In vivo effect of human chorionic gonadotropin on the migration of inflammatory cells in intact or castrated male and female guinea-pigs. A quantitative histological study. I. Study of intact male and female guinea-pigs. The migration of inflammatory cells and fibroblasts into dacron mesh tissue (Mersilene), immersed in a suspension of live BCG and implanted s.c. into intact male and female guinea-pigs treated with either commercial or purified human chorionic gonadotropin was investigated. Cell counts showed that in treated animals a significant reduction (about 70%) of whole cells occurred. The effect was maximal on neutrophil granulocytes (a reduction of 85%) by Day 5 after grafting. No significant differences appeared between males and females or between commercial and purified human chorionic gonadotropin.


In vivo effect of human chorionic gonadotropin on the migration of inflammatory cells in intact or castrated male and female guinea-pigs: a quantitative histological study. II. Study of castrated males and females. The effect of purified human chorionic gonadotropin (HCG) on the migration of inflammatory cells into dacron mesh tissue containing live BCG and implanted under the skin of castrated male and female guinea-pigs was investigated. Cell counts have shown that no significant difference appeared between controls and castrated guinea-pigs given HCG. This work suggests that the inhibiting effect of HCG on the mobility of neutrophil granulocytes is closely related to the presence of both male and female gonads.


Comparative studies of the metastatic potential of three transplantable rat mammary carcinomas of spontaneous origin. The metastatic potential of 3 spontaneously arising mammary carcinomas (Sp4, Sp15 and Sp22) has been examined when transplanted in the form of a viable cell suspension into the tissue of origin. Primary tumours were excised at different times after implantation and it was found that the metastatic potential of the immunogenic tumour Sp4 was directly correlated with the size of the primary tumour when excised. By contrast, the incidence of metastases from the non-immunogenic tumours Sp15 and Sp22 was similar irrespective of the size of the primary tumour on excision. The pattern of metastasis also differed between the tumours, although here there was no relation to immunogenicity. Thus, resection en bloc of large primary Sp4 or Sp15 tumours plus regional lymph nodes could be completely curative, signifying initial spread of tumours via the lymphatics and only subsequently via the blood stream. On the other hand, resection en bloc of primary Sp22 tumours plus regional lymph nodes at a similar stage of primary tumour development was never curative, signifying early spread via the blood stream. Other studies showed that the metastatic potential of mammary carcinoma Sp4 was an innate characteristic of the tumour and not related to the tissue of implantation since in addition to metastasizing from the mammary pad it metastasized when implanted either s.c. or intradermally in a region devoid of mammary tissue. Furthermore, a rat sarcoma Mc7 showed a negligible tendency to metastasize when implanted either in the mammary pad or in the s.c. tissue, where it had been induced with methylcholanthrene.


Resistance and susceptibility to the induction of rat adjuvant disease. Diverging susceptibility and severity achieved by selective breeding. Rats selected for their ability to develop or resist adjuvant disease were used to establish 2 inbred lines of rat over 20 generations. A resistant line was rapidly established with almost 100% non-responsiveness by the sixth generation. A line showing 100% susceptibility was also established very rapidly but throughout the course of the breeding programme the severity continued to increase in intensity to a level considerably above that to be seen in strains normally considered to be high responders. At the thirteenth generation and beyond, the susceptible line showed a marked sex difference in the secondary lesions, females being more severely affected than the males. The 2 lines of rat were also tested for their ability to develop experimental allergic encephalomyelitis (EAE) in selected generations. There was no clear correlation between the 2 diseases although those animals developing the most severe adjuvant disease also had the most severe EAE.


Enzyme content of hepatocellular lysosomes in the tumour-bearing rat. An increase in the number of hepatocellular lysosomes is detectable with the electron microscope in rats bearing a carcinogen-induced sarcoma but not in rats bearing a transplanted Walker tumour. Biochemical studies show an increase in lysosomal enzymes in the liver of both groups of rats, but the increase in the rats bearing a carcinogen-induced sarcoma was greater than that in rats bearing a Walker tumour. This difference is explained by the fact that only gross increases in lysosomal population are likely to be detected in the small samples examined with the electron microscope, but by biochemical methods whereby many grams of tissue are analysed, finer changes may be detected. The collective data to date indicate that an increase in hepatocellular lysosomes and/or lysosomal enzymes occurs in rats and man bearing a variety of tumours and to this there is, as yet, no known exception.


An investigation into the cytotoxicity of respirable dusts from British collieries. A series of respirable dusts from British collieries was collected and analysed for mineral content and physical characteristics. Where possible 2 samples of dust were collected from the same site at 8-month intervals. All dusts were tested for their cytotoxic potential using a permanent line of macrophage-like cells (P388D1). In addition, for some dusts, a haemolytic technique was used. With both techniques a positive overall correlation was found between cytotoxicity and the total ash content of the dusts. When the results from collieries producing high- and low-rank coals were considered separately, however, it was found that the ash content of high-rank dusts (r=0.75) showed a much closer correlation with cytotoxicity than low-rank dusts (r=0.40). With the cell test system the ash components, kaolin and mica (r=0.58) and to a lesser extent quartz (r=0.48) showed significant positive correlations with cytotoxicity for high-rank coal dusts but not for low. Using the haemolytic system, however, only the quartz content of the high-rank dusts showed a significant relationship (r=0.69) to levels of haemoglobin release. Both the results of mineralogical analysis of dust samples and cytotoxicity tests showed that the mineral content and cytotoxic potential of dusts collected from the same colliery, and even from the same underground site, at different times, varied considerably. A poor correlation was found between cytotoxicity and various measurements of pneumoconiosis risk but this may well be partly due to this great variation of dust composition with time. In general, the overall results of this study were in good agreement with those of previous work on coal dust toxicity in that both the rank and composition of colliery dusts were found to be of importance, whereas the role of quartz remained enigmatic.


Replenishment of alveolar macrophages in silicosis: implication of recruitment by lipid feed-back. The deposition of quartz in the pulmonary alveoli creates a major demand for macrophages to replace those destroyed, but local proliferation of monocytes appeared to be minimal and the role of systemic recruitment was therefore explored. Injected silica and lipids stimulated the phagocytic function of the mononuclear phagocytic system (MPS), whilst inhaled silica provoked lipid accumulation in the lung, thus suggesting that lipid might also induce a proliferative response in the marrow. Using marrow cultures, cells of the rat MPS were identified by size and phagocytic capacity for latex microspheres, and then subjected to kinetic analysis in litter-mate pairs by single and double labelling autoradiography, under normal conditions and after administration of lipid extracted from rat lungs consolidated by silica-induced alveolar lipo-proteinosis. Treatment of the results by a new device facilitated distinction of promonocytes from monocytes and thus afforded a more precise means of assessing MPS kinetics. The duration of DNA synthesis and the cell-cycle time of promonocytes were reduced and the rate of entry into DNA synthesis increased as a result of i.v. injection of lung lipid. These findings support the involvement of systemic recruitment of monocytes from the marrow by a positive feed-back mechanism when a powerful irritant persists in the lungs and the results are discussed in the overall context of silicotic fibrogenesis.


Contact allergic sensitivity to plants and the photosensitivity dermatitis and actinic reticuloid syndrome. Contact allergic sensitivity to oleoresin extracts from Compositae plants was found to be usually present in individuals suffering from the photosensitivity dermatitis and actinic reticuloid syndrome. It was demonstrated in forty-seven out of fifty-five examples of this syndrome. These results provide support for the view that contact allergic sensitivity is an important aspect of the state of chronic photosensitivity in the middle-aged and elderly male.


Reticulate erythema--a prodrome in hereditary angio-oedema. A previously undescribed short-lived non-puritic reticulate erythema has been noted in several members of unrelated families with hereditary angio-oedema. The eruption which may be widespread can last from 1 to 4 days and usually immediately precedes an overt attack of hereditary angio-oedema although this is not invariable, and swellings may occur in areas not previously involved by the erythema. Histology including immunohistology probably shows no specific changes.


The effect of various corticosteroids on the release of beta-glucuronidase from human leukocytes challenged with zymosan. Seven corticosteroids were tested for their effect on the release of beta-glucuronidase when human white cells were challenged with zymosan. Stabilization was noted in two areas of dilution with every steroid but destabilization occurred at high concentrations. However, those steroids that stabilized effectively at low concentrations were also those which were the most potent vasoconstrictors.


Neutron activation analysis of trace elements in skin. VIII. Selenium in normal skin. Selenium(Se) has been determined in skin by neutron activation analysis applying selective irradiation and cooling time, followed by measurement of the activity on a high resolution germanium lithium (Ge(Li)) detector. The levels  and distribution of Se in normal abdominal and plantar skin, together with other available evidence, lead us to believe that it is an essential trace element in man. It is suggested that Se has an essential role in skin keratinization.


Hyperglucagonaemia and necrolytic migratory erythema in cirrhosis--possible pseudoglucagonoma syndrome. Necrolytic migratory erythema is the distinctive cutaneous eruption seen with glucagon-producing tumours of the pancreas. Recognition of this eruption is important because it may lead to the early diagnosis of a glucagonoma. Recently, we saw a patient who had necrolytic migratory erythema, hyperglucagonaemia, and cirrhosis of the liver with no evidence of pancreatic tumour while alive or at autopsy. Serum glucagon levels during the period of observation and during an oral glucose tolerance test suggested that the hyperglucagonaemia was not due to an occult glucagon-producing tumour but may have been the result of advanced hepatic cirrhosis.


Dermatomyositis-like syndrome due to toxoplasma. A woman had the typical clinical, cutaneous, and muscular picture of dermatomyositis but without some of the biochemical alterations of that disease. The positive serological investigations for toxoplasmosis, the pressure in muscles of very numerous toxoplasms and their culture in mice, and the improvement after treatment with pyrimethamine and sulphametoxypyridine demonstrated that the signs of dermatomyositis had been caused by infection with toxoplasmosis.


Hypertrichosis due to minoxidil.

