Pigmented lesions in newborn infants.

1058 newborn infants were examined. Forty-one (3-9%) had clinically discernible pigmented lesions compatible with melanocytic naevi. Biopsy was performed on thirty-four of the forty-one and of these; eleven, representing 1-01% of the infants, proved to be melanocytic naevi. No giant (garment) naevi were seen in this series. Two of the eleven naevi pathologically examined showed histological changes similar to those that have been reported in some giant naevi, but the remaining nine were not only different from criteria usually assigned to giant naevi, but they also differed from the usual adult naevi, in that most were predominantly junctional. None of the melanocytic naevi in this series showed any suggestion of malignant change. In newborn infants it is often impossible clinically to distinguish naevi from other types of pigmented lesions, as only eleven out of the thirty-four pigmented lesions were melanocytic naevi. Seven of the eleven melanocytic naevi were under 1-5 cm in diameter. No pigmented lesions were found on the palms, soles or genitalia.


Subcutaneous fat necrosis of the newborn: Light and electron microscopic studies.

Specimens from two cases of subcutaneous fat necrosis of the newborn were studied by light and electron microscopy. Crystallization of the fat cell started with clearing of the central fat droplet, followed by the appearance of cylindrical crystals. The fat crystals were phagocytosed by cytoplasmic projections of macrophages as well as by foreign body giant cells. Calcium deposits were demonstrated within the central fat droplet of the degenerated fat cell of Case 2.


The effects of photosensitizers and ultraviolet irradiation on the biosynthesis and metabolism of prostaglandins.

Prostaglandin biosynthesis from arachidonic acid by skin microsomal fraction preparation was enhanced by UV-irradiation at wavelengths of 254 and 360 nm. In the presence of 8-methoxy psoralen (8 MOP) and coal tar, prostaglandin biosynthesis was further enhanced approximately 2-fold by UV-irradiation at 254 nm. Stimulation was less by UV-irradiation at 360 nm. 8-MOP enhanced the conversion of PGE2 into PGF2-9-ketoreductase prepared from skin high speed supernatant fractions. UV-irradiation at 254 nm and 360 nm with or without the photosensitizers had no effect on the activity of the PGE2-9-ketoreductase. These data therefore indicate that the action of UV-irradiation, 8-methoxy psoralen and coal tar on the skin may in part be due to their regulation of the biosynthesis and metabolism of prostaglandins in this tissue.


Metal corrosion by chloride in sweat. The problem of 'rusters' in industry.

Workers in the precision engineering industry who readily corrode metal are known as 'rusters'. We have described two patients with this condition, and review current ideas with regard to its cause, detection, prevention and treatment.


Proprietary hydrocortisone creams. Vasoconstrictor activities and bio-availabilities of six preparations.

Six proprietary hydrocortisone creams were evaluated for vasoconstrictor activities and bio-availabilities using an occluded blanching test. Statistical analysis showed a significant difference between the formulations. Dioderm, containing 0-1% hydrocortisone, was significantly more active than the 1% hydrocortisone creams--Alphaderm, Calmurid HC, Efcortelan and Vioform-Hydrocortisone. There was no significant difference between Dioderm and Dioderm C. Unlike creams containing more potent corticosteroids the hydrocortisone formulations apparently failed to produce steroid reservoirs in the stratum corneum as assessed by the blanching response.


Disseminated superficial actinic porokeratosis. Analysis of an affected family.

A family is analysed in which disseminated superficial actinic porokeratosis (DSAP) occurred in five members. All three children of one generation are affected. Clinical, histopathological and genetic aspects of DSAP are discussed. Disseminated superficial actinic porokeratosis appears to be a clinical variant of porokeratosis of Mibelli. Since DSAP is a genodermatosis inherited as an autosomal dominant it can occur in any geographical location; however excess sunlight can definitely exacerbate the condition.


Unilateral epidermal naevus resembling acanthosis nigricans.

A man born with a unilateral epidermal naevus on the right side of the abdomen developed at puberty symmetrical benign acanthosis nigricans in the body folds. The symmetrical eruption later disappeared completely. The unilateral epidermal naevus and the symmetrical eruption showed identical histological features, i.e. those of acanthosis nigricans. At the age of 32 he developed a muco-epidermoid cancer of the left parotid gland. A decision whether the association of unilateral epidermal naevi with malignant tumours occurs with unusual frequency requires (a) a long follow-up of the patient with the unilateral naevus, (b) a differentiation of the unilateral eruption from unilateral malignant acanthosis nigricans and (c) statistical evidence.


Alpha-thalassaemia trait in various racial groups in the United Kingdom: characterization of a variant of alpha-thalassaemia in Indians.

Patients whose red-cell indices are suggestive of thalassaemia trait, but who have a normal haemoglobin electrophoretic pattern, may be carriers of alpha-thalassaemia. A diagnosis of alpha-thalassaemia trait was made in 44 such patients, using the incorporation of [3H]leucine by reticulocytes to measure the relative rates of synthesis of the alpha- and beta-chains of adult haemoglobin. Patients with alpha-thalassaemia trait had a reduced rate of synthesis of the alpha-chains, with a mean alpha/beta specific activity ratio of 0.79+/-SD 0.07. The mean alpha/beta specific activity ratio of 20 control subjects was 1.06+/-SD 0.08. The diagnostic value of the haemoglobin H(Hb H) preparation was assessed in proven alpha-thalassaemia heterozygotes of various races. A high proportion of 'false negative' results in Indian and Negro heterozygotes indicated that the Hb H preparations is a highly unreliable screening test for use in a multi-racial population. There was no significant difference in the mean level of Hb A2 in alpha-thalassaemia heterozygotes (2.0+/0SD 0.6) compared with that of the control group (2.1+/0SD0.5). Comparison of data from patients with alpha- and beta-thalassaemia traits showed that alpha-thalssaemia trait is the milder disorder, in terms of its effects of red-cell morphology, red-cell indices and degree of globin chain imbalance. Amongst individual patients with alpha-thalassaemia trait, there was no correlation between the alpha/beta specific activity ratio and the red-blood-cell(RBC) d the red-blood-cell (RBC) count, mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH). This suggests that the alpha/beta ratio cannot be used to distinguish between carriers of a mild gene ('silent carriers') and carriers of a more severe disease. This is the first sutdy to characterize alpha-thalassaemia trait in Indians, in whom it appears to be a common disorder. Haematologically, alpha-thalassaemia trait in Indians is milder than that seen in Chinese and the fact that haemoglobin Bart's hydrops fetalis does not occur in Indians makes it likely that the genetics of Indian alpha-thalassaemia differ from those of the Chinese disease. A possible genetic model for Indian alpha-thalassaemia is discussed and the identification of the homozygote is seen as the first step in the determination of the underlying molecular defect.


Studies of the proporation and synthesis of haemoblogin C Philadelphia in red cells of heterozygotes, a homozygote, and a heterozygote for both haemoglobin G and alpha thalassaemia.

The proportion of Hb G Philadelphia (alpha68-Asn leads to Lys) in heterozygotes has been found to have a well-defined bimodal distribution around means of 33% and 46% Hb G. microcytosis and hypochromia are consistently associated with the latter group, who also have a decreased ratio of alpha/beta-chain synthesis in the peripheral blood, but these characters are not linked to the Hb-Galpha gene, because a parent with microcytosis and 46% Hb Galpha may have offspring with 33% Hb G without significant microcytosis. In one family a subject with Hb G and Hb G2 but no Hb A or Hb A2 is presumably a homozygote for alphaG. This subject has microcytosis and a decreased ratio of alpha/beta chain synthesis. In another family a subject with Hbs H, G and G2 but without Hbs A or A2 is heterozygous for both Hb G and alpha thalassaemia I. These findings are compatible with the hypothesis that the alphaG mutation occurs on a chromosome with only a single alpha-chain locus and that the expression in heterozygotes as 46% or 33% Hb G is determined by the homologous chromosome in trans having either one or two normal alphaA genes respectively. The significance of this polymorphism for chromosomes carrying alpha-chain genes is discussed.


Iron uptake and ferritin synthesis by peripheral blood leucocytes from normal subjects and patients with iron deficiency and the anaemia of chronic disease.

The ferritin content of monocytes, lymphocytes and polymorphs is reduced in patients with iron deficiency anaemia. In patients with the anaemia of chronic disease a reduced serum iron concentration is associated with an increase in the ferritin content of all peripheral blood leucocytes. Iron uptake by all cell types is related to transferrin saturation. In iron deficiency anaemia lymphocyte iron uptake is greatly increased, possibly relfecting intracellular iron depletion. In patients with active rheumatoid arthritis and carcinomatosis there is no alteration in leucocyte iron uptake or ferritin synthesis.


Chelate mediated transfer of iron from transferrin to desferrioxamine.

Desferrioxamine, widely used for the treatment of iron overload in Cooley's anaemia, binds iron so tightly that it should quantitatively remove iron from transferrin. Studies conducted in vivo and in vitro, however, have failed to demonstrate significant depletion of transferrin-bound iron by a stoichiometric excess of desferrioxamine. However, low molecular weight chelating agents, capable of forming ternary complexes with transferrin and ferric iron, can promote a rapid transfer of iron from transferrin to desferrioxamine. A possible mechanism for this facilitated exchange is offered.


Iron absorption in the rat: the search for possible intestinal mucosal carriers.

The biological relevance of four iron-containing fractions previously detected in rat intestinal mucosal cells has been studied. The distribution of iron in these fractions obtained by chromatography on Sepharose 6B has been examined after in vivo and in vitro incubation of mucosal cells with 59Ce. In addition, the effects of phenobarbitone, cycloheximide, iron-deficiency and iron-loading on the uptake and distrubution of iron within the four mucosal cell fractions was studied. The iron in fraction I was mostly bound to intracellular membrane particles. Fraction II was shown to be ferritin. Fraction III contained some transferrin and also a protein of molecular weight similar to transferrin but which was not precipitable by antitransferrin antiserum. Quantited with the results of 'chaser' experiments suggested that, in addition to ferritin, at least two of the fractions (I and III) were involved in the process of iron absorption by the mucosal cell.


Depressed phagocytic function exhibited by polymorphonuclear leucocytes from chronically iron deficient rabbits.

The production of chronic iron deficiency in rabbits was associated with impaired phagocytosis of opsonized Staphylococcus aureus by their polymorphonuclear leucocytes. These cells had a reduced ability to ingest the bacteria and the rate of ingestion could not be stimulated by autologous serum leucophilic gamma-globulin.


A comparison of platelet size, latelet count, and platelet 35S incorporation as assays for thrombopoietin.

