Papain inhibition by serum.

Sera from seven animal species (rat, cow, cat, dog, human, rabbit, and hamster) were tested and found to inhibit the papain-catalyzed hydrolysis of alpha-N-benzoyl-L-arginine-p-nitroaniline-HCl (L-BAPA). The relative concentration of inhibitor in each serum sample was expressed in terms of its papain inhibitory capacity (PIC) defined as the number of units of papain inhibited per ml of serum. Rat serum contained the highest concentration of inhibitor while hamster serum contained the lowest concentration. The inhibitor appears to be competitive with respect to L-BAPA and is heat labile and nondialyzable. Antipapain activity can be separated from antitrypsin activity by (NH4)2SO4 fractionation.


PO2 in the carotid body perfused and/or superfused with cell-free media.

We measured with a micro-O2 electrode the tissue PO2 (PtO2) in the cat carotid body (CB) to see whether it was adequately oxygenated when perfused or superfused with artificial, cell-free (c-f) solutions (pH = 7.4; temp = 35-38 degrees C). To obtain a relative measure of O2 consumption (VO2), we also measured the rate of disappearance of O2 following stoppage of the blood flow, and compared these disappearance curves with those during stoppage of the c-f perfusion solutions. In 14 cats normal (blood perfusion) PtO2 values ranged from 10 to 104 mmHG; chi = 72 +/- 4 (SE--as used throughout). During 3 h of c-f perfusion with air-equilibrated Locke's solution, PtO2 ranged from 62 to about 160 mmHg; chi = 133 +/- 4. When perfused with Fay's equilibrated with 98% O2-2% CO2 no PtO2 values in the CB were below 300 mmHg (4 cats). In eight additional cats the CB was cleared of blood then superfused with saline equilibrated with 50% O2 underneath and air-equilibrated saline over. Less than 5% of the PtO2 values found were below 5 mmHg. We conclude that most studies on the artificially perfused or superfused CB cannot be invalidated on the basis that the preparations were hypoxic. O2 disappearance curves taken during blood perfusion were significantly faster than during c-f perfusion indicating a marked reduction in VO2 with c-f perfusion.


Xenon and nitrogen single-breath washout curves in patients with airway obstruction.

In patients with chronic obstructive lung disease, we determined single-breath N2 and 133 Xe washout curves, and regional distributions of volumes (Vr) and of 133Xe boluses inhaled at residual volume (VIRV). Patients suffering from emphysema with minimal airway obstruction demonstrated large closing volumes and apicobasal distribution gradients, apparently because of a steep pulmonary recoil pressure-volume curve. In one subject with basal small airway disease there was no vertical gradient in regional residual volume; closing volume was increased with the 133Xe technique but almost absent with the N2 technique. Patients with moderate-to-severe airway obstruction had upward-sloping alveolar plateaus without distinct phase IV, and small apicobasal differences in Vr and VIRV. The latter resulted probably from increased regional differences in time constants counteracting the influence of gravity. Finally, patients with severe airway obstruction and basal emphysema demonstrated a rising N2 but a descending 133Xe plateau; the gradient for VIRV was normal, and reversed for Vr. This pattern was attributed to nongravitational differences in time constants causing a first in-first out distribution.


Maximal oxygen uptake during exercise with various combinations of arm and leg work.

Oxygen uptake (VO2) was determined in 10 males during the following types of maximal exercise (work time: about 5 min): uphill running, bicycling, arm work (cranking), and combined arm work and bicycling (A + L). The A + L exercise was performed in four different ways, the arms doing 10%, 20%, 30%, or 40% of the same total rate of work; and also with the maximal bicycle work load plus either maximal or submaximal arm work. VO2 was the same in running as in all types of A + L exercise, except when the arm work load was 10% and 40% of the total rate of work, where VO2 was 2.5% (P less than 0.05) and 9.4% (P less than 0.001) lower, respectively. Bicycle VO2 was lower than VO2 in running but equal to A + L VO2 when arm work intensity was 40% of the total rate of work. It is concluded that VO2 during maximal exercise a) to a certain extent depends on the exercising muscle mass, b) is lower than the oxygen-consuming potential of the muscles involved in A + L exercise, and c) in A + L exercise is influenced by the ratio of arm work to total rate of work and the subject's fitness for arm work and bicycling.