Hypertrichosis due to minoxidil. A case of hypertrichosis due to the hypotensive drug minoxidil is described. A review of the literature suggests that this complication appears in nearly all patients treated with this drug. The mechanism is unknown, but the similarity to the cases of hypertrichosis due to diazoxide, another potent vasodilator, suggests that increased cutaneous blood flow may be a factor.


Contact urticaria to rubber.

Contact urticaria to rubber. A case of contact wealing to rubber gloves attributed to natural latex from Hevea braziliensis is described.


Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.


Evaluation of equilibrium constants for the binding of N-acetyl-L-tryptophan to monomeric and dimeric forms of alpha-chymotrypsin. The binding of N-acetyl-tryptophan to the monomeric and dimeric forms of alpha-chymotrypsin in I = 0.2 acetate-chloride buffer, pH 3.86, has been studied quantitatively. Equilibrium sedimentation studies in the absence of inhibitor yielded a dimerization constant of 3.5 L/g. This value was confirmed by frontal gel chromatography of the enzyme on Bio-Gel P-30, which was also used to establish that N-acetyl-L-tryptophan binds preferentially to monomeric enzyme. From kinetic studies of competitive inhibition with N-acetyl-L-tryptophan ethyl ester as substrate, an equilibrium constant of 1300 M-1 was determined for the binding of N-acetyl-L-tryptophan to monomeric alpha-chymotrypsin. An intrinsic binding constant of 250 M-1 for the corresponding interaction with dimeric enzyme was calculated on the basis of these results and binding data obtained with concentrated (18.5 g/L) alpha-chymotrypsin. The present results refute earlier claims for exclusive binding of competitive inhibitors to monomer and also those for equivalence of inhibitor binding to monomeric and dimeric forms of alpha-chymotrypsin.


Effects of anions on the activation thermodynamics and fluorescence emission spectrum of alkaline phosphatase: evidence for enzyme hydration changes during catalysis. The effect of anions on the thermodynamic activation functions for a model enzyme, calf intestinal alkaline phosphatase (EC 3.1.3.1), have been studied in order to examine the role of protein hydration changes in establishing the energetics of enzyme catalysis. The influences of these anions on the activation volume (delta V) and activation free energy (delta G) reflected clear Hofmeister (lyotropic) series effects, in the order F- greater than Cl- greater than Br- greater than I- (order of increasing salting-out potential). A pronounced covariation was observed between the influences of these anions on Vmax, which is proportional to delta G, and on the negative activation volume of the reaction. Fluoride was able to counteract the influences of Br- and I- on both Vmax and delta V when combinations of these anions were employed. The effects of Br- and I- on Vmax and delta V were more pronounced at lower temperatures. The control delta V was increasingly negative at reduced temperatures. The effects of the neutral salts and propanol on delta V and delta G, as well as the effects of salting-in anions on the activation enthalpy and the negative activation entropy of the reaction, are consistent with a model which proposes that peptide groups or polar side chains on the native enzyme exergonically increase their exposure to solvent during the catalytic activation event. These conclusions are in accord with the known free energy, enthalpy, entropy, and volume changes which occur when model peptide groups are transferred between water and concentrated salt solutions. Consistent with the kinetic results, the fluorescence emission wavelength maximum of alkaline phosphatase increased in the presence of anions in the order F- greater than Cl- greater than Br- greater than I-. The salting-out ion (F-) and the salting-in ions (Br- and I-) shifted lambda max in different directions, and these lambda max shifts could be counterbalanced by using equimolar combinations of salting-in and salting-out anions. Control experiments with a model compound, N-acetyltryptophanamide, showed that the spectra shifts caused by the salts did not result solely from differential quenching by the anions of the solvent-exposed tryptophan(s) on the enzyme. Hofmeister additivity phenomena indicated that the solvent is at the basis of these salt-induced enzyme structural changes. It is concluded that changes in protein solvation during enzymic reactions contribute significantly to the thermodynamic activation parameters in both the native and the salt-perturbed enzyme.


Phospholipase D from savoy cabbage: purification and preliminary kinetic characterization. Phospholipase D has been purified 680-fold from an acetone powder of savoy cabbage in an overall yield of 30%. The purification involves solubilization of the acetone powder in a Ca2+-containing buffer and subsequent ammonium sulfate fractionation. Gel filtration on Sephadex G-200 and hydrophobic affinity chromatography using a gamma-aminopropane-agarose gel complete the purification. The two chromatographic steps were conducted in buffers containing 50% ethylene glycol, which was necessary in order to maintain stability of the enzyme. Purity was established on the basis of gel electrophoresis and ultracentrifugation. A preliminary kinetic characterization of the enzyme was carried out by using lecithins with short-chain fatty acids below the critical micelle concentration. A complex series of results were obtained which demonstrated the following. (1) The enzyme is quite sensitive to ionic strength, being inhibited at high ionic strength. (2) The pH optimum depends on the concentration of Ca2+ used in the assay. At 0.5 mM Ca2+ the pH optimum is 7.25, but it is 6.0 at 50 mM Ca2+. (3) The effect of substrate concentration at a given pH and ionic strength did not show simple hyperbolic kinetics but rather regions of parabolic and hyperbolic kinetics.


Isolation and characterization of argininosuccinate synthetase from human liver. This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.


Resonance Raman examination of axial ligand bonding and spin-state equilibria in metmyoglobin hydroxide and other heme derivatives. Resonance Raman spectra and excitation profiles have been obtained within the 5700-6300-A absorption band of purified sperm whale metmyoglobin hydroxide (MbIIIOH) solutions. A large enhancement occurs for a Raman peak at 490 cm-1 which is shown by isotopic substitution of 18O for 16O to be almost purely an Fe-O stretch. The Fe-O vibration in MbIIIOH occurs 5 cm-1 to lower energy than the corresponding vibration at 495 cm-1 in human methemoglobin hydroxide (HbIIIOH) [Asher, S., Vickery, L., Schuster, T., & Sauer, K. (1977) Biochemistry 16, 5849], reflecting differences in ligand bonding between Mb(III) and Hb(III). A larger frequency difference (10 cm-1) exists between MbIIIF and HbIIIF for the Fe-F stretch. We do not observe separate Fe-O or Fe-F stretches from the alpha and beta chains of either HbIIIOH or HbIIIF. Excitation profile measurements for MbIIOH indicate that the 5700-6300-A absorption band is composed of two separate absorption bands which result from a high- and a low-spin form of MbIIIOH. The spin-state-sensitive Raman band at 1608 cm-1 reflects the high-spin species and has an excitation profile maximum at about 6000 A while the low-spin Raman band occurs at 1644 cm-1 and shows an excitation profile maximum at 5800 A. The Fe-O stretch at 490 cm-1 has an excitation profile maximum at about 6000 A. The differences in frequency and Raman cross section between the Fe-X vibrations in MbIIIX and HbIIIX (X = OH-, F-) can be related to increases in the out-of-plane iron distance for the high-spin species of MbIIIX. The shift in the 1644-cm-1 MbIIIOH low-spin state Raman band indicative of the heme core size to 1636 cm-1 in HbIIIOH indicates a larger heme core size in HbIIIOH. Raman frequency shifts are used to estimate differences in bond strain energies between MbIIIX and HbIIIX (X = OH-, F-). Previous resonance Raman excitation profile data can be interpreted in terms of separate contributions from different spin-state species.


Amino acid sequence studies on the alpha chain of human fibrinogen. Complete sequence of the largest cyanogen bromide fragment. The largest fragment produced by complete cyanogen bromide digestion of the alpha chain of human fibrinogen contains 236 residues and has a calculated molecular weight of 23,949. The complete amino acid sequence of the fragment was determined by the isolation of peptides generated by plasmin, trypsin (including digestion of citraconylated material), staphylococcal protease, and chymotrypsin. In addition, some key subfragmentation was achieved by selective chemical cleavage at tryptophan residues. The fragment has an unusual amino acid composition, more than half of its residues being glycine, serine, threonine, and proline. There are very few nonpolar residues, although 7 of the alpha-chain's 10 tryptophans occur in this fragment. The fragment contains 2 cysteine residues located 30 residues apart which are connected by an intrachain disulfide bond in the native molecule. The tryptophans occur with a definite periodicity that highlights a series of 13-residue homology repeats. The fragment also contains the two principal alpha-chain cross-linking sites.


Amino acid sequence studies on the alpha chain of human fibrinogen. Exact location of cross-linking acceptor sites. Human fibrinogen was clotted under conditions that promote latent factor XIII activity and in the presence of a radioactive substitute cross-linking donor ([14C]glycine ethyl ester). The labeled fibrin was reduced and alkylated in the presence of 6 M guanidinium chloride. After dialysis and freeze-drying, the preparation was separated into its constituent polypeptide subunits by chromatography on (carboxymethyl)cellulose in the presence of 8 M urea. Under the incorporation conditions used, the radioactivity was limited to gamma chains (one donor molecule/chain) and alpha chains (two donor molecules/chain). The labeled alpha chains were digested with cyanogen bromide and fractionated on Sephadex G-50. All the radioactivity was found in a fragment previously designated H alpha CNI, the largest of the cyanogen bromide fragments in the alpha chain. The fragment was further fragmented by digestion with plasmin, trypsin, chymotrypsin, and/or staphylococcal protease. The incorporated radioactivity was found to reside in equal amounts at two different sites located 38 residues apart. These were determined to be positions 88 and 126 in H alpha CNI, which correspond to glutamine-328 and glutamine-366 in the alpha chain.


Amino acid sequence studies on the alpha chain of human fibrinogen. Overlapping sequences providing the complete sequence. The complete amino acid sequence of the alpha chain of human fibrinogen has been determined. It contains 610 amino acid residues and has a calculated molecular weight of 66,124. The chain has 10 methionines, and fragmentation with cyanogen bromide yields 11 peptides [Doolittle, R.F., Cassman, K.G., Cottrell, B.A., Friezner, S.J., Hucko, J.T., & Takagi, T. (1977) Biochemistry 16, 1703]. The arrangement of the 11 fragments was determined by the isolation of peptide overlaps from plasmic and staphylococcal protease digests of fibrinogen and/or alpha chains. In addition, certain of the cyanogen bromide fragments, preliminary reports of whose sequences have appeared previously, have been reexamined in order to resolve several discrepancies. The alpha chain is homologous with the beta and gamma chains of fibrinogen, although a large repetitive segment of unusual composition is absent from the latter two chains. The existence of this unusual segment divides the sequence of the alpha chain into three zones of about 200 residues each that are readily distinguishable on the basis of amino acid composition alone.


Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions. Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing  rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.


Intramembrane positions of membrane-bound chromophores determined by excitation energy transfer. A detailed theory has been derived to evaluate the efficiency of nonradiative transfer of electronic excitation energy between nonassociated membrane-bound chromophores. Two different approaches are presented and shown to lead to identical numerical results. In the first of these the efficiency of transfer is computed from the decay with time of the donor excited state. In the second approach, the efficiency is calculated directly, demonstrating that to a high degree of accuracy the array of acceptors can be represented as consisting of a single nearest acceptor plus a continuum of secondary acceptors. A general expression is derived for the dipole-dipole orientation factor as a function of the position of an acceptor. It is shown that, by invoking the range of orientations that must be present at the very least in a particular case, the expected values of transfer efficiency may be limited to a relatively narrow band of uncertainty about those predicted for total randomization. In the limit of total randomization, the theory reduces to functions of but two dimensionless parameters: an effective number of acceptors and a normalized distance of closest approach, a parameter which in turn is a function of an excluded surface area and the depth in the membrane of a donor relative to that of an acceptor. Finally, data analysis procedures are presented whereby one can determine the surface density of acceptors for a known geometry or, alternatively, determine the distance of closest approach for known surface densities.


Intramembrane position of the fluorescent tryptophanyl residue in membrane-bound cytochrome b5. We have developed a method to measure the intramembrane position of the fluorescent tryptophanyl residue in whole cytochrome b5 and the nonpolar membrane binding segment when these molecules are bound to phospholipid vesicles [Koppel, D.E., Fleming, P., & Strittmatter, P. (1979) Biochemistry (preceding paper in this issue)]. The method utilizes excitation energy transfer from the donor tryptophanyl residue in the protein to trinitrophenyl or danysl acceptor groups on the surface of the phospholipid bilayer. It was determined that that single fluorescent tryptophanyl residue in vesicle-bound cytochrome b5 and the nonpolar segment is located approximately 20-22 A below the surface of the bilayer. This position represents a minimum depth of penetration of this portion of the cytochrome in the membrane.


Equilibrium binding of [3H]tubocurarine and [3H]acetylcholine by Torpedo postsynaptic membranes: stoichiometry and ligand interactions. Studies are presented of the equilibrium binding of [3H]-d-tubocurarine (dTC) and [3H]acetylcholine (AcCh) to Torpedo postsynaptic membranes. The saturable binding of [3H]dTC is characterized by two affinities: Kd1 = 33 +/- 6 nM and Kd2 = 7.7 +/- 4.6 microM, with equal numbers of binding sites. Both components are completely inhibited by pretreatment with excess alpha-bungarotoxin or 100 microM nonradioactive dTC and competitively inhibited by carbamylcholine with a KI = 100 nM, but not affected by the local anesthetics dimethisoquin, proadifen, and meproadifen. The biphasic nature of [3H]dTC binding was unaltered in solutions of low ionic strength and by preparation of Torpedo membranes in the presence of N-ethylmaleimide, a treatment which yields dimeric AcCJ receptors. dTC competitively inhibits the binding of [3H]AcCH and decreases the fluorescence of 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane-2-trimethylammonium (Dns-Chol) in a manner quantitatively consistent with its directly measured binding properties. It decreases the initial rate of 3H-labeled Naja nigricollis alpha-toxin binding by 50% at 60 nM with an apparent Hill coefficient of 0.58. The stoichiometry of total dTC, AcCh, and alpha-neurotoxin binding sites in Torpedo membranes was determined by radiochemical techniques and by a novel fluorescence assay utilizing Dns-Chol as an indicator, yielding ratios of 0.9 +/- 0.1:0.9 +/- 0.2:1, respectively. The biphasic equilibrium binding function is not unique to dTC since other ligands inhibited [3h]acCh binding in a biphasic manner with apparent inhibition constants as follows: gallamine triethiodide (K11 = 2 microM, K12 = 1 mM); Me2dTC (K11 = 500 nM, K12 = 10 microM); decamethonium (K11 = 100 nM, K12 = 1.6 microM). Carbamylcholine, however, inhibited [3H]AcCh binding with a single KI = 100 nM. The observed competition between those ligands and [3H] AcCh cannot be completely accounted for by competitive interaction with two different affinities, and the deviations are discussed in terms of the positive cooperativity of the [3H] AcCh binding function itself. It is concluded that dTC binds only to the AcCh sites in Torpedo membranes and that those sites display two affinities for dTC but only one for AcCh.


Hybrid glycosaminoglycans synthesized by monolayers of chick embryo arterial fibroblasts. Monolayer cultures of arterial fibroblasts from 13-day chick embryonic aorta incorporated 35SO42- into glycosaminoglycans containing both glucuronic and iduronic acids. Bacterial chondroitinase ABC converted more than 98% of the 35SO4-labeled polymer to mono- or disaccharides, including (1) N-acetyl-D-galactosamine 4-sulfate, (2) delta 4,5-glucuronic acid 2- or 3-sulfate leads to N-acetylgalactosamine 6-sulfate, and (3) the unsaturated disaccharides normally obtained from chondroitin 4-sulfate and chondroitin 6-sulfate sequences. Chondroitinase AC converted only 77% of the 35SO4-labeled polymer to the same mono- and disaccharides and yielded, in addition, the following oligosaccharide products: (1) delta 4,5-glucuronic acid leads to N-acetylgalactosamine 4- or 6-sulfate leads to iduronic acid leads to N-acetylgalactosamine 6- or 4-sulfate; (2) N-acetylgalactosamine 4-sulfate leads to iduronic acid 2- or 3-sulfate leads to N-acetylgalactosamine 6-sulfate; (3) delta 4,5-glucuronic acid leads to N-acetylgalactosamine 4-sulfate leads to (iduronic acid leads to N-acetylgalactosamine 4-sulfate)2; (4) delta 4,5-glucuronic acid leads to N-acetylgalactosamine 4- or 6-sulfate leads to (iduronic acid leads to N-acetylgalactosamine 6- or 4-sulfate)2; (5) higher oligosaccharides containing iduronic acid and N-acetylgalactosamine 4-sulfate.


Gangliosides of human erythrocytes. A novel ganglioside with a unique N-acetylneuraminosyl-(2 leads to 3)-N-acetylgalactosamine structure. A novel ganglioside having both N-acetylgalactosamine and N-acetylglucosamine was isolated from human erythrocyte membranes. Its structure was characterized by successive exoglycosidase treatment, methylation analysis, and direct-probe mass spectrometry of permethylated intact and desialylated glycolipid. The core structure of the ganglioside was found to be N-acetylgalactosaminyl paragloboside with sialosyl substitution by 2 leads to 3 linkage at the terminal GalNAc residue: NeuNAc alpha 2 leads to 3 GalNAc beta 1 leads to -3Gal beta 1 leads to 4 GlcNAc beta 1 leads to 3 Gal beta 1 leads to 4Glc leads to Cer. The ganglioside was characterized by the presence of a ceramide with myristic acid as the major component.


Affinity-directed cross-linking of membrane-bound acetylcholine receptor polypeptides with photolabile alpha-bungarotoxin derivatives. Photolabile derivatives of [125I]-alpha-bungarotoxin that retain specific binding to Torpedo californica acetylcholine receptor have been utilized as structural probes of the receptor complex of polypeptide components in its membrane-associated form. The derivatized toxins contained aryl azide side chains poised to form covalent cross-links to both associated and adjacent polypeptides following toxin-receptor complex formation. The results demonstrate that, depending on the possible radius of extension of the photoactivated group from the parent toxin, either (1) both the polypeptide to which the toxin derivative binds and an adjacent polypeptide can be derivatized upon photolysis or (2) only the adjacent polypeptide is labeled. The results lend strong support to the notion that the nicotinic receptor from T. california is composed of a complex of different polypeptides.


Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 2. Reactivation of irreversibly denatured aggregates. Noncovalent aggregation is a side reaction in the process of reconstitution of oligomeric enzymes (e.g., lactic dehydrogenase) after preceding dissociation, denaturation, and deactivation. The aggregation product is of high molecular weight and composed of monomers which are trapped in a minium of conformational energy different from the one characterizing the native enzyme. This energy minimum is protected by a high activation energy of dissociation such that the aggregates are perfectly stable under nondenaturing conditions, and their degradation is provided only by applying strong denaturants, e.g., 6 M guanidine hydrochloride at neutral or acidic pH. The product of the slow redissolution process is the monomeric enzyme in its random configuration, which may be reactivated by diluting the denaturant under optimum conditions of reconstitution. The yield and the kinetics of reactivation of lactic dehydrogenase from pig skeletal muscle are not affected by the preceding aggregation-degradation cycle and are independent of different modes of aggregate formation (e.g., by renaturation at high enzyme concentration or heat aggregation). The kinetics of reactivation may be described by one single rate-determining bimolecular step with k2 = 3.9 x 10(4) M-1 s-1 at zero guanidine concentration. The reactivated enzyme consists of the native tetramer, characterized by enzymatic and physical properties identical with those observed for the enzyme in its initial native state.