Average platelet size, platelet count, and 35S-incorporation into platelets were compared as methods for the measurement of thrombopoietin-stimulated thrombopoiesis. In mice injected with rabbit anti-mouse platelet serum (RAMPS) average platelet size was shown to be increased as mice were recovering from thrombocytopenia. Also, 35S-measurements on platelets of these mice showed significant increases in cpm/average platelet 2-4 days after RAMPS treatment. Significant increases in 35S-incorporation into the total circulating mass of platelets were found on days 3-4. In normal mice or mice in rebound-thrombocytosis injected with thrombopoietin, platelet size remained unchanged, whereas the platelet cound and 35S-incorporation into platelets were shown to be significantly increased. Moreover, a dose-response experiment in mice pretreated with RAMPS showed a slight increase in platelet count as the dose of TSF was increased, but platelet sizes were unaltered. The % 35S-incorporation into platelets showed a significant linear dose-response, i.e. as the dose of thrombopoietin was increased, as increase in % 35S-incorporation into platelets was observed. These data indicated that of the three indirect measurements of thrombopoietin, the % 35S-incorporation into mouse platelets was the most sensitive, followed by platelet counting; the least sensitive measurement of thrombopoiesis was change in platelet size.


Activation of platelets by platelet activating factor (PAF) derived from IgE-sensitized basophils. IV. PAF does not activate platelet factor 3 (PF3).

Platelet activating factor (PAF) derived from antigen-stimulated, IgE-sensitized rabbit basophils acts on platelets to induce aggregation and secretion of their content of granule-bound vasoactive amines. Despite this, PAF did not activate platelet factor 3. In contrast, collagen induced aggregation, secretion and PF3 activation in the washed platelets. Other stimuli (ADP, C3b, thrombin) also initiated both secretion and PF3 activation. A wide dose range of PAC, including those giving maximal secretion and aggregation, were ineffictive in making PF3 available and the possibility that PAF inhibited PF3, or its generation, was also excluded. It is concluded that PAF is a unique stimulus for platelets and that secretion and aggregation are not necessarily accompanied by PF3 generation.


Fragmentation haemolysis in patients with severe diabetic angiopathy.

Haemolytic anaemia associated with prominent red cell fragmentation is described in seven patients with long-standing diabetes mellitus. A common freature in the patients was severe microangiopathy as detected by retinal examination and microscopic examination of the kidneys. Renal or pancreatic islet malfunction per se is not involved in the haemolytic syndrome, since red cell abnormalities persisted in one patient for over a year following successful renal and pancreatic transplantation--this, despite the maintenance of normal renal and carbohydrate homeostasis. The kinetics of fragmentation was sutdied by tranfusing snormal type O cells into this type A patient. With reisolation of these cells by the Ashby-technique, rapid and porgressive red cell fragmentation was demonstrated by: (a) membrane lipid loss; (b) osmotic fragility increase; and (c) increase in mean cell haemoblobin concentration. This studies indicate that a red-cell-fragmentation haemolytic anaemia may occur in long-standing diabetes mellitus, related to the angiopathy of this disease and not to insulin deficiency or renal malfunction.


Binding of thrombin to human platelets and its possible significance.

Thrombin binds tightly to human platelets. The binding reaction is dependent on the thrombin concentration used. At a physiologically significant thrombin concentration, there are about 500 binding sites per platelet with an apparent dissociation constant of 0.02 u/ml. Autoradiography studies of platelets treated with labelled thrombin showed that the thrombin was located on the platelet surface. Separation of the subcellular fractions of platelets treated with labelled thrombin by density gradient centrifugation revealed that the membrane area contained over 80% of the radioactivity initially applied. Furthermore, isolated platelet membranes dind thrombin similar to intact platelets. These data suggest that the receptors for thrombin are located on the platelet membrane. Cytochalasin A, cytochalasin B or prostoglandin E1 did not have any effect on thrombin binding although these agents inhibited platelet aggregation. Thus, binding of thrombin is not sufficient for aggregation of platelets and other steps are involved. These agents do not affect the induction of stimulation but interfere at a later step in the trhombin-platelet interaction. On the other hand, hirudin completely inhibited binding of thrombin to platelets. It appears that the platelet receptor recognizes that part of the thrombin molecule on its surface which hirudin. Binding studies with serotonin loaded platelets showed a close correlation between thrombin binding and the release reaction.


Factor VIII and human platelet aggregation. III. Further studies on aggregation of humna platelets by neuraminidase-treated human factor VIII.

When highly purified human factor VIII is submitted to agarose gel chromatography in the presence of 0.5 M CaCl2, the procoagulant activity (low molecular weight factor VIII, LMW-F VIII) is separated from the void volume protein (Vo-VIII). Upon incubation of human factor VIII with purfied neuraminidase, a very stable platelet aggregating activity develops in the "Vo-VIII' fraction, not in the "LMS-FVIII' part. Evidence is provided that the generated aggregating activity is a property of the 'carrier protein' for LMW-F VIII. Desialylated factor VIII retains its antigenic reactivity, its procoagulant or ristocetin cofactor properties and the capacity of its subunits to dissociate and recombine. Neuraminidase-treated human factor VIII, in contrast to intact bovine factor VIII or intact human factor VIII in the presence of restocetin, does not induce aggregation of EDTA-platelet rich plasma, of congenitally afibrinogenaemic platelet rich plasma, nor of washed platelets.


Plasma prolactin levels during pregnancy.

Plasma prolactin levels in women who were between 8 and 40 weeks pregnant were determined by a homologous double antibody radioimmunoassay method. There were 980 samples from 839 uncomplicated and 213 samples from 116 complicated pregnancies. Prolactin levels in normal pregnancies varied from 6 ng/ml during early pregnancy to 210 ng/ml near term. A few values beyond the normal range were found in various groups of complicated pregnancies. About 14 per cent of samples from patients with threatened abortion and 7 per cent from patients with hypertension were above the upper normal range. The proportion of values below the 10th centile in patients with a low urinary oestrogen excretion was significantly higher than that in the normal population. Plasma prolactin levels did not seem to be a valuable guide to maternal or fetal well-being.


Six years' experience of the prediction of severity in rhesus haemolytic disease.

Prediction charts used in the management of patients suffering from rhesus haemolytic disease are presented and the accuracy investigated for two methods of amniotic fluid analysis. In 468 samples from 141 patients there were 7-9 per cent errors of prediction when both methods were used together but in only four cases (2-8 per cent) was the error thought to have possibly contributed to wrong management.


The effect of oral ritodrine on maternal and fetal carbohydrate metabolism.

The reported growth-promoting effects of the beta-sympathomimetic compound, ritodrine, have been investigated. The carbohydrate tolerance of eight pregnant women was found to be unaffected by treatment with oral ritodrine over a ten-week period. A further observation that the carbohydrate metabolism of the newborn infants of these women was within normal limits tends to discount possibility that any growth-promoting action ritodrine may have on the fetus is mediated through a diabetogenic effect on the mother.


Further clinical evaluation of the selenomethionine uptake test.

A review of the selenomethionine uptake ratio as a measure of placental function is presented. Attempts at the early identification of the poorly growing fetus were unsuccessful, and the previously published close correlation of results with clinical findings in later pregnancy has not been confirmed. The theoretical basis of the test has been re-examined. Although modification much reduced the incidence of technically unsatisfactory readings, other difficulties prevented the test from being clinically useful. To some extent the aim of the test has been superseded by concurrent developments and advances in care of the premature baby.


Measurement of intervillous and myometrial blood flow by an intravenous 133Xe method.

A new 'non-invasive' method for measuring intervillous and myometrial blood flow is presented. After 2 mCi of 133Xe in physiological saline was injected intravenously the patient held her breath for 20 seconds. The tracer entered the placenta as a short bolus and its removal was followed with a scintillation detector. Intervillous and myometrial blood flow per unit volume was calculated from the two-exponential curve. The mean +/-SD intervillous flow in normal pregnancy was 135+/-49 ml/minute/100 ml, the corresponding half time being 0-56+/-0-16 minutes. The myometrial flow was 7-7+/-2-5 ml/minute/100 g. The method gave reproducible results and took only 20 minutes to perform. The dose of radiation to the mother was less than 1 mrad.


Buccal prostaglandin E2 for induction of labour.

The administration of buccal prostaglandin E2 (PGE2) was evaluated in 30 patients; effective uterine stimulation occurred in 90 per cent of those receiving a dose of 1 mg hourly. Two patients had a prolonged contraction on a single occasion during labour. The unpleasant taste of the tablets and a high incidence of nausea and vomiting indicated that buccal prostaglandins had no advantages over other methods of administering oxytocic agents.


Early postpartum tubal ligation under epidural analgesia.

Tubal ligation under epidural analgesia in the early puerperium (within 10 hours of delivery) was attempted in 51 patients who had an epidural block established during labour. The results are described. No major difficulties were encountered.


Altered patterns of thyroid hormones in serum and urine in pregnancy and during oral contraceptive therapy.

Serum total, percentage free fraction and absolute serum free hormone concentration of thyroxine and triiodothyronine were measured in control, pregnant and oral contraceptive users, together with the daily urinary losses of unconjugated thyroid hormones. Increased urinary losses of both hormones, in particular thyroxine, were apparent in pregnancy and these could not be explained in terms either of an increased filtered load of hormone or the presence of proteinuria. The possible existence of filterable small-molecular weight hormone-binding substances in the urine of pregnant patients is discussed. It is concluded that assay of urinary thyroid hormones during pregnancy is of limited diagnostic value because of overlap with thyrotoxic values.


The scanning electronmicroscopy of human cervical mucus in the non-pregnant and pregnant states.

The ultrastructure of the high viscosity phase of human cervical mucus was studied by scanning electronmicroscopy. In the normal woman, sequential mucus samples taken throughout the menstrual cycle showed a honeycomb-like structure with interconnecting channels separated by thin membraneous walls. The diameter of the channels in the mucus was 2 to 6 mum in the early follicular phase, 30 to 35 mum in the late follicular phase and 4 to 6 mum in the luteal phase. During the first trimester of pregnancy and also at the beginning and end of the menstrual cycle a compact, dense form of mucus structure was observed. In women using either copper-releasing or pharmacologically inert intrauterine contraceptive devices (IUCDs), the basic structure was similar to that seen in normal women. The channel diameters were normal in women using the inert IUCD but the mucus from women using a copper-releasing IUCD showed a slight decrease in the late follicular phase channel diameter which was dependent on the time the device had been in place. Globular appendages to the membraneous walls of the mucus were observed in the presence of a copper-releasing IUCD.


Factors influencing absorption of subretinal fluid.

