terminal, carbohydrate-free, approx. 50 residue fragment. Their amino acid and carbohydrate composition, and size, are in close agreement with the sequence data of Tomita, M., Furthmayr, H. and Marchesi, V.T. (Biochemistry (1978), 17, 4756--4770). The fragments represent three well delineated portions of the glycoprotein molecule.


Precocious differentiation of chick embryo pancreas in vitro. Roles of prednisolone, insulin and L-thyroxine.

The hormonal requirement for functional differentiation of chick embryo pancreas were investigated by using organ cultures in chemically defined medium. The hormones tested were prednisolone, insulin and thyroxine, and the parameters examined were alpha-amylase (EC 3.2.1.1) and chymotrypsinogen (EC 3.4.4.5) activities, and the ultrastructure of the tissues. Addition of prednisolone alone to explants from 14-day-old chicken embryo pancreas for 3 days increased the activities of amylase and chymotrypsinogen in the tissues by 3.4- and 6.6-fold, respectively, those of tissues before cultivation. Neither thyroxine or insulin alone, nor both hormones together affected pancreatic exocrine differentiation. Thyroxine enhanced the effect of prednisolone on both enzymes, but insulin did not. When the explants were cultured in the medium containing all three hormones, maximum enzyme activities were observed; amylase or chymotrypsinogen activity being 7- or 18-fold, respectively, that of tissues before cultivation. But these three hormones were not simultaneously necessary. Morphological differentiation was also observed in explants cultuvated in medium containing these three hormones. These results suggest that glucocorticoids are essential for normal differentiation of chick pancreas during the late fetal period, possibly with insulin and thyroxine, and also support the idea that pancreatic enzymes are controlled separately.


Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes.

In chicken thymocytes isolated from 15--40 day-old chickens, after a 2 h incubation at 37 degrees C, insulin stimulated amino isobutyric acid uptake (maximal response: 40--50% of increase at 1 microgram insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 microgram insulin/ml). Binding of 125I-labelled chicken insulin to thymocytes was rapid and higher at 15 degrees C than at 37 degrees C. At steady state, (90 min at 15 degrees C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of 125I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10(8) cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15 degrees C. Degradation of 125I-labelled chicken insulin in the incubation medium was negligible at 15 degrees C but very noticeable at 37 degrees C. Therefore, the low level of insulin binding at 15 degrees C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.


Pattern of glycosaminoglycans and glycoproteins associated with nuclei of regenerating liver of rat.

1. Hyaluronic acid was detected as the largest glycosaminoglycan component in the glycosaminoglycan fraction from purified nuclei of regenerating livers as in the case of normal livers (Furukawa, K. and Tarayama, H. (1977) Biochim. Biophys. Acta 499, 278--289). However, the nuclear content of glycosaminoglycans tended to decrease after partial hepatectomy, reaching one-third of the normal liver level at 24--30 h after partial hepatectomy. On the other hand, two new polyanionic components were detected in the glycosaminoglycan fraction from regenerating liver nuclei. 2. One of these new components seems to be a sulfated glycopeptide. The 35SO4 incorporation into this component was stimulated biphasically after partial hepatectomy; the first stimulation occurring immediately after partial hepatectomy and the second stimulation occurring almost in parallel to the DNA synthesis. 3. Another polyacnionic component which also increases in the nuclear content after partial hepatectomy lacks hexuronic acid, sialic acid and 35SO4 and yet it is intensely stained by Alcian Blue. Preliminary investigations revealed the presence of hexose, ribose and phosphate as the major components. 4. In contrast to the primary localization of hyaluronic acid in the chromatin fraction and also in the nonhistone chromosomal protein fraction from it, these new polyanionic components were detected mainly in the karyosol fraction.


Collagen synthesis by cultured skin fibroblasts from siblings with hydroxylysine-deficient collagen.

It has been previously shown that dermis from subjects with hydroxylysine-deficient collagen contains approximately 5% of normal levels of hydroxylysine and sonicates of skin fibroblasts contain less than 15% of normal levels of collagen lysyl hydroxylase activity. However, cultures of dermal fibroblasts from two siblings with hydroxylysine-deficient collagen (Ehlers-Danlos Syndrome Type VI) compared to fibroblasts from normal subjects synthesize collagen containing approximately 50% of normal amounts of hydroxylysine. The lysyl hydroxylase deficient cultures synthesize both Type I and Type III collagen in the same proportion as control cultures. Both alpha 1(I) and alpha 2 chains are similarly reduced in hydroxylysine content. Collagen prolyl hydroxylation by normal collagen lysyl hydroxylation is the same with or without ascorbate supplementation. In mutant cells the rate of prolyl hydroxylation measured after release of inhibition by alpha, alpha'-dipyridyl is the same as in control cells. The rate of lysyl hydroxylation is reduced in mutant cells but only to approximately 50% of normal.


Preliminary studies on the composition and rheological properties of the extracellular polysaccharide synthesized by Pseudomonas PBI (NCIB 11264).

A microorganism isolated from a carbohydrate-rich industrial effluent synthesized an exocellular slime polysaccharide composed of glucose and galactose in a molar ratio of 7.45 +/- 0.68 : 1, and two non-carbohydrate substituents acetate (3--4%) and pyruvate (5--9%). Contamination by rhamnose and mannose was detectable in crude polysaccharide samples. Solutions of the polysaccharide were pseudoplastic, but not thixotropic, and formed gels in the presence of certain trivalent cations.