Conformations of dinucleoside phosphates in aqueous solution. The conformations of dinucleoside phosphates have been reexamined by semiempirical potential energy calculations. Conformations I, II, and III, proposed by Lee & Tinoco [Lee, C. H., & Tinoco, I., Jr. (1977) Biochemistry 16, 5403], are possible species after refinement of their structures by potential energy minimization. These three conformers can represent three types of dinucleoside phosphate species in solution. Dhingra et al. [Dhingra, M. M., Sarma, R. H., Giessner-Prettre, C., & Pullman, B. (1978) Biochemistry 17, 5815] had concluded that conformations of type II and III were unlikely or impossible. They favored conformations g-g- (equivalent to I), g+g+,g+t, and tg+; the last three conformations have little stacking and are calculated to be energetically less favorable by more than 5 kcal/mol. Common structures of the types I, II, and III are found for dinucleoside phosphates with different purine-pyrimidine sequences. The sequence dependence of the potential energy of these three conformers has been calculated. The experimental nuclear magnetic resonance data of dinucleoside phosphates are consistent with these three conformations.


Cloning of synthetic deoxyribonucleic acid that codes for embryonic cardiac myosin light-chain polypeptide. Double-stranded complementary deoxyribonucleic acid (cDNA) transcribed in vitro from a partially pure myosin light-chain messenger ribonucleic acid (mRNA) of the chick embryonic heart was cloned in Escherichia coli strain chi 1776 by using the HindIII cleavage site in the plasmid pBR322. The insertion of essentially full length DNA was achieved by repeated selection of large-size cDNA transcripts. Of the 12 transformants that contained large-size DNA inserts, the clone pML10 insert was 950 base pairs in length, almost the same size as myosin light-chain mRNA (980 nucleosides). The clone pML10 was identified by hybridization with a highly pure cDNA probe and by hybrid-arrested translation assay. pML10 was further characterized by partial restriction enzyme mapping. The availability of a cloned DNA probe for myosin light-chain facilitates the analysis of the mechanism underlying the induction of cardiac muscle specific gene transcription in presumptive heart-forming cells of the chick blastoderm.


Thermal stability and protein structure. Amino acid sequences have been compared for thermophilic and mesophilic molecules of ferredoxin, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase. It is shown that Gly, Ser, Ser, Lys, and Asp in mesophiles are generally substituted by Ala, Ala, Thr, Arg, and Glu, respectively, in thermophiles. These exchanges suggest that thermal stability can be achieved by the addition of many small changes throughout the molecule without significant change in the backbone conformation. Their overall effect is primarily to increase internal and decrease external hydrophobicity as well as to favor helix stabilizing residues in helices. These substitutions minimize interruption of function or internal residue packing arrangements. Although the analysis has been confined to the above-mentioned molecules, the observed stabilizing principles may be more generally applicable.


Complete amino acid sequence of a papain-solubilized human histocompatibility antigen HLA-B7. 1. Isolation and amino acid composition of fragments and of tryptic and chymotryptic peptides. As a part of the overall strategy for determining the complete covalent structure of the papain-solubilized portion of the heavy chain of the human histocompatibility antigen HLA-B7, the protein was dissected into various fragments by a combination of partial acid hydrolysis and cyanogen bromide cleavage. After purification by chromatographic procedures, these fragments have been used as a source for tryptic and chymotryptic peptides. Thirty-three major tryptic and twenty-two major chymotryptic peptides were purified in nanomole amounts and their amino acid compositions determined. These peptides account for the whole extent of the polypeptide chain with the exception of the amino-terminal CNBr pentapeptide. They provide the basis for the formal alignment of the acid cleavage and cyanogen bromide fragments of the molecule as well as the source material for the elucidation of the primary structure of the HLA-B7 heavy chain.


Complete amino acid sequence of a papain-solubilized human histocompatibility antigen, HLA-B7. 2. Sequence determination and search for homologies. The complete amino acid sequence of the papain-solubilized heavy chain of a human histocompatibility antigen (HLA-B7) has been elucidated. It consists of a polypeptide of 271 residues (31 333 daltons). A single glycan moiety is attached to an asparagine residue at position 86 by an N-glycosidic bond. Two intrachain disulfide bonds, arranged linearly, involve half-cystine residues at positions 101 and 164 and at positions 203 and 259. They form two loops of 62 and 55 residues, respectively, separated by 38 residues. Computer analysis of the sequence suggests the existence of internal homology between the amino-terminal portion (residues 1--90) and the region of the first disulfide loop (residues 91--180). There is a significant homology between the second disulfide loop region of the chain (residues 182-271) and immunoglobulin (Ig) constant domains and beta 2-microglobulin [Orr, H. T., Lancet, D., Robb, R. J., López de Castro, J. A., & Strominger, J. L. (1979A) Nature (London) (in press)]. However, no such homology to Ig is apparent in the amino-terminal or in the first disulfide loop regions.


Effect of estrogen on ovalbumin gene expression in differentiated nontarget tissues. By use of cloned DNA fragments as probes, low levels of ovalbumin RNA sequences (structural and intervening sequences) were detected in nuclear RNA extracts of nontarget tissues, such as liver, spleen, brain, and heart of chicks. The expression of the ovalbumin gene sequences was hormone dependent. In estrogen-stimulated chicks, a low level of ovalbumin RNA sequences, ranging from 0.2 to 0.7 molecule per cell, was present in nontarget tissues while less than 0.01 molecule per cell could be found in the same tissues of unstimulated chicks. A significant amount of the ovalbumin mRNA sequences was also found in polysomes of liver and brain. The ovalbumin mRNA sequences could be translated into proteins which were only localized in a few cells among the entire population of liver cells as determined by an immunocytochemical assay. These results suggest that there are some cells in liver, spleen, heart, and brain which can respond to hormone stimulation and produce ovalbumin mRNA and its translational product.


Isolation and characterization of bovine plasma prekallikrein (Fletcher factor). Prekallikrein (Fletcher factor) has been purified from bovine plasma approximately 25 000-fold with an overall yield of 14%. Purification steps included ammonium sulfate fractionation and column chromatography on heparin-agarose, DEAE-Sephadex, CM-Sephadex, benzamidine-agarose, and arginine methyl ester-agarose. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. Bovine plasma prekallikrein is a glycoprotein with a molecular weight of 82 000 as determined by sedimentation equilibrium centrifugation. It contains 12.9% carbohydrate, including 6.2% hexose, 4.5% N-acetylglucosamine, and 2.2% N-acetylneuraminic acid. Prekallikrein is a single polypeptide chain with an amino-terminal sequence of Gly-Cys-Leu-Thr-Gln-Leu-Tyr-His-Asn-Ile-Phe-Phe-Arg-Gly-Gly. This sequence is homologous to the amino-terminal sequence of human factor XI (plasma thromboplastin antecedent). Both prekallikrein and kallikrein require kaolin to correct Fletcher factor deficient plasma. Kallikrein, however, has a specific activity 3.5 times greater than prekallikrein. Prekallikrein does not correct plasma deficient in factor XII (Hageman factor), factor XI, or high molecular weight kininogen (Fitzgerald factor).


Conformations and electronic structures of oxidized and reduced isoalloxazine. The conformations of oxidized and reduced isoalloxazine have been examined by a molecular orbital method, PRDDO (partial retention of diatomic differential overlap). The angle theta of fold about the N...N line of the central ring is zero for the planar oxidized form, but a bend of theta = 10 degrees requires only 2 kcal/mol. On the other hand, the reduced form is nonplanar (theta approximately 15 degrees), and the barrier for reversal of this bend is 4 kcal/mol, comparable with that in simple amines. Molecular properties and reactivity are interpreted in terms of charge and orbital distributions, and localized molecular orbitals have been derived by the method of Boys.


Spin equilibrium and quaternary structure change in hemoglobin A. Experiments on a quantitative probe of the stereochemical mechanism of hemoglobin cooperativity. The molecular mechanism of hemoglobin cooperativity was studied kinetically by flash photolysis on mixed-state hemoglobins which consist of three ferrous carboxy subunits and one hybrid ferric subunit including fluoromet, azidomet, cyanatomet, and thiocyanatomet. The effects of conformational transitions on the hybrid subunit were detected by kinetic absorption spectroscopy after the CO was fully photodissociated from the binding sites by a large pulse of light from a tunable dye laser. The hemoglobin conformational transition rate was observed to depend on its state of ligation. At 22 degrees C, pH 7, and 0.1 M phosphate, the deoxy R yields T conformational change rate is 4 x 10(4)s-1. The rate decreases to 1.4 x 10(4)s-1 for singly ligated hemoglobin. The R yields T conformation change alters the energy separation between high- and low-spin states for azidomet, cyanatomet, and thiocyanatomet subunits by about 700, 300, and 300 cal/mol, respectively. There are two possible implications of this result: (1) the iron atom spin state is not the only major factor in the determination of its position with respect to the heme plane or (2) the change with conformation of the protein force exerted by the proximal histidine on the iron atom (for an iron to heme-plane displacement of less than 0.3 A) is less than 50% of that expected from simple models in which this motion is responsible for cooperativity.


Decarboxylation of bovine prothrombin fragment 1 and prothrombin. Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at 110 degrees C for several hours. The fully decarboxylated fragment 1 product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of 2 mM Ca2+. There is no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated fragment 1 species generated by heating for shorter periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the 10 gamma-carboxyglutamic acid residues in fragment 1, including both high- and low-affinity metal ion binding sites. Prothrombin itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.


Interaction of local anesthetics with Torpedo californica membrane-bound acetylcholine receptor. The effects of local anesthetics on the rate of the agonist-induced increase in ligand affinity of membrane-bound acetylcholine receptor from Torpedo californica were examined. The rate of the transition in receptor affinity was determined by following the time-dependent increase in inhibition of iodinated alpha-bungarotoxin binding caused by 1 microM carbamylcholine. At concentrations below those that directly inhibited the binding of iodinated alpha-bungarotoxin, dibucaine increased the rate of the transition to a high-affinity state and tetracaine decreased this rate. The measured rate constants were 0.026 +/- 0.008 s-1 in the presence and 0.010 +/- 0.002 s-1 in the absence of dibucaine while tetracaine decreased the rate to 0.006 +/- 0.002 s-1 as compared to a control value of 0.012 +/- 0.003 s-1. A parallel was observed between the effectiveness of a compound in increasing or decreasing the rate of the agonist-induced transition in affinity and the change in its apparent inhibition constant in the presence of carbamylcholine (increase or decrease) measured by the displacement of tritiated perhydrohistrionicotoxin. This parallel could be explained by assuming (a) that local anesthetics bound directly to the specific histrionicotoxin binding site or (b) that they bound to a different site and the observed effects were caused by conformational changes.