In 200 cases of retinal detachment successfully treated without drainage of subretinal fluid complete reattachment of the retina was achieved in the first postoperative week in 76 per cent of cases. Delay in subretinal fluid absorption in the remaining 24 per cent of cases was directly related to the duration of the retinal detachment but was not influenced by the patient's age, refractive error, or the characteristics of the detachment.


Trabeculectomy in a Black American glaucoma population.

Trabeculectomy was performed on 51 Black patients with uncontrolled glaucoma. The operation was successful in 80 per cent of cases followed-up for an average of 18 months. Success seemed to be independent of whether the superficial scleral flap was or was not sutured, although subconjunctival drainage, as indicated by the appearance of a bleb, seemed to be the predominant mechanism whereby the procedure functioned. The success rate in this series is the highest reported for glaucoma filtering procedures in Black glaucoma populations. Trabeculectomy may now be the operation of choice in uncontrolled glaucoma occurring in Black patients.


Abnormal pupillary responses in myasthenia gravis. A pupillographic study.

Using an open-loop stimulus (Maxwellian view), the direct pupillary response to light and its derivative curves (velocity and accleration of pupillary reactions) were recorded by infrared video pupillography in seven patients with myasthenia gravis and in three normal subjects. Responses before and after intravenous injection of 5 mg edrophonium hydrochloride were determined. Five of the patients had never taken anticholinesterase drugs except for diagnostic injections of edrophonium while the other two patients had been treated with oral preparations of corticosteroids. Analyses of the pupillary responses showed reduced amplitude, amximal velocity, and maximal acceleration of pupillary constriction in the myasthenic patients. On the other hand, changes in parameters of pupillary dilatation were minimal. Abnormal values returned towards normal within five minutes after edrophonium injection in all patients except in one individual who was being treated with corticosteroids at the time of examination. These results suggest that involvement of the iris sphincter may be common in patients with myasthenia gravis and this can be demonstrated by analysing the pupillary reaction and its derivative curves in response to an open-loop stimulus.


Optic nerve metastasis from a mediastinal carcinoid tumour.

A 25-year-old man with a rapidly progressive metastatic carcinoid tumour arising in the mediastinum developed a metastasis to the optic nerve. This is the first instance in the literature of an ocular metastasis from this variant of carcinoid tumour. The ocular pathology, ultrastructure, and recent information on the derivation of this tumour, believed to originate in the thymus, are presented.


Cilia incarnata.

In connexion with three case reports of cilia incarnata (examples of both cilium incarnatum externum and cilium incarnatum internum), the aetiology and the signs and symptoms of this infrequently recognized anomaly are described.


Recovery of operator DNA binding activity from denatured lactose repressor.

Lactose repressor monomer subunits, prepared with sodium dodecyl sulfate, have been renatured-reassociated to yield tetrameric repressors active in binding to operator DNA. The majority of moleculesin renatured repressor populations have operator binding properties indistinguishable from the stock, undenatured repressors from which they were prepared. Renaturation of a mixture of monomers from wild-type (Q) and a tight binding repressor yields a population of active repressors containing molecules with operator binding characteristics intermediate to those of the "parental" repressor types.


Site of action of ricin on the ribosome.

The extent of the inhibitory effect of ricin in polyphenylalanine synthesis by eukaryotic ribosomes is strongly dependent upon the concentration of ribosomes and the elongation factors EF 1 and EF2. Maximal inhibition by ricin, in this assay is observed when either ribosomes or the two elongation factors are added under limiting conditions, whereas ricin-treated ribosomes support protein synthesis at saturating concentrations of elongation factors and ribosomes. Similarly, the enzymatic binding of Phe-tRNA to ribosomes is drastically blocked in ricin-treated ribosomes when low EF 1 concentrations are added to the reaction mixture, but there is no inhibition when EF 1 is at saturating concentrations. Furthermore, formation of the complex EF 2-guanosine triphosphate-ribosome, using free ribosomes pretreated with ricin, is strongly inhibited at limiting concentrations of EF2, but is not affected at saturating concentrations of this factor. However, ricin does not inhibit the EF 2-dependent translocation of peptidyl-tRNA by polysomes, although the toxin is very active in preventing amino acid incorporation by polysomes. Our results suggest that the damaging effect of ricin on the ribosome causes a decreased affinity for both elongation factors EF 1 and EF 2. Thus, the toxin inhibits the enzymatic binding of aminoacyl-tRNA to ribosomes. The lack of inhibition of translocation by ricin suggests that the toxin cannot interact with ribosomes with substrate bound to the acceptor site. Essentially similar results are observed with ricin, abrin, ricin A chain, abrin A chain, and ricinus agglutinin A chain. A possible effect of the toxins on initiation and/or termination is further discussed.


Polyribonucleotides containing thiopurines: synthesis and properties of poly (6-thioguanylic acid).

The synthesis of poly(2-amino-6-chloropurinylic acid) [poly(n2cl6Pu)] by the polynucleotide phosphorylase catalyzed polymerization of 2-amino-6-chloro-9-(beta-D-ribofuranosyl)purine 5'-diphosphate and its chemical conversion to poly(6-thioguanylic acid) [poly(s6G)] is described. Poly(s6G) was found to form a relatively unstable complex with poly(C), the properties of which were incompatible with those previously reported for the same complex prepared by another method [Darlix, J.L., Fromageot, P., and Reich, E. (1973), Biochemistry 12, 914]. It was found that poly(s6G) could be thermally converted to a copolymer of which with poly(C) was strikingly similar to that reported earlier for poly(s6G)-poly(C).


Temperature-sensitive DNA polymerase induced by a bacteriophage T5 mutant: relationship between polymerase and exonuclease activities.

DNA polymerase induced by bacteriophage T5ts53, a mutant with temperature-sensitive polymerase, was purified to about 95% purity as judged by dodecyl sulfate gel electrophoresis. The 3' leads to 5' exonuclease associated with the polymerase had higher activity than that associated with the parent wild-type enzyme. It was more stable to heat than the polymerase, and it degraded primer-template even in the presence of 4 dNTP's at higher temperature. However, the evidence presented shows that the inhibition of DNA synthesis by higher temperature was primarily due to defects in polymerase function rather than to overactive exonuclease. The presence of primer-template DNA stabilized the polymerase to heat. Purified ts53 polymerase was also shown to discriminate against incorportion of BrdUMP, especially at higher temperature. This is an agreement with observations made in vivo with ts53-infected bacteria.


The folding and quaternary structure of trimeric 2-keto-3-deoxy-6-phosphogluconic aldolase at 3.5-A resolution.

An X-ray crystallographic structure determination has been carried out on 2-keto-3-deoxy-6-phosphogluconic (KDPG) aldolase at 3.5-A resolution using the multiple isomorphous replacement method with three heavy atom derivatives along with anomalous dispersion contributions from two of the derivatives. Crystals grown from ammonium sulfate-phosphate buffered (pH 3.5) solutions were: cubic, a= 103.40 (4) A, space group P213. KDPG aldolase consists of trimeric heterologous assemblages utilizing crystallographic threefold symmetry. The overall profile of the oligomeric structure viewed down the threefold axis resembles that of a ship propeller while the subunits are approximate irregular oblate ellipsoids (25 X 45 X 45 A). The folding of most of the polypeptide chain was traced unambiguously. Secondary structural features consist of nine helical regions (75 residues, 35%) and a pair of two parallel chains. The subunit contains a long empty channel which is about 9 X 9 X 30 A with one of the pair of parallel chains forming part of the wall. Three mercury binding sites are located in this channel. These might correspond to the two readily accessible and one of the two buried cysteine residues of each subunit. The channel terminates with another cavity of about 8 X 10 X 25 A near the surface of the oligomeric structure. The regions of the subunits near the threefold axis are characterized by a high degree of secondary structural organization and these make close intersubunit contacts. Quarternary interactions are due mainly to side-chain interactions of helices.


Complete amino acid sequence of the myoglobin from the Atlantic bottlenosed dolphin, Tursiops truncatus.

The complete amino acid sequence of the major component myoglobin from the Atlantic bottlenosed dolphin, Tursiops truncatus, was determined by specific cleavage of the protein to obtain large peptides that are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the 2 methionine residues and 4 peptides were obtained by cleaving the methyl acetimidated protein with trypsin at the 3 arginine residues. By subjecting 4 of these peptides and the apomyoglobin to automatic Edman degradation, over 80% of the covalent structure of the protein was obtained. The remainder of the primary structure was determined by further digestion of the central cyanogen bromide peptide with trypsin and staphylococcal protease. This myoglobin differs from that of the sperm whale, Physter catodon, at 15 positions, from that of the California gray whale, Eschrichtius gibbosus, at 14 positions, from that of the common porpoise, Phocoena phocoena, at 6 positions, and from the myoglobin of the Black Sea dolphin, Delphinus delphis and the Amazon River dolphin, Inia goeffrensis, at 5 and 7 positions, respecitvely. All substitutions observed in this sequence fit easily into the tertiary structure of sperm whale myoglobin.


Circular dichroism studies on glycogen phosphorylase from rabbit muscle. Interaction with the allosteric activator adenosine 5'-monophosphate.

Circular dichroism (CD) spectra of glycogen phosphorylase from rabbit muscle have been measured in the presence of various ligands, particularly in the near-ultraviolet wavelength region. Phosphorylases a and b gave similar positive CD spectra as each other, in the 250-310-nm region. The differences in CD between the a and b forms, as well as the CD changes induced by binding of substrate and other ligands except nucleotides to the enzyme, are all relatively small. Binding of AMP and other nucleotides to phosphorylases a and b, and NaBH4-reduced phosphorylase b, however, induces much larger CD spectral changes than the above. The difference CD curve obtained by subtracting the phosphorylase b curve from that of the enzyme- AMP complex is smooth, with a positive maximum at 266 nm and a negative at 289 nm. The results with various other nucleotides show that the induced Cotton effects are dependent on the base chromophore of the nucleotides. The rotational strength of the induced Cotton effect in phosphorylase b by AMP increases under various conditions, under which the affinity of the enzyme for AMP is enhanced, e.g., the addition of glucose 1-phosphate, inorganic phosphate, fluoride ion, divalent metal cations, and spermine, low temperatures, and conversion of the enzyme to the a form. On the contrary, these factors little affect the induced Cotton effects by IMP, GMP, and dAMP. Amylodextrin gave no effect on the extrinsic Cotton effect by binding of AMP plus Mn2+ to phosphorylase b, while it did retard the AMP-induced tetramerization of the enzyme. It is suggested that the interaction of nucleotides with phosphorylase involves stacking between the base ring of the bound nucleotides and an aromatic amino acid residue at the allosteric site of the enzyme, and that, in the high affinity form of the enzyme for AMP, particular bondings are newly formed between the enzyme and the nucleotide allowing the heterotropic cooperativity.