Uteroplacental blood flow during hyperthermia with and without respiratory alkalosis.

The effect of induced maternal hyperthermia (1.5 degrees C rise over 60 min) on the uterine and umbilical circulations was examined in two groups of chronically instrumented pregnant sheep. Hyperventilation occurred in both groups. In the group in which the resultant respiratory alkalosis was untreated (N = 5), uterine blood flow decreased to 53 +/- 3% (mean +/- SE; P less than 0.01) of control at maximal maternal temperature. Umbilical blood flow also decreased 30 +/- 6% (P less than 0.01) below control levels. In the other group, normocapnia was maintained (N = 4). Uterine blood flow decreased in this group to 75% +/- 2% (P less than 0.01) of control levels, but umbilical blood flow did not significantly change. There was no significant change in maternal or fetal mean arterial pressure during hyperthermia in either group. Thus, maternal hyperthermia per se results in a significant decrease in uterine blood flow primarily through an effect on uterine vascular resistance, but without a concomitant change in umbilical blood flow.


Effect of ascorbic acid on rate of heat acclimatization.

There is some indication in the literature that ascorbic acid (vitamin C) may reduce the physiological responses to heat stress. Consequently, the effect of ascorbic acid ingestion on heat-strain indicators has been studied on a group of 60 mining recruits undergoing climatic room acclimatization. Of the 60 men, 19 received a daily dose of 250 mg ascorbic acid; 21 a daily dose of 500 mg ascorbic acid; and 20 received a placebo daily. Measurements of rectal temperature, heart rate, and hourly sweat rate were made on all subjects during the 4 h of heat exposure per day for 10 days. The wet bulb temperature was 32.2 degrees C, the dry bulb 33.9 degrees C, the air movement 0.4 m/s, and the work rate 35 W. The results indicate that the rate and degree of acclimatization, as assessed by 4th-h rectal temperature, is enhanced by ascorbic acid supplementation and that no differences in response could be shown between daily dosages of 250 and 500 mg of vitamin C.


Platelet antiserum inhibits hypoxic pulmonary vasoconstriction in the dog.

The literature suggests that platelets might help mediate the pulmonary vascular pressor response to hypoxia. This study evaluated the hypoxic response in dogs rendered acutely thrombocytopenic by the administration of platelet antiserum. Between 30 and 90 min after the antiserum the pulmonary vasoconstrictor response to hypoxia was virtually abolished, but subsequently returned at a time when the number of circulating platelets remained very low. The prior administration of meclofenamate completely preserved the hypoxic response, though the platelet count still fell precipitously. We conclude that circulating platelets are not necessary for hypoxic vasoconstriction. It is possible that the reaction between platelets and antiserum evokes the synthesis of a dilator prostaglandin which might be responsible for the temporary inhibition of the pressor response to hypoxia but this remains to be proven.


Influence of muscular development, obesity, and age on the fat-free mass of adults.

Body fat and the fat-free mass (FFM) were estimated in 36 men and 43 women deliberately chosen to represent a variety of physical types; these were 1) young sedentary, 2) "muscular," 3) younger obese, 4) older obese, and 5) older nonobese individuals of both sexes. The body fat and the FFM were estimated from measurements of body density (by total immersion in water, measurement being made of the residual volume of air present in the lungs at immersion) and from measurements of total body potassium (using a whole-body monitor to assess the natural 40K isotope present in the body). The muscular men and women and the younger obese men and women had a considerably greater FFM and thus had greater quantities of potassium than the corresponding sedentary groups. There were significantly different estimates of the FFM calculated from density and from total body K in three groups, the sedentary young men, the muscular, and the younger obese women. The density and the potassium content of the FFM appear to decline with obesity and aging. Muscular development is associated with a decrease in the density but an increase in the potassium content of the FFM.


Effects of water immersion on renal hemodynamics in normal man.