Physical studies of cell surface and cell membrane structure. Determination of phospholipid head group organization by deuterium and phosphorus nuclear magnetic resonance spectroscopy. Phospholipid head group conformations in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), DPPC-cholesterol, and DPPE-cholesterol dispersions, in excess water above the pure lipid gel to liquid-crystal phase transition temperature, have been calculated by using comparisons between experimental 2H and 31 P NMR spectral parameters and theoretical results obtained from a plausible model of head group motions. The new calculations are compared with results obtained in previous studies [Seelig, J., Gally, H. U., & Wohlgemuth, R. (1977) Biochem, Biophys. Acta 467, 109--117; Brown, M. F. & Seelig, J. (1978) Biochemistry 17, 381--384; Seelig, J., & Gally, H. U. (1976) Biochemistry 15, 5199--5204] and are shown to agree qualitatively under certain highly restrictive conditions. Under more general conditions, it is shown that many possible solutions are generated but that these may often be separated into a small number of likely conformations in which the head group torsion angles are restricted to specific ranges rather than to a discrete set of values. There is no NMR evidence, however, to support the notion that there are only single conformational solutions to the NMR measurements for the above phospholipid systems.


Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate. The steady state. Ammonia is known to inhibit the steady-state rate of oxidation of L-glutamate catalyzed by glutamate dehydrogenase. We reported previously [Brown, A., Colen, A. H., & Fisher, H. F. (1978) Biochemistry 17, 2031] kinetic evidence supporting the formation in the initial rapid phase of a complex which is composed of enzyme, reduced coenzyme, alpha-ketoglutarate, and ammonia. We show here that the effects of ammonia on the steady-state reaction can be correlated with transient-state kinetic effects related to the concentration of that ammonia-containing complex. These results indicate the existence of alternate reaction pathways which become important at high ammonia concentrations. These new pathways provide an additional route for the release of NADPH from the enzyme surface. The expanded mechanism shows that the noncompetitive product inhibition by ammonia can occur without the simultaneous presence of ammonia and L-glutamate on the enzyme. This mechanism also accommodates the observed substrate inhibition by L-glutamate.


Salt-induced conformational changes in skeletal myosin light chains, troponin-C, and parvalbumin. Evidence for salt-induced changes in myosin light chains [dissociated by treatment with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)], troponin-C (TnC), and parvalbumin was obtained from chymotryptic digestion, circular dichroism, fluorescence, and difference absorption studies. High salt (0.6 M NaCl) protects the DTNB light chain from proteolysis, increases its alpha-helical content, and quenches the tryptophan fluorescence. These effects are similar to the changes induced by Ca2+ but smaller in magnitude. TnC is affected by monovalent cations in a similar manner. Changes in the alpha-helical content resemble the effect of Ca2+. The enhancement of tyrosine fluorescence reflects conformational changes in the Ca2+-Mg2+ binding sites. The increase in the fluorescence of dansylaziridine-labeled TnC suggests perturbation of Ca2+-specific sites by salt. Cancellation of this effect by Mg2+ binding to the high-affinity sites is indicative of site-site interactions. In Whiting parvalbumin, salt-induced a perturbation of tryptophan absorption similar in nature to the Ca2+ effect.


Visible light-induced DNA crosslinks in cultured mouse and human cells. Cool-white fluorescent light induces crosslinks in DNA when proliferating cells are exposed at 37 degrees C for 20 h to 4.6 J/m2/s in culture medium supplemented with fetal bovine serum. Using the Kohn alkaline elution technique, we now find that: 1. Increased light intensity increases DNA crosslinks. 2. The crosslinking is medium-mediated. 3. Oxygen enhances the crosslinking. 4. The extent of crosslinking is decreased at high cell density. 5. The crosslinks can be removed by digestion with proteinase K (0.02 to 0.50 mg/ml). 6. Human cell lines including those derived from adult prostate, fetal lung (IMR-90) and mixed fetal tissues are susceptible to light-induced crosslinks. 7. Crosslinkage is not decreased by addition of catalase to the medium and the effective wavelength is probably between 450 nm and 490 nm. From these results we conclude that the mechanism of light-induced crosslinks differs from that of light-induced chromatid breaks and that the major lesion observed is protein-DNA cross-linkage rather than DNA strand breaks.


Metabolism of O6-alkyldeoxyguanosines and their effect on removal of O6-methylguanine from rat liver DNA. O6-Methyldeoxyguanosine and O6-ethyldeoxyguanosine are weak inhibitors (of approximately equal potency) of the removal of O6-methylguanine from methylated DNA by a rat liver enzyme in vitro. When administered to rats, O6-ethyldeoxyguanosine retarded the removal from liver DNA of the O6-methylguanine which had been produced by pretreatment with dimethylnitrosamine, but the effect was short lived. O6-Methyldeoxyguanosine was much less effective. When cells in culture were grown in a medium containing radioactive O6-methylguanine or O6-methyldeoxyguanosine there was negligible incorporation of the methylated base into DNA, but substantial conversion to guanine which was incorporated. When these substances were injected into rats after partial hepatectomy, a very small incorporation of O6-methylguanine into DNA apparently occurred. Both O6-ethyldeoxyguanosine and O6-methyldeoxyguanosine were dealkylated by rat liver extracts, but the methylated derivative was metabolized much more rapidly. O6-Methylguanosine and O6-ethylguanosine were also dealkylated by rat liver extracts, but the corresponding bases were not attacked. This reaction was probably carried out by the adenosine deaminase in the extracts because it could be prevented by addition of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent adenosine deaminase inhibitor, and could also be effected by purified calf intestinal adenosine deaminase. The Km for the demethylation of O6-methyldeoxyguanosine by calf intestinal adenosine deaminase was comparable to that for adenosine, whereas the Km for O6-ethyldeoxyguanosine was ten times greater. The V for O6-methyldeoxyguanosine was about 11% that for adenosine, but that for O6-ethyldeoxyguanosine was only 0.3%. The higher Km and the slower V for O6-ethyldeoxyguanosine may contribute to the slower dealkylation of this nucleoside by liver extracts and could account for its greater effect on slowing O6-methylguanine excision from DNA in vivo.


RNA synthesis and RNA polymerase activities in germ cells of developing rainbow trout testis. Spermatogenesis is a complex developmental process which sequentially generates several different germ cell types. These cell types from rainbow trout (Salmo gairdnerii) testis were separated by sedimentation in serum albumin gradients and characterized on the basis of their physical properties, chronological appearance, and protein synthesis. The rate of RNA synthesis, the types of RNA made, and the RNA polymerase activities present were determined for each cell type. The rate of RNA synthesis decreased from a high level in spermatogonia and spermatocytes to a low level in early spermatids and was absent in late spermatids and mature spermatozoa. Newly synthesized RNA in spermatogonia and spermatocytes consisted of a variety of molecular weight species, including 18 S and 28 S ribosomal RNAs. The synthesis of high molecular weight RNAs, especially ribosomal RNAs, decreased drastically in early spermatids, leading to the synthesis of only small molecular weight RNAs. RNA polymerase I and II were present in all cell types but the activities of both showed large decreases between spermatocytes and middle spermatids. Both RNA polymerase activities were almost absent from spermatozoa. The activities of RNA polymerase I and II from unfractionated testis cells at different stages of hormone-induced spermatogenesis were quantitated by fractionation of the solubilized extract on DEAE-cellulose. Both polymerases showed major decreases in activity which began near the chronological mid-point of development. For polymerase I the decrease in activity was over 400 fold, for polymerase II over 200 fold. The number of RNA polymerase II molecules per testis cell, quantitated by the binding of [3H]amanitin to cell extracts, also decreased markedly during spermatogenesis. The reduction in polymerase II activity was accompanied by a parallel 200-fold decrease in[3H]amanitin binding. The reduction in polymerase activity appears, therefore, to be due to an actual reduction in the cellular content of RNA polymerase II molecules. These results suggest that transcription in maturing testes is regulated, at least in part, by the concentrations of the RNA polymerases.


Initiation of mammalian protein synthesis. The multiple functions of the initiation factor eIF-3. The protein synthesis initiation factor eIF-3 (a multicomponent protein complex) was labelled with 32P by phosphorylation with a protein kinase present in a partially purified 'hemin-controlled repressor' preparation. The interaction of the labelled factor with the 40 S ribosomal subunit during the course of initiation was followed. It binds to the 40 S subunit in the absence of other initiation factors and inhibits the Mg2+-dependent reassociation of the 40 S with the 60 S ribosomal subunit. It stimulates the binding of the ternary complex (eIF-2, GTP, Met-tRNAf) to the 40 S subunit, and earlier work (Trachsel, H., Schreier, M.H., Erni, B. and Staehelin, T. (1977) J. Mol. Biol. 116, 745-767) also showed it to be essential for the subsequent binding of mRNA. The factor is released from the 40 S initiation complex during the 60 S subunit joining reaction.


Chromosome diminution in Ascaris suum. Two-fold increase of nucleosomal histone to DNA ratios during development. The swine intestinal nematode, Ascaris suum, eliminates chromatin material from its primordial somatic cells during early embryogenesis. A technique for isolation of nuclei from pre- and post-diminution stage embryos has been developed and these isolated nuclei were used in investigations of nuclear events during diminution. The amount of DNA per nucleus determined by diphenylamine assays and isotope dilutions was 0.66 pg and 0.29 pg in pre- and post-diminution nuclei, respectively. Thus, A. suum loses 56% of its nuclear DNA during diminution. The loss of nuclear DNA enabled in vivo examination of histone to DNA ratios as a function of changes in DNA quantities. Ascaris histones were identified by acid extractability and tryptic fingerprint comparison with rat liver histones. Measurement of histone quantities was accomplished using linearity of Coomassie blue binding to histones separated in dodecyl sulfate gels. Ascaris nucleosomal histones levels were relatively constant in pre- and post-diminution nuclei. However, nucleosomal histone to DNA ratios approximately doubled during diminution.