Site of action and biphasic effect of neutral salts in the phosphorylase kinase reaction.

The inhibition of phosphorylase kinase catalytic activity by 0.1 M neutral salts was predicted by the Hofmeister series of anions. The site of action of the salts was determined by the following evidence to be on the phosphorylase kinase molecule directly, rather than on its protein substrate. (1) Nonactivated kinase was more sensitive to salt inhibition than the activated form. (2) Ca2+ partially overcame the inhibition of nonactivated kinase. (3) Inhibition by Cl- occurred with either phosphorylase or a tetradecapeptide containing the convertible seryl residue as substrate. (4) Phosphorylation of nonactivated phosphorylase kinase by protein kinase was markedly inhibited by NaNO3, but this salt had little effect on the phosphorylation of histone by protein kinase. The influence of neutral salts on phosphorylase kinase activity was biphasic. Although activity was inhibited at low salt concentrations, it actually was stimulated as the salt concentration was increased. A similar biphasic response to various salt concentrations was observed in the velocities of autophosphorylation of phosphorylase kinase. The lag in the rate of product formation seen during the activity assay was less pronounced at inhibitory salt concentrations and was abolished at stimulatory salt concentrations. How the influence of salts relates to autophosphorylation and the lag is considered.


A detailed structural comparison between the charge relay system in chymotrypsinogen and in alpha-chymotrypsin.

An improved 2.5-A electron density map of chymotrypsinogen was calculated by incorporating heavy-atom anomalous scattering effects and a new model of the molecule was constructed. Phases from x-ray structure factors (R = 0.43) computed from this model were then used in the calculation of another electron density map against which the model was further refined. The catalytic Ser-195 side chain in the new model is in the "down" or "acyl" orientation and its Ogamma atom is in position to form a normal hydrogen bond with Nepsilon2 of His-57. In contrast, the corresponding hydrogen bond in alpha-chymotrypsin (Birktoft, J.J., and Blow, D.M. (1972), J.Mol. Biol. 68, 187) is severely distorted, probably as a consequence of a 1.5-A shift in the relative positions of the two cylindrical folding domains composing most of the molecule. We suggest that this activiation induced distortion of the charge-relay, hydrogen-bonding system plays an important role in the genesis of enzymic activity, in accord with an earlier proposal by Wang concerning the role of bent hydrogen bonds in enzyme catalysis (Wang, J.J. (1970), Proc. Natl. Acad. Sci. U.S.A. 66, 874).


Microtubule-associated proteins and the stimulation of tubulin assembly in vitro.

Microtubules, purified by cycles of assembly and disassembly in vitro, are composed of tubulin and several microtubule-associated proteins (MAPs). When the MAPs were separated from the tubulin by phosphocellulose chromatography, the tubulin by phosphocellulose chromatography, the tubulin no longer assembled at 37 degrees C as measured by turbidity. If the MAPs and tubulin were recombined and warmed to 37 degrees C, microtubules assembled. MAPs stimulated tubulin assembly by affecting both the initiation and elongation processes. The effect on initiation was indicated by results showing an increase in initial rate and a decrease in average microtubule length as the MAP:tubulin ratio was increased. The initiation and elongation activities of the MAPs at 4 degrees C during which time the initiating activity decreased while the ability to affect the total amount of assembly remained constant. The decrease in initiating ability was correlated with the loss of the two major components of the MAP fraction, MAPs 1 and 2.


Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 1. Single component phosphatidylcholine liposomes.

The fluorescence depolarization associated with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene is used to monitor the changes in fluidity accompanying the gel-liquid crystalline phase transition in synthetic phosphatidycholine dispersions. The parameters of the phase transition are determined for both large, multilamellar liposomes and small, single-lamellar vesicles. These parameters are compared with those obtained using other techniques. In addition, the data are interpreted in terms of two limiting molecular models, which in turn offer insight into the structural differences between multilammelar liposomes and small vesicles.


Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 2 Two-component phosphatidylcholine liposomes.

The fluorescence deplorarization associated with the hydrophobic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene is used to monitor changes in fluidity accompanying the gel-liquid crystalline phase transition in phosphatidylcholine dispersions. In this way, the parameters of the phase transition are determined for both large, multilamellar liposomes and small, single-lamellar vesicles composed of three mixtures of phosphatidycholines: dimyristoyl-dipalmitoyl, dimyristoyl-distearoyl, and dioleyl-dipalmitoyl. Phase diagrams for these mixed-lipid vesicles are constructed by plotting the delimiting temperatures of the phase transition vs. the lipid compostion of the vesicle. The phase diagrams are interpreted to suggest that the miscibilities of the lipids studied are markedly different in small, single-lamellar vesicles and large multilamellar liposomes. These results are discussed in terms of the effects of high curvature on the structure of biological membranes.


HeLa cell DNA polymerase gamma: further purification and properties of the enzyme.

DNA polymerase gamma has been purified over 60 000-fold from HeLa cells which contain no detectable type C viral particles. This purified enzyme shows a specific activity of 25 000 units/mg of protein which is comparable to the known specific activity of homogeneous preparations of human alpha and beta polymerases. The isolated enzyme shows apparent molecular weights ranging from 160 000 to 330 000 according to the method of analysis. The enzyme exhibits optimal activity for copying poly(A) in the presence of 50 mM KPO4 and 130 mM KCl and, under these conditions, copies poly(A) 20 times more rapidly than activated DNA. These assay conditions permit a clear distinction between the gamma-polymerase and DNA polymerase beta which is markedly inhibited by phosphate at this concentration. A comparison of the copying of activated DNA, poly(dA) and poly(A) by DNA polymerases alpha, beta, and gamma under optimal assay conditions for each enzyme is presented. Studies with synthetic and natural nucleic acid templates also show the gamma-polymerase to behave differently that the reverse transcriptases of avian myeloblastosis virus or Rauscher leukemia virus.


The distirubtion of active genes (globin) and inactive genes (keratin) in fractionated chicken erythroid chromatin.

Repeatable fractionation of sheared chromatin from purified populations of chicken erythroid cells has been achieved, based on the Bio-Gel procedure of Janowski et al. ((1972) Karolinska Symp. 5, 112). For reticulocytes, 3-5% of chromatin DNA is excluded from Bio-Gel A-50 m (peak I) and over 90% elutes in the included volume of the column(peak II). Peak I material has a higher protein/DNA ratio than peak II chromatin and the two fractions have melting profiles characteristic of "active" and "inactive" chromatin, respectively. In cells prelabeled with [3H]uridine or [3H]leucine there was very pronounced preferential association of radioactivity with the "active" peak I chromatin. The distribution of "active" (globin) and "inactive" (keratin) gene sequences in the DNA of fractions from peak I and peak II chromatin was determined with complimentary DNA (cDNA) probes to chicken globin mRNA and chicken feather keratin mRNA. While slight enrichment for globin gene sequences was found in peak I (relative to DNA in these fractions), some 80% of the total globin hybrid formed was found in peak II fractions. Experiments with the keratin cDNA probe showed that these genes were equally distributed in both chromatin fractions rather than being confined to the "inactive" peak II material. The hybridization data in particular question the validity of claims for fractionation of chromatin into "active" and "inactive" material.


Nature of the Thermal pretransition of synthetic phospholipids: dimyristolyl- and dipalmitoyllecithin.

The hydrated synthetic lecithins, dimyristoyl and dipalmitoyllecithins, undergo two thermal transitions, a broad low enthalpy "pretransition" prior to the sharp first-order "chain-melting" transition. Both phospholipids exhibit the same temperature-dependent structural changes associated with the thermal pretransition. At low temperatures, below the pretransition, a one-dimensional lamellar lattice is observed. The hydrocarbon chains are fully extended and tilted with respect to the plane of the lipid bilayer. The hydrocarbon chain packing displays a temperature dependence and the angle of tilt of the hydrocarbon chains decreases with increasing temperature, reaching a minimum value of 30 degrees at the pretransition temperature of both lecithins. The pretransition is associated with a structural transformation from a one-dimensional lamellar to a two-dimensional monoclinic lattice consisting of lipid lamellae distorted by a periodic ripple. The hydrocarbon chains remain tilted in the temperature range intermediate between the pretransition and chain-melting transition. The cell parameters of this two-dimensional lattice exhibit a compositional dependence. The a parameter (proportional to the lamellar repeat distance) increases with increasing water content, while the b parameter (a measure of the ripple periodicity) decreases with increasing water content. At the chain-melting transition, the hydrocarbon chains of the phospholipid melt and assume a liquid-like conformation and the lattice reverts to one-dimensional lamellar. These structural changes observed for dimyristoyl- and dipalmitoyllecithins may be a common feature of all synthetic lecithins exhibiting a thermal pretransition. The appearance of the pretransition and accompanying two-dimensional may arise from specific interactions between the choline moiety of the polar head group and the structured water matrix surrounding it.


Elucidation of lectin receptors by quantitative inhibition of lectin binding to human erythrocytes and lymphocytes.

The binding to normal and sialidase-treated human erythrocytes and lymphocytes of four 125I-labeled lectins [Maackia amurensis hemagglutinins (MAM and MAH), Ricinus communis hemagglutinin (RCH), and Bauhinia purpurea hemagglutinin (BPH)] was studied in detail. The quantitative inhibition assays against the lectin binding to the cells were also performed with various glyco-proteins and glycopeptides as inhibitors. The comparison of the inhibition constants of the inhibitors thus obtained with the association constants of the lectins to the cells permitted estimation of the relative receptor activities of cell surface glyco-proteins toward the lectins.


Visual-pigment spectra: implications of the protonation of the retinal Schiff base.

Various models of visual-pigment spectra are critically discussed in terms of the spectral properties of protonated Schiff bases and the common structural features of most proteins. The opsin apoprotein is capable of regulating visual pigment wavelengths in ways that are difficult or impossible to reproduce in model systems. Theories based on solvent effects of the spectra of protonated Schiff bases may be misleading. Careful parameterization using known polyene spectra allows accurate calculation of the spectral properties of protonated Schiff bases. It is shown that an isolated protonated Schiff base of retinal should absorb near 600 nm and that blue-shifted spectra seen in solution arise from associated counterions or solvent molecules. We conclude that the most plausible specific model of chromophore-protein interactions is one in which the protonated Schiff base is closely associated with its counterion and where additional negatively charged or polar groups are positioned by the protein in the vicinity of the ring half of the chromophore. Pigment absorption maxima, bandwidths, and the A2-A1 pigment absorption differences arise naturally from these simple models of pigment spectra.


Hummel-Dreyer gel chromatographic procedure as applied to ligand-mediated association.

A mass transport theory of the gel chromatographic procedure of Hummel and Dreyer for measuring the binding of small ligand molecules to macromolecules has been formulated for ligand-mediated macromolecular association. It is concluded that the Hummel-Dreyer procedure is the method of choice for quantitating ligand binding in such systems. Guidelines are drawn for unambiguous interpretation of the nonclasical elution profiles in terms of the number of binding sites on the macromonomer and their intrinsic binding constant. Most of these guidelines also apply to static equilibrium methods such as equilibrium dialysis.


Poly(uridylic acid) sequences in messenger ribonucleic acid of HeLa cells.

The poly(uridylic acid [poly(U)] sequences of 30 to 40 nucleotides found in the heterogeneous nuclear ribonucleic acid (RNA) Of HeLa cells are also present in the poly(adenylic acid) [poly(A)] containing messenger RNA (mRNA) of these cells. Twenty-five percent of the total poly(U) sequences in the cell are found in the cytoplasm after HeLa cells have been labeled for 2.5 h with 32P. This value is close to 40% if only poly(A) containing RNA molecules of the cell are considered. The distribution of poly(U) sequences parallels that of poly(A) sequences in different size classes of cytoplasmic messenger RNA. From the concentrations and lengths of the two sequences it can be estimated that about 20% of the poly(A) containing mRNA molecules could contain one poly(U) sequence. Several lines of evidence have been developed to rule out poly(U) containing heterogeneous nuclear RNA (HnRNA) as the source of the poly(U) sequences in cytoplasm. Poly(U) sequences are also found in RNA molecules of the nucleus and cytoplasm which do not contain poly(A). Although poly(U) sequences are most abundant in nuclear RNA species lacking poly(A), they are well represented in a class of RNA molecules in cytoplasm which lack poly(A), but which otherwise resemble the polysomal mRNA of HeLa cells.


Chromatographic and functional comparison of human placenta and HeLa cell tyrosine transfer ribonucleic acids.

Chromatographic and functional properties of tyrosine isoaccepting transfer ribonucleic acids (tRNAs) from placenta and HeLa cells were analyzed and compared. RPC-5 chromatography separated four major isoacceptors from each source, with those from HeLa cells eluting generally later than those from placenta. There was some overlap: HeLa tRNA1Tyr eluted in a position between placenta tRNA3Tyr and tRNA4Tyr; and HeLa tRNA2Tyr and placenta tRNA4Tyr eluted in similar positions with the HeLa isoacceptor eluting slightly later than the placental isoacceptor. Thus there are no isoacceptors common to both sources. The function of the individual isoacceptors was compared in a rabbit-reticulocyte, cell-free, protein-synthesizing system for both the rate of incorporation of tyrosine into the polypeptide chain and the site of incorporation in alpha-globin. Two isoacceptors were compared simultaneously in the same reaction, and overlapping comparisons were made to relate each isoacceptor to all the others. There were no significant differences in the rates of incorporation among the isoacceptors, nor were there any differences in the sites of incorporation. All eight isoacceptors donated tyrosine equally well into the three tyrosine containing tryptic peptides of alpha-globin. Whatever the structural differences among the tyrosine isoacceptors are, they do not affect the function of the tRNA in this protein-synthesizing system.


Structural difference between alpha-paramyosin and beta-paramyosin of Mercenaria mercenaria.

A method is described for extraction of alpha-paramyosin in amounts comparable to that formerly attained for beta-paramyosin (15-25 mg/g of muscle). A modification of the procedure for sodium dodecyl sulfate gel electrophoresis is described that permits the separation on coelectrophoresis of alpha-paramyosin (207 000 daltons) and beta-paramyosin (200 000 daltons). The alpha- and beta-paramyosins also can be distinguished by gel electrophoresis at pH 2.3 and by differences in solubility in the region of 0.2-0.4 ionic strength at neutral pH. Evidence is presented that the segment lost from alpha-paramyosin during degradation to beta-paramyosin came from the C-terminal end. This evidence is based on determinations of N- and C-terminal amino acids and on the size of segments obtained after chemical cleavage at the sites of Cys residues. It has been observed earlier that the solubility characteristics of beta-paramyosin at neutral pH are determined by the C-terminal one-third of the molecule and the present results indicate that the additional small segment of about 3.5% of the total mass that is present in the C-terminal end of alpha-paramyosin accounts for the marked difference in solubility of the two forms.


beta Structure of aqueous staphylococcal enterotoxin B by spectropolarimetry and sequence-based conformational predictions.

Conformations of the globular protein staphylococcal enterotoxin B have been examined experimentally by ultraviolet circular dichroism (CD) and visible optical rotatory dispersion (ORD). Chen-Yang-Chau analysis (Chen, Y.-H., Yang, J.T., and Chau, K. H. (1974), Biochemistry 13, 3350) of the far-ultraviolet CD spectrum of native enterotoxin B revealed (assuming an average helix length of 11 residues) 9% alpha helix, 38% beta structure, and 53% random coil. A fourfold increase in alpha-helix was observed for enterotoxin exposed to 0.2% sodium dodecyl sulfate, behavior typical for globular proteins of low helical content. Values of -40 to -50 for the Moffitt-Yang parameter b0 calculated from visible ORD suggested 6-13% alpha helix in native enterotoxin. Application of a new predictive model (Chou, P. Y., and Fasman, G. D. (1974), Biochemistry 13,222) to the amino acid sequence of enterotoxin B indicated 11% alpha helix, 34% beta structure, and 55% coil in native enterotoxin. The excellent agreement for the amount of alpha and beta conformation utilizing different optical and predictive methods indicates beta structure as the dominant secondary structure in native enterotoxin B. Most of the beta structure is predicted by Chou-Fasman analysis to reside in two large regions of antiparallel beta sheet involving residues 81-148 and residues 184-217. Such highly cooperative regions of anti-parallel beta sheet account for the slow unfolding of enterotoxin B in concentrated guanidine hydrochloride and rapid folding of guanidine hydrochloride denatured enterotoxin B to native conformation(s) (Warren, J.R., Spero, L., and Metzger, J. F. (1974), Biochemistry 13, 1678). A more than twofold increase in alpha-helix content with a small diminution in beta structure was detected by CD and ORD upon acidification of aqueous enterotoxin to pH 2.5. Thus, the beta structure of enterotoxin B appears to resist isothermal denaturation and constitutes a stable interior core of structure in the enterotoxin molecule.


Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines.

beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.


Binding of adenosine 5'-diphosphate to creatine kinase. An investigation using intermolecular nuclear Overhauser effect measurements.

Measurements of the nuclear Overhauser effect (NOE), which is a nuclear magnetic resonance (NMR) double resonance technique, for the H-2 proton on ADP have been used to identify the amino acid residue binding ADP at the active site of creatine kinase. Application of a strong radio-frequency field H2 at a frequency of 0.9 ppm or 1.7 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate results in a negative NOE for the H2-2 proton resonance of ADP in its complex with creatine kinase. The magnitude of the NOE for the ADPH-2 proton depends on the ratio of ADP to creatine kinase binding site concentration; the dependence indicates that there is rapid exchange between free and bound ADP. Comparable values of the NOE for the H-2 proton of adenosine 5'-mono-, 5'-di-, and 5'-triphosphate and inosine 5'-diphosphate in binary complexes with creatine kinase show that the binding site for these nucleotides is the same. The large negative NOE for the H-2 proton of ADP is maintained for the various binary, ternary, quaternary, and pentenary complexes of creatine kinase with ADP formed by addition of the activator Mg(II), the other substrate creatine, and the planar anion nitrate which is an inhibitor. These results indicate that the conformational changes known to occur upon addition of the other ligands do not involve the entire active site. In particular, the environment around the nucleotide is unperturbed. Inactivation of creatine kinase by reaction with iodoacetamide causes considerable conformational changes. However, as indicated by the large negative NOE for the H-2 proton of ADP in a binary complex with the inactivated enzyme, the environment around the base is altered minimally. Experiments were performed to identify the proton groups on the enzyme, resonating at 0.9 and 1.7 ppm, which interact with the ADPH-2 proton. An NOE was not observed when the aromatic protons of the enzyme were irradiated with the strong radiofrequency field H2 implying that aromatic protons are not near the H-2 proton of ADP in the enzyme complex. The H-2 proton of 1-N6-ethenoadenosine 5'-diphosphate, an analogue of ADP with the 1-nitrogen and 6-nitrogen blocked from potentially hydrogen bonding, still exhibits a large NOE in the nucleotide-enzyme complex. The indication is that the protons promoting the H-2 proton NOE are not on an amino acid residue which binds ADP by hydrogen binding. Creatine kinase was inactivated by reacting the single essential arginyl residue per subunit with diacetyl. No NOE for the H-2 proton of ADP in the presence of the inactivated enzyme was observed. Observation of the H-2 proton resonance of the inhibitor adenosine in the presence of the enzyme revealed no NOE in contrast to the observations with the adenine nucleotides.


Active-site-directed inactivation of aromatase from human placental microsomes by brominated androgen derivatives.

Several brominated androgen derivatives were tested for their ability to inactivate microsomal aromatase from term human placenta. In the experimental protocol, the microsomal homogenate was incubated either with androstenedione or a brominated derivative of androstenedione (16alpha-bromo-6-ketoandrostenedione, 16alpha-bromoandrostenedione, 7alpha-(3'-bromoacetoxypropyl)androstenedione, 6alpha-bromoandrostenedione, or 6beta-bromoandrostenedione) and reduced nicotinamide adenine dinucleotide phosphate in a nitrogen saturated buffer composed of glycerol, ethylenediaminetetraacetic acid, and dithiothreitol in tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4) under nitrogen at 4 degrees C with shaking. After the incubation period, the microsomes were recovered by centrifugation and washed once before determining aromatase specific activity. The brominated androgen derivatives which inactivated aromatase were 7alpha-(3'-bromoacetoxypropyl)androstenedione and 6alpha-bromoandrostenedione. The structures of 6alpha- and 6beta-bromoandrostenedione were unequivocally established by single crystal x-ray diffraction techniques. The extent of the enzyme inactivation by 6alpha-bromoandrostenedione was linearly proportional to the logarithm of its concentration. The evidence that this inactivation occurs at the aromatase active site is that androstenedione, when coincubated with 6alpha-bromoandrostenedione, protected aromatase from this inactivation. Progesterone provided much less protection than androstenedione. Furthermore, both 6alpha- and 6beta-bromoandrostenedione are competitive inhibitors of androstenedione aromatization, as determined by a Lineweaver-Burk plot, and 6alpha-bromoandrostenedione gives the same type I cytochrome P-450 binding spectrum with placental microsomes as androstenedione. These data suggest that 6alpha-bromandrostenedione is effective as an active-site-directed inhibitor of placental microsomal aromatase.