Nuclear protein kinase activities during the cell cycle of HeLa S3 cells. To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S3 cells, a nuclear phospho-protein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.


Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures. Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.


Chromatin condensation. Possible dehydrating and stabilizing factors. The effect of Na+, Mg2+, spermidine and spermine on the dehydration of chromatin gel and precipitation of soluble chromatin has been compared. Considerable differences have been found in the relative ratios within the studied group (Na+, Mg2+, spermidine and spermine) between the ability to dehydrate (1 : 32 : 53 : 67) and to precipitate (1 : 53 : 800 : 2000) chromatin. On the basis of the dependence of precipitation on initial chromatin concentration it has been suggested that the observed effect as contributed considerably by interparticle aggregation is a relatively good measure of the ability of cation to stabilize higher order structures of chromatin through direct crosslinking or induction of hydrophobic associations at selected sites. In contrary to that the method estimating the direct dehydration measures the overall dehydrating effect of a cation exerted on the whole chromatin. It has been suggested on the basis of the above comparative data that the in vivo regulation of the degree of overall chromatin hydration should occur through changes in concentration of free small inorganic cations. Larger organic polycations like polyamines should be mainly involved in stabilization of the higher order chromatin structures. The stabilizing role of large polyanions like RNA has been ruled out. It has also been found that the unwinding of chromatin DNA results in considerable chromatin hydration.


The biosynthesis of nitrogenase MoFe protein polypeptides in free-living cultures of Rhizobium japonicum. The biosynthesis of the constituent polypeptides of nitrogenase component I (Rj 1) in free-living cultures of Rhizobium japonicum (strain 110) was investigated under different growth conditions. Cells were pulse-labelled and the proteins analysed by one and two-dimensional gel electrophoresis. The positions of the constituent Rj 1 polypeptides were identified by co-electrophoresis with purified Rj 1 isolated from bacteroids of soybean nodules, and by comparison with an immunoprecipitate from a culture induced for nitrogenase. The synthesis of the proteins preceded any detectable enzyme activity and increased with time, reaching a maximum after 3 days. At this time, between 6 and 8% of the total sodium dodecyl sulfate-soluble protein synthesized was Rj 1. Exposure to air led to a dramatic decrease in the rate of Rj 1 synthesis, with almost complete regression after 20 min. In the presence of KNO3, there was no nitrogenase activity, but the proteins were present in similar amounts (7%) as the control culture. When mannitol and glycerol were used as the sole carbon sources, the amount of Rj 1 synthesized was extremely low.


9-beta-D-Arabinofuranosyl-8-n-butylaminoadenine, a C-8 substituted nucleoside in the anti conformation. Crystallographic and NMR studies. The protein NMR spectrum of 9-beta-D-arabinofuranosyl-8-n-butylaminoadenine shows an unusually low-field 5'-hydroxyl proton resonance, which has been interpreted in terms of an anti glycosidic conformation together with an 05' ... N8 intramolecular hydrogen bond. Confirmatory evidence for this was obtained by an X-ray crystallographic study; in the crystal, the glycosidic angle chi is 52.7 degrees and the sugar pucker is C3' endo-C4' exo.


Similar distributions of repaired sites in chromatin of normal and xeroderma pigmentosum variant cells damaged by ultraviolet light. Excision repair of damage from ultraviolet light in both normal and xeroderma pigmentosum variant fibroblasts at early times after irradiation occurred preferentially in regions of DNA accessible to micrococcal nuclease digestion. These regions are predominantly the linker regions between nucleosomes in chromatin. The alterations reported at polymerization and ligation steps of excision repair in the variant are therefore not associated with changes in the relative distributions of repair sites in linker and core particle regions of DNA.


Metabolism of prostaglandins, prostaglandin analogs and thromboxane B2 by lung and liver microsomes from pregnant rabbits. Liver microsomes from pregnant rabbits converted prostaglandins F2 alpha, E1, and E2 to their 20-hydroxy metabolites along with smaller amounts of the corresponding 19-hydroxy compounds. Prostaglandins E1 and E2 were also reduced to prostaglandins F1 alpha and F2 alpha, respectively, and prostaglandin E1 was isomerized to 8-isoprostaglandin E1. The above products were also identified after incubation of prostaglandins with liver microsomes from non-pregnant rabbits. In this case, the yield of 20-hydroxy metabolites was much lower. Thromboxane B2 and a number of prostaglandin F2 alpha analogs were also hydroxylated by lung and liver microsomes from pregnant rabbits. The relative rates of hydroxylation by lung microsomes were: prostaglandin E2 approximately prostaglandin F2 alpha approximately 16,16-dimethylprostaglandin F2 alpha approximately 13,14-didehydroprostaglandin F2 alpha greater than thromboxane B2 greater than 15-methylprostaglandin F2 alpha approximately 17-phenyl-18,19,-20-trinorprostaglandin F2 alpha approximately ent-13,14-didehydro-15-epiprostaglandin F2 alpha. Similar results were obtained with liver microsomes except that thromboxane B2 was a relatively poorer substrate for hydroxylation.


Effect of even- and odd-numbered saturated fatty acids on glycerolipid synthesis in rat liver slices. Effect of even- and odd-numbered saturated fatty acids, ranging from lauric to stearic acids, was studied on the de novo synthesis of glycerolipids in rat liver slices. For all fatty acids tested, a marked synthesis of saturated glycerolipids was observed except for phosphatidylethanolamine. When compared at the fixed concentration (2 mM), myristic acid caused a peak synthesis of saturated glycerolipids, and the presence of longer or shorter even- and odd-numbered fatty acids resulted in their lesser formation. The formation of saturated species of triacylglycerol and phosphatidylcholine closely followed the mode of synthesis of saturated diacylglycerols, though dipentadecanoyl-and dipalmitoylglycerols appeared to be less converted to the corresponding saturated triacylglycerols in comparison to the other saturated diacylglycerols. Very little formation of saturated diacylglycerols occurred when lauric, tridecanoic and stearic acids were tested. The majority of lauric and tridecanoic acids incorporated into saturated diacylglycerols was shown to be chain-elongated prior to esterification.


Biosynthesis of triacylglycerols in the intestines of rainbow trout (Salmo gairdnerii) fed marine zooplankton rich in wax esters. Intestinal preparations from rainbow trout fed a diet rich in wax esters incorporated [1(-14)C]hexadecanoic acid and [1(-14)C]hexadecanol into triacylglycerols at the same rate. The ratio of the number of H atoms from C1 of hexadecanol to the number of molecules of hexadecanol incorporated into triacylglycerols was 1.6 : 3.0. [U-14C]Glucose was incorporated much faster into the glycerol moiety of triacylglycerols than was [U-14C]aspartic acid. We conclude that the oxidation of absorbed fatty alcohol to fatty acid and its subsequent incorporation into triacylglycerols is closely linked with the reductive formation of triacylglycerol-glycerol from glucose. The ability of trout intestines to metabolise fatty alcohol to triacylglycerols was the same in fish fed wax esters as in those fed triacylglycerols.


Decreased incorporation of L-[U-14C]serine into phosphatidylserine by polymorphonuclear leukocytes during phagocytosis. A decreased rate of L-[U-14C]serine incoroporation into phosphatidylserine of polymorphonuclear leukocytes exposed to starch granules was observed. L-[U-14C]serine uptake was also depressed under identical conditions. The degree of reduction in specific radioactivity of phosphatidylserine was parallel to that of L-[U-14C]serine uptake. Both uptake and efflux of 45Ca2+ were enhanced in cells with starch granules, but no significant change in cellular calcium levels was detected. These results suggest that the reduced L-[U-14C]serine incorporation into phospholipids may be attributable to decreased availability of this amino acid. The involvement of Ca2+ fluxes in phosphatidylserine synthesis in intact leukocytes cannot, however, be excluded.


Identification of phosphatidyl-1-aminopropane-2-ol in rat liver after administration of 1-aminopropane-2-ol. Phosphatidyl-1-aminopropane-2-ol has been detected in rat liver after intraperitoneal administration of 1-aminopropane-2-ol hydrochloride. The new glycerophospholipid was chromatographically identified by comparison with chemically and enzymatically synthesized phosphatidyl-1-aminopropane-2-ol and by gas-liquid chromatography of the polar group as trifluoroacetyl derivative.


Characterisation of cadmium-binding proteins from mussels, Mytilus edulis (L), exposed to cadmium. Three Cd2+-binding proteins have been purified and partially characterised from the digestive gland of the bivalve mollusc, Mytilus edulis, after exposure to Cd2+. The major protein, which was judged to be pure on polyacrylamide gel electrophoresis, showed many of the characteristics of mammalian metallothionein; having a high -SH content, few aromatic amino acids and a high A250/A280 nm ratio which disappears on acidification. It also contains Zn and Cu, but differs in its higher apparent molecular weight of about 25 000 and high glycine content (12-19%). The two additional Cd2+-binding proteins had lower cysteine contents and different molar proportions of Cd2+, Zn2+ and Cu2+.


The occurrence of cadmium in sub-cellular particles in the kidney of the marine mussel, Mytilus edulis, exposed to cadmium. The use of electron microprobe analysis. In mussels (Mytilus edulis) chronically exposed to cadmium, 85% of the Cd2+ was found to be associated with membrane-limited granular structures when elemental analyses were carried out on cryo-sectioned tissue by electron probe X-ray microanalysis. These granules also contained high concentrations of sulphur and phosphorus as well as other metalions, including Ca2+, iron and Zn2+. In contrast, after homogenisation and fractionation by differential centrifugation, the major proportion of the Cd2+ was found in the cytoplasmic fraction. However, many lysosomes were also ruptured by this treatment. Gel filtration chromatography of this fraction indicated the presence of a Cd2+-binding component of similar molecular weight to the metallothionein purified from the digestive gland of the same animals. It is therefore proposed that metallothionein may be associated with particulate structures which would thus reduce its cellular toxicity.