Cholesterol exchange between fat cells, chylomicrons and plasma lipoproteins.

Isolated rat adipocytes were incubated with serum lipoproteins or lymphchylomicrons which contained 14c-labeled cholesterol. The specific activity of lipoprotein free cholesterol decreased and that of cellular free cholesterol increased linearly up to 7 h. At this time the cell cholesterol specific activity was only 11% of that of medium cholesterol indicating that the rate of exchange was slow. The specific activity of lipoprotein esterified cholesterol remained unchanged while that of cells showed a slight increase suggesting esterification of incorporated free cholesterol. No detectable change of the lipoprotein or cellular cholesterol concentration occurred indicating that the uptake of radioactive free cholesterol was due to exchange without net movement of sterol.  The radioactive cholesterol was incorporated into both membrane fraction and the fat droplet of the adipocytes. The rate of cholesterol exchange was temperature-dependent but it was not influenced by the metabolic state of the cells and not by addition of metabolic inhibitors. Trypsin or pronase treatment of the cells were without influence on the rate of the exchange and denaturation of the plasma lipoproteins with formalin increased the rate of exchange. These results indicate that the exchange of cholesterol is a physical chemical process, which is not linked to energy metabolism of the cells, and which is not mediated by either specific lipoprotein receptors on fat cell membranes or pinocytic uptake of lipoproteins. The rate of free cholesterol exchange showed a linear correlation with the concentration of lipoprotein particles in the medium. The relative transfer rate was highest for chylomicrons and decreased in order chylomicron remnants greater than very low density lipoprotein greater than low density lipoprotein greater than high density lipoprotein. A saturation of the system could be obtained only with high density lipoprotein.


A new rat liver phospholipid exchange protein.

The soluble fraction from several mammalian tissue homogenates is known to stimulate phospholipid exchange between cell membrane fractions or artificial vesicles. All phospholipid exchange proteins purified to data exhibit an acidic isoelectric point. Using an assay that measures the transfer of [32P] phosphatidylcholine from liposomes to beef heart mitochondria, we report the presence of a new phospholipid exchange protein with a basic isoelectric point (8.4) in rat liver cytosol.  A purification procedure, consisting of pH adjustment to 5.1, gel filtrations on Sephadex G 75 and DE 52 cellulose, isoelectric focusing between a pH of 5 and 10, and gel filtration on Sephadex G-50, yielded a fraction with high phosphatidylcholine exchange activity per mg of protein. This fraction exhibits a major band and two minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.  The molecular weight of the major band (18 700) is close to that for basic exchange protein fraction obtained by gel filtration (17 000). The distribution of basic and acidic exchange proteins differs markedly in various tissues and animal species.  About 50 and 35% of phosphatidylcholine exchange activity from rat liver and rat intestine respectively are due to basic phospholipid exchange proteins. In contrast, no basic exchange protein was found in beef heart and only a small amount in beef liver. In the latter organ, less than 10% of phosphatidylcholine exchange activity was due to a basic phospholipid exchange protein fraction.


Evidence for carrier proteins in bile acid synthesis. The effect of squalene and sterol carrier protein and albumin on the activity of 12alpha-hydroxylase.

The possibility that carrier proteins are involved in bile acid synthesis was investigated using rat liver homogenates. The 105 000 X g supernatant fraction was found to contain heat stable proteins that bound the bile acid precursor, 7alpha-hydroxy-4-cholesten-3-one, and increased the amount of 7alpha, 12alpha-dihydroxy-4-cholesten-3-one formed by the microsomal enzyme, 12alpha-hydroxylase. Subsequent studies were carried out to determine if squalene and sterol carrier protein or albumin, two lipid binding proteins present in the 105 00 X g supernatant fraction of rat liver homogenates, may be responsible for the effects seen with this fraction. Squalene and sterol carrier protein bound several water insoluble bile acid precursors, including 7alpha-hydroxy-4-cholesten-3-one, and increased the apparent activity of 12alpha-hydroxylase. Squalene and sterol carrier protein, however, did not bind either cholic acid or chenodeoxycholic acid. Rat serum albumin also bound 7alpha-hydroxy-4-cholesten-3-one and increased the apparent activity of 12alpha-hydroxylase. Kinetic analysis indicated that the apparent stimulation of 12alpha-hydroxylase by squalene and sterol carrier protein and albumin was due to increased solubilization of the substrate, 7alpha-hydroxy-4-cholesten-3-one. Thus, these studies indicate that bile acid precursor carrier proteins are present in the 105 000 Xg supernatant fraction of rat liver homogenates and suggest that squalene and sterol carrier protein or albumin may participate as carrier proteins in bile acid synthesis.


Phospholipases A1 and A2 in lamellar inclusion bodies of the alveolar epithelium of rabbit lung.

1. A lamellar body-enriched fraction was prepared from rabbit lung and characterized by electron microscopy, surface activity studies, phospholipid assay and marker enzymes. 2. Both phospholipases A1 and A2 were found to be present in lamellar bodies. After these had been ruptured both enzymes were found to be principally in the soluble phase. 3. The possible roles for phospholipases in lamellar body development and in the respiratory distress syndrome of the newborn are discussed.


Metabolism of different monoacylphospholipids in isolated hepatocytes and the intact rat.

The 1-[3H] palmitoyl, 2-[3H] oleoyl, and 2-[14C] linoleoyl derivatives of sn-glycero-3-phosphoethanolamine and the corresponding derivatives of sn-glycero-3-phosphocholine were injected intraportally to rats and their incorporation into liver lipids was studied 15 min thereafter. Both the uptake by the liver and the degree of acylation was higher for the unsaturated compounds. The uptake of lysophosphatidylethanolamine was higher than that of lysophosphatidlycholine. The metabolism of 1-lysophosphatidylethanolamine was also studied in isolated hepatocytes. The degree of hydrolysis was much more prominent than in vivo. After injecting 2-[14C] linoleoyl derivatives, a large part of the 14C was recovered in the dienoic phospholipids. Subfractionation by reversed-phase partition chromatography showed that the isotope was located in the palmitoyllinoleoyl and stearoyl-linoleoyl fraction. The 100 X stearoly/(palmitoyl + stearoyl) ratio was 84 in dienoic phosphatidylethanolamine and 59 in dienoic phosphatidylcholine. This preference for stearic acid is significantly larger than in other pathways yielding dienoic phospholipids. It can be concluded that the monoacylphospholipid acyltransferase reactions operating at positions 1 or 2 yield different saturated acyl chain profiles in phosphatidylethanolamine and phosphatidylcholine of a specific unsaturation. This may be important in the regulation of the fatty acid composition of the membrane phospholipids.


Phospholipid acyl group stability in cultured fibroblasts. Differences between human cell lines of fetal and adult origin.

Human fibroblasts of both fetal and adult origin incorporated [1-14C] acetate primarily into phospholipid acyl groups (70-80% of total radioactivity). When these labeled cells were replated in non-radioactive medium, there was continuous loss of 14C from steroids, triacylglycerols and non-lipid material. In contrast, after some initial loss, cell lines of fetal origin completely retained 14C in cellular phospholipids during continued cell division. Unlike cells of fetal origin, fibroblasts of adult origin continued to lose radioactivity from their phospholipid acyl groups during growth in unlabeled medium. Loss of radioactivity from [1-3H] acetate incorporated into phospholipids of adult cells cannot be attributed to cell death since it was not accompanied by any loss of previously incorporated [ME-14C] thymidine. If cellular phospholipids were labeled with [U-14C] glycerol, both fetal and adult fibroblasts continued to lose radioisotope from the cells during growth in nonradioactive medium. Thus, there is turnover of the phospholipid molecules themselves in fetal human fibroblasts grown in vitro, but their acyl groups are retained within cellular phospholipids. In this respect, fibroblasts of fetal origin resemble established cell lines such as the L fibroblast. Fibroblasts of adult origin do not exhibit this complete conservation of their phospholipid acyl groups.


Transmission electron microscopy of biliary mixed lipid micelles.

One potential artifact problem in election microscopy of micellar systems is that structural changes and aggregation could be induced by high vacuum dehydration in sample preparation prior to viewing and photomicrography. This could account for an earlier report on "macroassemblies" of biliary lipid, a finding which has been discrepant with the predominance of other evidence on the size, configuration and behavior of biliary micelles. In the present work, this possibility was explored and no evidence for such macroassemblies demonstrated when freeze-drying sample preparation was used; whereas, with conventional technique, the "macroassemblies" were again observed. It is concluded that freeze-drying or freeze-etching sample preparation is required to avoid this form of artifact in electron microscopy of micellar systems. The present findings are compatible with a spherical-equivalent or oblate ellipsoid configuration for biliary mixed micelles.


Cyclic peroxides and the thiobarbituric assay.

Several monocyclic peroxide compounds and two acyclic hydroperoxides have been tested for activity in the thiobarbituric acid test. All cyclic peroxides tested which have beta dioxygen functionality on non-tertiary carbons gave positive thiobarbituric acid tests at 532 nm. (epsilon = 10(3) --10(4)) Two acyclic unsaturated hydroperoxides which were derived from gamma-linolenic acid also gave positive thiobarbituric acid tests. In addition to the 532-nm-absorbing species, all peroxidic compounds tested showed a transient absorption at 450 nm during the thiobarbituric acid test. The species responsible for this 450 nm absorption appears to be an intermediate in a series reaction sequence. This intermediate is converted, in time, to the 532-nm-absorbing species. Thiobarbituric acid assay of the crude autoxidation product mixture of gamma-linolenic acid also shows this transient 450-nm-absorbing species. Added ferric ion enhances the 532 nm abosrbance of the thiobarbituric acid assay of cyclic peroxides.


Reaction of Tris with aldehydes. Effect of Tris on reactions catalyzed by homoserine dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase.

Tris buffer was observed to produce an apparent inhibition of the homoserine dehydrogenase (EC 1.1.1.3)-catalyzed reduction of aspartic beta-semialdehyde and an apparent inhibition of the glyceraldehyde phosphate dehydrogenase (EC 1.2.1.9)-catalyzed oxidation of glyceraldehyde 3-phosphate. In each case, the apparent inhibition was found to be due to a lowering of the substrate concentration as a result of a reversible reaction between the free base form of Tris and the substrate, an aldehyde. The product of the reaction was tentatively identified as an imine on the basis of its spectral properties. The inhibition of these two enzymatic reactions by Tris was employed to investigate the kinetics of the reaction of Tris with their substrates. Assuming that these aldehydes exist entirely as the free aldehyde in aqueous solution, equilibrium constants of 369 +/- 12M-1 and 68 +/- 1.5M-1 were determined at 25 degrees C for the reaction of the free base form of Tris with glyceraldehyde 3-phosphate and asparitic beta-semialdehyde, respectively. Correcting for the existence of the hydrated form of glyceraldehyde 3-phosphate in aqueous solution, an equilibrium constant of 1.1-10(4) M-1 was obtained for the reaction of this aldehyde with the free base form of Tris. Forward and reverse direction rate constants for the reaction of Tris with glyceraldehyde 3-phosphate were determined at pH 7.45 and pH 8.5, and both were found to be pH-dependent.


Studies on the nature of different molecular forms of glucose-6-phosphate dehydrogenase purified from human leukocytes.

Several molecular forms of human glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) corresponding to different stages of post-synthetic modifications have been purified from human leukocytes. The various enzyme forms were different in their specific activity, their kinetic properties and their isoelectrofusing pattern. The molecular weight of the subunits of the different forms was not modified. The changes in the electrofocusing pattern were not due to modifications of the N-terminal ends, the oxidation of thiol groups or the non-covalent fixation of an acid molecule upon the enzyme. Carboxypeptidase B cleaved a C-terminal lysine from the different enzyme forms and shifted the isoelectric point of the different enzyme active bands towards the acid pH. The different enzyme forms studied here seemed to result from the action upon 'native glucose-6-phosphate dehydrogenase' of 'modifying factors' especially abundant in some leukemic granulocytes. The modifying factors did not seem to be consumed during the 'modification' of glucose-6-phosphate dehydrogenase. Moreover, the storage for one year of unmodified enzyme resulted in changes in its electrofocusing pattern similar to those quickly induced by the 'modifying factors'. Consequently it appears that the modifying factors are catalysts of the modification of special residues of glucose-6-phosphate dehydrogenase. The hypothesis that this modification involves the deamination of asparagine or glutamine residues is put forward.


Purification and properties of rat lung soluble glutathione peroxidase.

Gluthathione peroxidase (gluthatione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) has been purified approximately 2700-fold from rat lung soluble fraction. The purified enzyme was shown to be homogeneous by sodium dodecyl sulfate/urea polyacrylamide gel electrphoresis. Selenium-75 tracer cochromatographed with the enzyme activity, indicating that rat lung soluble gluthathione peroxidase is a selenium enzyme. The enzyme had an approximate molecular weight of 80000 and contained four identical subunits. The optimal activity of the enzyme was at between pH 8.8 and 9.1. The enzyme had general specificity toward hydroperoxides, and high specificity for reduced glutathione. The kinetic behavior or the purified lung soluble glutathione peroxidase followed a ping-pong-like mechanism; the enzyme first reduced the lipid hydroperoxide substrate to the corresponding hydroxy fatty acid, then was regenerated to the native form by reduced glutathione.


Transaminase of branched chain amino acids. XI. Leucine (methionine) transaminase of rat liver mitochondria.

An aminotransferase (transaminase) which is active for leucine and methionine, but not for valine or isoleucine, was purified from rat liver mitochondria. The purified preparation appeared homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was shown to be 55 000 by gel filtration. It differed from enzyme II (leucine aminotransferase, EC 2.6.1.6) in the supernatant fraction, another transaminase which is also specific for leucine and methionine, in molecular weight, Km values for substrates, electrophoretic mobility, chromatographic behavior and heat stability. From comparison with related transaminases it was concluded to be a new enzyme and named mitochondrial leucine (methionine) transaminase.


Phosphoglycerate kinase from young and old Turbatrix aceti.

Phosphoglycerate kinase (ATP:3-phospho-D-glycerate-1-phosphotransferase, EC 2.7.2.3) from young and old Turbatrix aceti has been purified to homogeneity. The "old" enzyme exhibits a marked reduction in specific activity both in crude homogenates and in pure form when compared to preparations from young nematodes. The specific activities for pure "young" and "old" enzymes are 650-750 and 300-400 units/mg, respectively. All other properties of "young" and "old" enzymes were nearly identical, including molecular weight (43 000), Km, behavior on columns, thermal stability and mobility during gel electrophoresis at three pH values. The results are discussed in terms of the possible mechanism of formation of "altered" enzymes. In addition, certain properteis of the nematode phosphoglycerate kinase are compared with those of the enzyme from yeast and rabbit muscle.


Purification and characterization of two forms of urokinase.

Affinity chromatography on agmatine-Sepharose was used for the separation of two active forms of urokinase (EC 3.4.99.26) from partially purified human urinary urokinase. The approximate molecular weight of the heavier form was 47 000 and of the lighter 33 400. Both forms were homogeneous by sodium dodecyl sulfate gel electrophoresis and by 3H-labeled diisopropylphosphorofluoridate and 14C-labeled p-nitrophenyl-p'-guanidinobenzoate incorporation studies. The 33 400 mol. wt. form had a single chain, and the 47 000 mol. wt. form had two chains (33 100 and 18 600 mol. wt.) linked by disulfide bonds. The specific activity of the heavier form was 104 000 CTA units/mg protein, compared with 226 000 units/mg for the lighter form but the activities per mmol of active site (molar activities) of the two forms were almost identical (9.6-10(9) and 10.2-10(9) CTA units/mmol). Isoelectric focusing on gels showed that the 47 000 material contained one major subform with a pI of 8.60 and a minor subform with a pI of 8.90, while the 33 400 material had three major subforms with pI values of 8.35, 8.60 and 8.70, respectively, and a minor subform with a pI of 8.05. 3H-labeled diisopropylphosphorofluoridate incorporation studies revealed an active-site serine residue in the heavy chain.


The gamma chain of the lamb.

The sequence of the gamma-chain of lamb (Finn-Marino) haemoglobin has been investigated. Total amino acid analysis and analysis of the products of cyanylation, together with analysis of tryptic, peptic, thermolysin and Staphylococcus aureus (strain V8) protease peptides and with dansyl-Edman degradation enabled almost the entire sequence to be elucidated. The differences between this sequence and that of the gamma-chains of man, cattle, goat and Rambouillet sheep are discussed.


Molecular properties of rat intestinal alkaline phosphatase.

Rat intestinal alkaline phosphatase is an heterogeneous glycoprotein that contains three protein sub-forms separable by electrophoresis. The molecular weight for the glycoprotein (i.e. the average for the three sub-forms) is 157 000-160 000. Three protein sub-forms are detectable on sodium dodecyl sulphate-polyacrylamide gel electrophoresis that migrate at rates corresponding with molecular weights of 64 000, 79 000 and 92 000. Treatment of native alkaline phosphatase with 6 M guanidine - HC1 or buffer at pH 3.0 results in a product with a molecular weight of 78 000 and 70 000, respectively. Thus it is concluded that each of the three sub-forms is a dimer of identical or closely similar subunits. Limited proteolysis results in the production of new enzymically active sub-forms separable by electrophoresis. Using a bacterial protease it is possible to convert intestinal alkaline phosphatase into a form with a molecular weight of 132 000 without causing any significant change in kinetic properties. Electrophoresis of this new form on sodium dodecyl sulphate polyacrylamide gel suggests that it is composed of 66 000-dalton subunits. The native enzyme contains at least 20% by weight of carbohydrate that probably contributes to microheterogeneity of a second degree superimposed on that stemming from the presence of three protein sub-forms. Treatment with various glycosidases has no effect on electrophoretic behaviour, however. It is suggested that the three sub-forms possibly represent different stages of a maturation process that operates by limited proteolysis of a single parent protein.


Bilirubin and biliverdin binding to rat Y protein (ligandin).

The binding of bilirubin to poly(L-lysine) produces an optically active complex at pH 10.1. Circular dichroism spectra of these complexes are distinguishable from those generated by binding of bilirubin to the high affinity sites on albumin. Comparison of the circular dichroism spectra of bilirubin bound to the hepatic protein ligandin with those of bilirubin complexed with albumin or polylysine indicates that binding of bilirubin to ligandin occurs at two types of sites. These are distinguishable on the basis of their spectral properties, one resembling the high affinity site of bovine serum albumin and the other resembling polylysine. Complexes of biliverdin with albumin and ligandin bear similarities to the bilirubin-protein complexes. The native protein itself has an ordered structure which consists of 41% alpha-helix and is not altered by the binding of bilirubin.


The binding of 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate by human antithrombin III.

The binding of 1-anilino-8-naphthalenesulfonate to human antithrombin III was studied by fluorescence enhancement of the fluorophor and fluorescence quenching of the protein emission. Two molecules of 1-anilino-8-naphthalenesulfonate were found to bind per antithrombin molecule with an average dissociation constant of 4.4-10(-5) M. The binding of heparin to antithrombin was studied by ultraviolet difference spectroscopy. The stoichiometry of the heparin binding indicated 1.8 binding sites with an average dissociation constant of 4.3 - 10(-6) M. Further the fluorometric competition experiments with 1-anilino-8-naphthalenesulfonate, heparin, salicylate and caprylate indicated two different classes of anion binding sites on the antithrombin molecule.


Changes in the ultraviolet and circular dichroism spectra of aquo-, hydroxy-, azido-, and cyanocobalamin when bound to human intrinsic factor or human transcobalamin I.

The ultraviolet and the circular dichroic spectra of aquo-, hydroxy-, azido-, and cyanocobalamin free or bound to purified human intrinsic factor or purified human transcobalamin I were recorded. Except for azidocobalamin-transcobalamin I, the molar absorption for the gamma1-band in the ultraviolet spectra increased with a factor between 1.2 and 1.4 when the cobalamins were bound to protein. The ultraviolet spectrum of azidocobalamin-transcobalamin I changed so that the gamma2-band became the most intense. Minor changes were observed in the other ultraviolet spectra. The most prominent change in the CD-spectra of protein bound cobalamins was the increase in negative elipticity above 400 nm. This increase was most intense for cyano- and azidocobalamin-transcobalamin I. The study emphasizes that the mechanism by which human intrinsic factor and human transcobalamin I bind cobalamins differ.