High magnetic field Mössbauer studies of deoxymyoglobin, deoxyhemoglobin, and synthetic analogues. Mössbauer spectra of deoxymyoglobin, deoxyhemoglobin, and the synthetic analogues, iron (II) 2-methylimidazole meso-tetraphenylporphyrin, and iron (II) 1,2-dimethylimidazole meso-tetraphenylporphyrin have been observed in high magnetic fields and over a wide range of temperature. At temperatures greater than 20 K all materials exhibit remarkably similar spectra, with anisotropic internal magnetic fields decreasing as 1/T. All have negative quadrupole interaction, and both this and the magnetic anisotropy imply that the orbital of the odd electron is prolate in the ground quintet, with little unquenched orbital angular momentum. At 4.2 K the spectra differ, suggesting different detailed structure within the quintet. In contrast to the proteins, the 2-methyl model exhibits spectra at 4.2 K which imply that the lowest spin state has high susceptibility in a single direction.


Conformational studies on somatostatin. II. The C-terminal hexapeptide fragment. The results of a conformational study on the C terminal hexapeptide of Somatostatin are presented. Semi-empirical energy calculations and high resolution NMR methods have been used to obtain information on the conformational properties of SRIF9-14 in [2H6]dimethylsulfoxide and 2H2O. It is concluded from the energy calculations that the peptide has an averaged conformation in which semi extended and folded structures are important. Only some of the folded conformations can explain the chemical shift differences between the amino acid residues Thr10 and Thr12 as a ring current shift by the Phe11 aromatic ring on Thr10. The nonequivalence is more pronounced in dimethyl-sulfoxide (0.23--0.15 ppm) where it decreases with increasing temperature towards the temperature independent value in 2H2O (0.03 ppm). This suggests that the folded conformations are somewhat predominant in dimethylsulfoxide solutions. In 2H2O the semi extended and folded structures are statistically equally important and the peptide is more flexible. A comparison with a study on the smaller fragments SRIF10-12 and SRIF10-13 which have similar conformational properties, demonstrates the usefulness of the fragment approach in conformational studies of peptides.


Human adenosine deaminase. Stoichiometry of the adenosine deaminase-binding protein complex. In many human tissues adenosine deaminase exists as a large molecular weight complex (large form) composed of adenosine deaminase and an adenosine deaminase binding protein. The molar ratio of adenosine deaminase to binding protein in this large form complex appears to be 2:1, respectively, based on several observations. Scatchard-type analysis of the binding of 125I-labeled adenosine deaminase to purified binding protein indicates that 2.15 mol of adenosine deaminase are bound to 1 mol of binding protein. Chemical cross-linking of 125I-labeled adenosine deaminase-binding protein complex (large form) with glutaraldehyde produces 6 cross-linked species with molecular weights consistent with the proposed 2 to 1 stoichiometry. Sedimentation equilibrium analyses reveal a native molecular weight of 300 890 for the adenosine deaminase-binding protein complex (large form), 37 500 for small form adenosine deaminase, and 213 300 for the binding protein. A 2:1 molar ratio of adenosine deaminase and binding protein in the large form complex is most consistent with these molecular weight estimates.


Characterization of a yeast phase specific protein from a fungus, Histoplasma capsulatum. We have characterized an unusual yeast phase specific protein from Histoplasma capsulatum. The protein, which we have called protein 6, is produced by the yeast cells which have been derepressed for sulfite reductase, and it can account for more than 40% of the total extract protein. Synthesis of both sulfite reductase and protein 6 is subject to cysteine repression. However, sulfite reductase activity is maximal in logarithmically growing cells whereas protein 6 is synthesized de novo and accumulated by stationary phase cells. The following are the major physicochemical properties of protein 6: (1) the native protein has a molecular weight of about 15 000; (2) electrophoresis on a sodium dodecyl sulfate polyacrylamide gel yielded a single band with a molecular weight 7600; (3) protein 6 is capable of reducing the dye, nitroblue tetrazolium, and cytochrome c, a property that has been found to be shared by a number of trypsin inhibitors, and (4) the molecule is negatively charge and is relatively resistant to proteolysis. The amino acid composition of protein 6 has been determined.


Intermolecular cross-links in collagen of human placenta. 1. There are four reducible cross-links in human placenta collagen. The principal one is dehydrodihydroxylysinorleucine. the next in abundance is dehydrohydroxylysinorleucine. The minor components are dehydrolysinonorleucine and dehydrohistidinohydroxymerodesmosine. 2. With increasing length of gestation, the collagen incorporates more tritium radioactivity into its reducible cross-links, with its maximum incorporation at the second trimester. The uptake declines thereafter. 3. Alkaline hydrolysis reveals that both dehydrodihydroxylysinonorleucine and dehydrohydroxylysinonorleucine occur in vivo partly as glycosylated complexes.


Resolution of the major hemorrhagic component of Trimeresurus flavoviridis venom into two parts. 1. The major hemorrhagic component (HR1) in the venom of Trimeresurus flavoviridis was purified further by gel filtration on Sephadex G-200, superfine, resulting in its resolution into two parts, 1A and 1B. 1A possessed proteolytic activity towards casein, while 1B was almost free from such activity. Both components were associated with lethal toxicity. 2. The purified preparations of 1A and 1B were homogeneous as judged by several criteria. The molecular weights of the purified principles determined by dodecyl sulfate gel electrophoresis were approximately 60 000. 1A shows anomalous behaviour on ultracentrifugation and gel filtration owing to concentration-dependent polymeric interaction. The purified components were acidic glycoproteins with isoelectric points of 4.4 and they contained neutral sugar, amino sugar and sialic acid altogether amounting to 17-18% on the total weight basis. 3. The two hemorrhagic components were closely related, if not identical, immunologically.


Formation of nuclei during delay time prior to aggregation of deoxyhemoglobin S in concentrated phosphate buffer. The delay time prior to aggregation of deoxyhemoglobin S was found to be shortened significantly if monomeric deoxyhemoglobin S was added near the end of the expected delay time. This suggests the formation of a preaggregation complex of deoxyhemoglobin S, probably so-called "nuclei", near the end of the delay time. In fact, the molecular weight of deoxyhemoglobin S as measured by a low angle laser light scattering photometer increased exponentially during the delay time prior to the visible aggregation of deoxyhemoglobin S. These results support the nucleation-controlled aggregation mechanism for the aggregation of deoxyhemoglobin S in concentrated phosphate buffer.


The amino acid sequence of the phospholipase A2 isoenzyme from porcine pancreas. The primary structure of porcine pancreatic isophospholipase A2 (EC 3.1.1.4) has been investigated. The sequence of procine isophospholipase differs from the sequence of porcine phospholipasy by four substitutions; viz. Ala12 leads to Thr; His17 leads to Asp leads to; Met20 leads to Leu and Glu71 leads to Asn.


Proton NMR study of model substrate binding in hemoproteins. Intercalation of mercuric triiodide in sperm whale met-aquo myoglobin. 1H-NMR spectra have been recorded for sperm whale met-aquo myoglobin intercalated with xenon, cyclopropane, mercuric triiodide and auric triiodide. All four agents are known to intercalate on the proximal side of the heme over pyrrole A. The complexes of xenon and cyclopropane exhibit insignificant shifts for all four heme methyls, suggesting that these species fit into an existing hole without causing significant perturbations on the heme cavity. Mercuric and auric triiodide, on the other hand, induce substantial changes in the hyperfine-shifts for the heme methyls. Based on the previously assigned methyl peaks in met-aquo myoglobin, we find that methyl-1, closest to the intercalating agent, is affected most, with the influence decreasing with distance from the binding site. These results indicate that determination of the relative perturbations on the assigned heme methyl shifts due to substrate binding can be utilized to ascertain the substrate-heme stereochemistry in high-spin ferric hemoproteins such as peroxidases.


Protamines and related proteins from spermatozoa of molluscs. Characterization and molecular weight determination by gel electrophoresis. The spermatozoa of most species of molluscs contain a mixture of proteins with compositions related to those of histones and protamines. The various components present in the spermatozoa of Cryptochiton stellerii, Donax trunculus, Mactra corallina and Gibbula divaricata have been isolated and characterized. The results obtained, taken together with those previously found (Subirana, J.A., Cozcolluela, C., Palau, J. and Unzeta, M. (1973) biochim. Biophys. Acta 317, 364--379), show that in all the molluscs studied one or more arginine-rich components are present. The molecular weight of these proteins varies vary much in different species and is usually much greater than in conventional fish protamines. Conventional histones, as well as lysine-rich proteins of low molecular weight, have also been found in ripe spermatozoa of several species. The molecular weights have been estimated by gel electrophoresis, using polymerized iridine as a standard.


Purification and characterization of 33 kilodalton protein of spinach chloroplasts. A protein was prepared from spinach chloroplasts in a highly purified form. The isoelectric point of the protein was 5.2. The apparent molecular weight was estimated to be 33 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea, and 34 000 by gel filtration column chromatography with Sephadex G-100. The protein was provisionally named '33 kilodalton protein' according to the molecular weight. The absorption spectrum of the protein did not show any absorption band in the visible region. No histidine was found in the amino acid analysis of the protein. The 33 kilodalton protein was released from the thylakoid membrane by EDTA-treatment and also by sonic oscillation. The protein was bound to System II particles, but not to System I particles.


Stability of fibronectin biological activity following chemical modification. Fibronectin isolated from human plasma functions in vitro as a mediator of adhesion and spreading of trypsinized fibroblasts on native or denatured collagen. As a means of elucidating structural characteristics which might contribute to fibronectin's biological activity, we have modified and digested the protein with several chemicals. Following various treatments, the protein was utilized to mediate cell adhesion and spreading on collagen to determine which alteration disrupted its activity. Fibronectin remained functionally intact after partial or complete reduction and alkylation, oxidation of 59% of the carbohydrates with sodium periodate, citraconylation, carbodiimide-catalyzed amide formation, and oxidation of 35.2 residues of tryptophan/molecule with N-bromosuccinimide. Dinitrofluorobenzene treatment, which phenylated ten residues/molecule of fibronectin, successfully inactivated fibronectin's in vitro biological function. Effective modification of the protein was determined by appropriate analytical procedures. Since fibronectin retained its biological function after several treatments that presumably affected its molecular conformation, we concluded that its secondary or tertiary structure appears not to be essential for its in vitro activity, or alternatively that the protein possesses a biologically active domain relatively resistant to chemical modification.


Isolation and characterization of hydroxyproline-containing proteins secreted by a murine carcinoma cell culture. A collagenous protein was isolated from a murine carcinoma cell culture, which has been shown to synthesize basement membrane. The molecular weight of this protein was estimated to be 155 000. It eluted from carboxymethyl-cellulose in the region near the alpha 1 and beta 11 components of calf skin collagen. 63--69% of the peptide-bound prolines were hydroxylated, and the 4-/3-hydroxyproline ratios ranged from 12 : 1 to 14 : 1. About 95% of the hydroxylysines in the peptide were glycosylated, and almost all of them were in the glucosylgalactosyl dissacharide form. Judging from the posttranslational characteristics, this collagenous protein is probably of basement membrane type.


The relative affinity of transferrin and albumin for zinc. The relative affinity of transferrin and albumin for zinc has been measured by competitive dialysis at a low zinc concentration in 0.15 M NaCl, 50 mM HEPES, 0.1 mM trisodium citrate (pH 7.2). There were small differences between albumins and larger ones between transferrin preparations, but all albumins bound zinc more firmly than any transferrin did. It is known that transferrin is largely responsible for the uptake of zinc from an intestinal membrane in rats, but much of the metal is subsequently transferred to albumin. The current results show that both in humans and in rats (a) no special mechanism is needed to provide energy for this transfer, and (b) full equilibration would lead to virtually complete transfer in contrast with what actually occurs in vivo.


Specific sulfonation of tyrosine, tryptophan and hydroxy-amino acids in peptides. 1. ClSO3H in trifluoroacetic acid rapidly converts serine and threonine into O-sulfate ester derivatives while tyrosine and tryptophan are converted into arylsulfonic acids. 2. H2SO4 in trifluoroacetic acid reacts more slowly with serine, threonine and tyrosine while is not able to modify tryptophan. 3. All other amino acids are perfectly stable under the above reaction conditions. 4. Peptides containing susceptible amino acid residues are specifically converted into the corresponding sulfonated derivatives in high or quantitative yield.


The presence of gamma-carboxyglutamic acid-containing protein in atheromatous aortae. It has been established that a gamma-carboxyglutamic acid-containing protein is present in rat aortae after long term atherogenic diet administration. A similar protein was proven to be present in turkey tibial tendons that are predisposed to undergo physiological calcification. The molecular weight and amino acid composition of both proteins were identical. They contained six glutamic acid residues per molecule, three of which were gamma-carboxylated. The proteins studied were also identical in their N-terminal sequence over six residues. This sequence was fully coincident with that published for osteocalcin (Price, P.A., Poser, J.W. and Raman, N. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3374--3375). In the region corresponding to residues 20--26 in osteocalcin, a single replacement of valine for isoleucine was found in turkey tendon protein. From the physiological point of view it should be mentioned that the level of the gamma-carboxyglutamic acid containing protein in atherogenic diet fet rat aortae exceeds that found normally in bone or in tissues predisposed for physiological calcification.


Basis of thermostability in pig heart lactate dehydrogenase treated with O-methylisourea. Acetamidination of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with ethyl acetimidate resulted in an increase of thermostability, and covalent bridge formation between pairs of lysine residues is observed. Guanidination with O-methylisourea of the enzyme also increases the thermostability, but such a bridge seems not to be formed. Increased thermostability of guanidinated enzyme is considered to be due to the shift of the pK values of the lysine residues from 10.5 to 12.5 after guanidination. Modification experiments with carbodiimide reveals that the enzyme contains 4.6 pairs of neighboring lysine and carboxyl residues per subunit, and amide bonding between 3.2 pairs results in an increase of thermostability. Guanidination of 4.6 Lys/subunit of the enzyme yields an enzyme derivative with considerably increased thermostability. Salt bridge formation between the 4.6 pairs of neighboring carboxyl and guanidinated lysine residues per subunit might make a major contribution to the increased thermostability of the guanidinated enzyme.


Partial sequence data for the L-(+)-lactate dehydrogenase from Streptococcus cremoris US3 including the amino acid sequences around the single cysteine residue and at the N-terminus. The following amino acid sequence information has been determined for the fructose 1,6-bisphosphate-dependent lactate dehydrogenase from Streptococcus cremoris US3: the C-terminal amino acid, the N-terminal sequence of the first 20 amino acids and the sequence of a 53-residue tryptic peptide containing the only cysteine residue in the protein. The enzyme was cleaved by alkali at the cysteine residue following reaction first with 5,5'-dithiobis(2-nitrobenzoic acid) and then with K14CN. This treatment yielded two cleavage products as well as some higher polymers and some uncleaved enzyme. The radioactive cleavage product was purified and its size indicated that the cysteine residue is 80 residues from the C-terminus. Comparisons of the sequences determined for the S. cremoris enzyme with those already known for dogfish lactate dehydrogenase indicate that the two enzymes are only distantly related since the sequence homology between them is limited and of borderline statistical significance.


The primary structure of the asialo-carbohydrate units of the first glycosylation site of human plasma alpha 1-acid glycoprotein. The elucidation of the structures of the carbohydrate units linked to glycosylation site I of human plasma alpha 1-acid glycoprotein is described. These carbohydrate units can be grouped into compounds with bi- (class A) and triantennary (class B) structures and the triantennary structure with a fucose residue (class BF) (Fig. 1). The structural variability of the carbohydrate units of glycosylation site I and also of glycosylation sites II to V (Fournet, B., Montreuil, J., Strecker, G., Dorland, L., Haverkamp, J., Vliegenthart, J.F.G., Binette, J.P. and Schmid, K. (1978) Biochemistry 17, 5206--5214) accounts largely for the microheterogeneity of alpha 1-acid glycoprotein.


The susceptibility to tryptic hydrolysis of peptide bonds involving epsilon-N-methyllysine. Using isolation of soluble peptides as a means of comparing the efficiency of tryptic hydrolysis of various forms of the phase-1 flagellin of Salmonella typhimurium, it was found that peptides terminating in N-methyllysine required a longer period of exposure to enzyme for their detection than did equivalent peptides terminating in lysine.


N-Terminal homology in three cysteinyl proteases from Papaya latex. Sequences to residue 17 have been determined for the three Papaya cysteinyl proteases, chymopapain and papaya peptidase A and B. Extensive homologies were found for these three enzymes and with papain and bromelain. These results suggest that the five sulphydryl enzymes discussed derive from a common ancestral gene.


Interaction of alpha-actinin, filamin and tropomyosin with F-actin. The abilities of alpha-actinin, filamin and tropomyosin to bind F-actin were examined by cosedimentation experiments. Results indicated that smooth muscle alpha-actinin and filamin can bind to actin filaments simultaneously with little evidence of competition. In contrast, tropomyosin exhibits marked competition with either filamin or alpha-actinin for sites on actin filaments.


Calcium and magnesium binding by parvalbumin. A proton magnetic resonance spectral study. Proton magnetic resonance spectroscopy has been used to monitor the kinetics and nature of the conformational transitions induced by binding of calcium and magnesium to carp parvalbumin. Rate constants have been determined for the exchange between the cation dependent conformational states of the protein in solution. These data enable a description of the kinetics and mechanism controlling the calcium flux in vivo during contraction.


Binding of calcium by fragment 38-108 of pike parvalbumin. Cyanogen bromide splitting of pike parvalbumin III was carried out and fragment 38-108 containing two calcium-binding domains was isolated. The obtained fragment binds calcium rather well (Kd = 1.6.10(-5) M), possesses a secondary structure, its CD spectrum changes after removal of calcium.


NMR studies of primary and secondary sites of parvalbumins using the two paramagnetic probes Gd (III) and Mn (II). The binding of cations by parvalbumins was studied by the proton relaxation enhancement (PRE) method using the paramagnetic probes Gd(III) and Mn(II). Gd(III) appears as a specific probe of the primary sites CD and EF with the following binding parameters: n = 2, KdGd = 0.5 x 10(-11) M and epsilon b = 2.3. The low value of epsilon b is the result of a nearly complete dehydration of the protein bound ions. Competition experiments between Gd(III) and various diamagnetic cations show the following order of affinity for the EF and CD sites: Mg2+ less than Zn2+ less than Sr2+ less than Ca2+ less than Cd2+ less than La3+ less than or equal to Gd3+. Mn 2+ is a specific probe of a secondary site with the following binding parameters: n = 1, KdMn = 0.6 x 10(-3) M and epsilon b = 17. The high value of epsilon b suggests that the protein bound Mn(II) has retained most of its hydration shell. Competition experiments between (Mn(II) and different cations show similar affinities for this site: Ca2+ less than or equal to Mg2+ less than or equal to Cd2+ less than or equal to Mn2+. This secondary site is located near the EF primary site.


The use of amino acid sequence analysis in assessing evolution. The thirteen year history of assessing evolution by amino acid sequence analysis has made apparent the limitations imposed upon this system by the finite nature of the characters. This finiteness exists on several levels and ultimately expresses itself as parallelism, back mutation and the retention of primitive characters in the sequences of proteins from present day species and the putative ancestral protein chains. Sequence analysis shares these problems with other molecular approaches, but because it is concerned both with the nucleotide substitutions in the genome and with the functional roles of proteins, it has unique advantages. For example, the large fluctuation in the rate of fixation of mutations in a protein's evolution can be detected and used to point out the unreliability of any molecular clock for estimating divergence dates. Moreover, when consideration is given to studies which assign functional significance to specific amino acid sites in a protein, changes in function during the descent of a protein can be appreciated and their significance correlated with organismal evolution.