Copper(I) and copper(II) in complexes of biochemical significance studied by X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopic measurements of copper complexes of biochemical significance were carried out to permit the conclusion whether or not copper is present in the Cu(I) or Cu(II) state. Only one single homogeneous signal in the X-ray photoelectron spectra of the Cu(I) 2p1/2 and 2p3/2 levels was seen regardless whatever Cu(I) complex was used. By contrast one more or less split satellite in addition to the main 2p copper signal appeared when Cu(II) complexes were studied. The extent of satellite splitting was dependent on the nature of the ligands coordinated with Cu(II). Thus, a strong splitting was observed in the spectra of Cu-(trifluoroacetylacetonate)2 and Cu-(biuret)2Cl2 were Cu(II) is exclusively bound to oxygen having a formal double bond. No such splitting was seen in Cu(II) chelates where the metal was bound to single bonded oxygen and/or nitrogen. It excited great interest to see that in the antiferromagnetically coupled Cu(II) complexes Cu2-(succinate)2-4H2O, Cu-(HCCO)2, CuO and in the completely diamagnetic Cu2-(u,3-diphenyltriazene)4 complex Cu(II) could be detected. The reaction of Cu(I) and Cu(II) with the thiol sulphur of either cysteine, penicillamine or alpha-mercaptopropionylglycine yielded Cu(I) complexes. During the X-ray exposure of the different samples photoreduction of Cu(II) was not observed. For example, the satellite structure of the copper 2p levels using Cu(II)-cystine remained unchanged during the measurement.


Fluorescence study of N-(3-pyrene)maleimide conjugated to rabbit skeletal F-actin and plasmodium actin polymers.

A fluorescent probe N-(3-pyrene)maleimide was conjugated to rabbit skeletal F-actin at the site of most reactive sulfhydryl group (Cys-373). Its fluorescence anisotropy decay showed a single correlation time of 560 ns at 25 degrees C, which is in a very good agreement with the correlation time of the dansyl-L-cysteine group conjugated to the same site of F-actin reported very recently [Wahl, Ph., Mihashi, K, and Auchet, J-C. (1975) FEBS Lett. 8, 164-167]. Actin from plasmodia of myxomycates, Physarum polycepharum, was also conjugated with N-(3-pyrene) maleimide and the fluorescence anisotropy was compared with rabbit skeletal F-actin using the classical steady excitation method. It was found that the internal mobility of the magnesium polymer of plasmodium actin is remarkably larger than both plasmodium F-actin and rabbit skeletal F-actin.


Protein fluorescence and electronic energy transfer in the determination of molecular dimensions and rotational relaxation times of native and coenzyme-bound horse liver alcohol dehydrogenase.

The intrinsic fluorescence lifetimes of horse liver alcohol dehydrogenase (EC 1.1.1.1) and pig heart isocitrate dehydrogenase (EC 1.1.1.42) have been determined to be 5.36 ns and 4.84 ns, respectively. When reduced coenzyme is bound, the fluorescence lifetime of alcohol dehydrogenase is reduced to 4.98 ns while that of isocitrate dehydrogenase remains unchanged. Oxidized coenzymes have no effect on fluorescence lifetimes of alcohol and isocitrate dehydrogenases. This virtual constancy of protein fluorescence lifetimes has allowed the conclusion to be reached that in protein-ligand complexes with equilibrium constants in the range of 10(4)-10(6) M(-1), the static mode of quenching is substantial. The observation of resonance energy transfer in alcohol dehydrogenase-NADH complex facilitates the determination of the distance between tryptophan and the reduced nicotinamide ring involved in the transfer as 30.6 A, compared to the effective molecular radius of 36.2 A for alcohol dehydrogenase. The increased rotational relaxation times of coenzyme-bound alcohol dehydrogenase relative to the unliganded form (sigmah = 72 ns) indicate in this protein structural fluctuations occurring in the time range of nanoseconds.


Factors affecting the luminescence of tryptophan at 77 K.

The effects of solvent polarity, heavy atoms and sulphur-containing compounds on the fluorescence and phosphorescence of indole and tryptophan have been investigated. The low-temperature luminescence of a group of dipeptides containing tryptophan is also reported. The phosphorescence to fluorescence quantum yield ratio and the phosphorescence lifetime are shown to be particularly sensitive to environmental factors. The relevance of these results to the interpretation of protein luminescence spectra is briefly discussed.


The effect of trypsin on bovine transferrin and lactoferrin.

The iron-saturated and iron-free (apo) forms of bovine transferrin and lactoferrin were digested with trypsin and the digests analysed by column chromatography and electrophoresis. Both of the iron-saturated proteins were more resistant to proteolysis than the corresponding apoproteins, and iron-transferrin was more resistant than iron-lactoferrin. Digestion of iron-transferrin yielded two iron-binding fragments with molecular weights of 32 000 and 38 500 whereas apotransferrin yielded only the larger fragment. In digests of lactoferrin, up to five different fragments with molecular weights ranging from 25 000 to 52 700 were detected, there being no obvious qualitative difference between digests of iron-lactoferrin and apolactoferrin. The susceptibility of apolactoferrin to tryptic digestion was only slightly reduced when apolactoferrin was complexed with beta-lactoglobulin, suggesting that complex-formation is not a mechanism for protecting lactoferrin against intestinal degradation. There was no immunological cross reaction between bovine transferrin or its digestion products against anti-lactoferrin antiserum, or vice-versa.


Studies on the sulfhydryl groups in type III collagen.

The Type III collagen molecule, [alpha](III)]3, is comprised of three alphal(III) chains each of which contains two cysteinyl residues. Free sulfhydryl groups, however, could not be detected in the denatured, trimeric gamma-component of Type III collagen as judged by the failure to form derivatives with the alkylating reagents iodo-[14C]acetic acid and 4-vinylpyridine. This absence of sulfhydryl group reactivity did not appear to result from disulfide-binding between Type III collagen and noncollagenous peptides. Collectively, the results indicate that all of the six sulfhydryl groups of the Type III collagen molecule participate in interchain disulfide-bonding.


One electron reduction of metmyoglobin and methemoglobin and the reaction of the reduced molecule with oxygen.

We have used the pulse radiolysis technique to reduce with solvated electrons (see article) a single Fe(III) site in methemoglobin and metmyoglobin. The reduction process was followed spectrophotometrically and the reactions rate constants were measured: (see article) =6.5 +/- 1-10(10) M-1-S-1. (see article)=2.5 +/- 0.3-10(10) M-1-S-1. Approx. 60% of the (see article) have reacted with the hemin group, and the rest of the (see article) have probably reacted with the globin moiety. We followed the reaction of the reduced proteins to yield the oxyderivatives and measured the rate constants of the oxygenation process k reduced methemoglobin + O2 = 2.6 +/- 0.6-10(7) M-1-S-1 and k myoglobin + O2 = 1.8 +/- 0.2-10(7) M-1-S-1, all the rate constants were measured at pH = 6.8, I = 0.004, T = 22 +/- 2 degrees C. The high rate constant for reduced methemoglobin indicates that one-site-reduced methemoglobin is probably in the R state, as predicted for methemoglobin from X-ray analysis. The spectra of the reduced and oxygenated species were measured under similar conditions at gamma = 450-650 nm. We were able to follow slight changes in the micro-second time scale, these changes were attributed to conformational changes. We were not able to detect any reaction between the radical (see article) and the hemin group (which would result in a complex such as heme O-2). This may be due to kinetic reasons.


Renaturation of formyltetrahydrofolate synthetase from urea and guanidinium chloride solutions.

Formyltetrahydrofolate synthetase from Clostridium cylindrosporum and Clostridium acidi-urici was denatured in 6 M urea and 4 M guanidinium chloride. Viscometric, fluorimetric and ultracentrifugal measurements were used to determine that the protein is completely unfolded under these conditions. The polypeptide chains refold upon dilution of the denaturant-protein solutions to give final concentrations of 0.5 M urea or 0.1 M guanidinium chloride. In the presence of NH4+, but not in its absence, the refolded proteins associate to produce the catalytically active tetramer. Refolding and reassociation were followed by measuring changes in protein fluorescence and by determination of sedimentation constants. Under most conditions 80% of the enzymic activity is recovered.


Isolement et caractérisation de deux subunites constitutives des glycoproteines de structure du tissu sous cutané de lapin.

Structural glycoproteins have been extracted by 8 M ureau from the insoluble residue remaining after collagenase digestion of rabbit dermis and purified by Sepharose 4 B chromatography. After reduction and alkylation, Dowex 1 x 2 chromatography allowed separation of two structural glycoproteins (D1 and D2) in an homogenous state as shown by chromatographic and electrophoretic behaviour as well as N terminal amino acid determination. These two glycoproteins have a molecular weight of about 16 000. Their amino acid compositions (very similar), are characterized by a high level of dicarboxylic amino acids and the absence of hydroxyproline and hydroxylysine. The less acidic glycoprotein (D1) has glycine for N terminal amino acid and contains 10.4 percent of bound carbohydrates. The glycoprotein D2 contains 5.1 percent of bound carbohydrates and its N terminal amino acid is glutamic acid.


Covalently closed minicircular DNA associated with Acetabularia chloroplasts.

When Acetabularia cliftonii chloroplast DNA (p = 1.706 g/cm3) is centrifuged in an ethidium bromide-CsCl gradient, the lower band is enriched for DNA with a buoyant density of 1.712 g/cm3 containing small covalently closed circular molecules. The minicircles measure 4.15 +/- 0.30 mum in the closed conformation and 4.35 +/- 0.20 mum in the open conformation. They are not of nuclear or bacterial origin, and appear to exist as independent entities within the chloroplast, although a mitochondrial origin cannot be completely ruled out. No 40-45 mum circles, as found in other chloroplasts, were found in either ethidium bromide-CsCl fraction. None were found in total chloroplast DNA by any of a number of methods tried. Linear molecules up to 200 mum were measured in chloroplast lysates. The main chloroplast genome may consist of very large circular molecules which are broken by even small shear forces.


Control of transfer RNA synthesis in the presence of inhibitors of protein synthesis.

The rate of synthesis of transfer RNA in suspension cultures of Chang's liver cells, has been examined in the presence of various inhibitors of protein synthesis with different modes of action. Inhibitors of polypeptide chain elongation such as cycloheximide and emetine stimulated the rate of synthesis of transfer RNA at concentrations that inhibited protein synthesis by 60-90%. Trichodermin, an inhibitor of the elongation and termination steps in protein synthesis, had as effect similar to that of cycloheximide and emetine. On the other hand verrucarin, an inhibitor of initiation, and puromycin, an analogue of the aminoacyl terminus of tRNA, had little effect of the synthesis of transfer RNA at low concentrations. At high concentrations these compounds inhibited transfer RNA synthesis. Inhibitors of protein synthesis can be divided in two groups based on their effect on the polysom pattern. The first group increased the number of large polysomes, while verrucarin and puromycin lead to a break down of large polysomes and to an accumulation of monosomes and small polysomes. Thus, there appears to be a correlation between the effect of these inhibitors of protein synthesis on the aggregational state of the polysomes and their effect on the rate of transfer RNA synthesis.