Although previous studies have demonstrated that water immersion to the neck (NI) results in a significant natriuresis and diuresis, the mechanisms are incompletely delineated. Since recent studies have demonstrated that NI induces a marked increase in cardiac index, it is possible that, as a consequence, renal hemodynamics may be augmented markedly during NI thereby mediating the encountered renal changes. The present study was undertaken to delineate the effects of NI on renal plasma flow and glomerular filtration rate as assessed by the clearance of PAH (CPAH) and inulin (CIn), respectively. Nine normal male subjects were studied on two occasions, control and NI. The conditions of seated posture and time of day were identical. Immersion did not alter CIn or CPAH at a time when sodium excretion was increasing markedly. The constancy of CPAH during NI suggests that renal blood flow is unaltered and that the natriuresis of NI is mediated independently of alterations in overall renal perfusion. The sluggish decline of a naturiuresis during recovery is consistent with the presence of a humoral factor contributing to the encountered natriuresis.


Effect of gas physical properties and flow on lower pulmonary resistance.

We measured lower pulmonary resistance (Rlp) in eight dogs and three men breathing gas mixtures having different densities (p) and similar viscosities (mu). Rlp increased with gas density and with flow rate (V). In the dogs, these effects were not observed in lung segments subtended from 4-mm-ID bronchi; in more central airways, resistance varied approximately as (mup V)0.5. These results are compatible with Poiseuille flow in peripheral airways, and, in central airways, with flow resistance described by the equation of boundary layer growth. Rather than two discrete flow regimes, it is likely that flow patterns undergo a continual metamorphosis as Reynolds' numbers (Re) decrease between trachea and alveoli. Accordingly, the airways pressure-flow relationship is not described by any single fluid dynamics equation, but may be explained by the general equation, P = Kmu2-apa-1Va, where a reflects the proportion of inertial to viscous pressure losses and varies between 1 and 2 according to Re. Rohrer's equation described the observed pressure-flow relationships and predicted the change in Rlp with gas physical properties, suggesting a physical basis underlying this adequate mathematical description.


Tissue oxygen tension in rabbits measured with a galvanic electrode.

A galvanic electrode which generates current in response to oxygen was used to measure tissue PO2 in 10 rabbits. Sixteen electrodes were calibrated in vitro at PO2 = 0 mmHg and PO2 = 100 mmHg before implantation into muscle and peritoneal cavity. Electrode calibration was checked after removal 7-20 days later; mean +/- SE reading at PO2 = 0 was 1.5 +/- 1.83 mmHg and at PO2 = 100 was 98.1 +/- 6.49 mmHg. Continuous recordings of tissue PO2 showed little fluctuation in unanesthetized rabbits resting quietly. Tissue PO2 was lower than arterial or venous PO2 during air breathing but frequently exceeded venous PO2 during O2 breathing. Highly significant correlations (P less than 0.001) were found between blood and tissue PO2 over a wide range of values obtained during air breathing and during 10%, 50%, and 100% O2 breathing. Thus, the galvanic electrode provided a reliable measure of tissue PO2 and maintained its stability in vivo for periods as long as 20 days.


Correction for cardiac output calculations by the nitrous oxide technique.

Calculations of cardiac output using the N2O technique as described by Becklake et. al. (J. Appl. Physiol. 17: 579, 1962) result in spurious values when barometric pressure or body temperature deviates from 760 Torr or 37.0 degrees C, respectively. Nitrous oxide (N2O) uptake (VN2O) must be corrected to standard pressure, dry at 37.0 degrees C.


A new method of measuring skin humidity.

A device suitable for the long-time measurement of relative skin humidity is described. This is a simple circuit with a resistance bridge for lithium chloride sensors connected to a digitally steered logic circuit, which causes dried air to stream intermittently through a measuring chamber placed on the skin in such a way that the relative humidity over the skin is maintained at a constant level. The number of switching time periods is proportional to the relative humidity (secretion performance) of the skin and can be counted, recorded, or directly fed into a digital calculator. The apparatus, including a two-channel version with skin temperature recording, has proved useful in sleep investigations under extreme climatic conditions.


A development of the oxyhemoglobin dissociation curve analyzer.

The development and improvement of an oxyhemoglobin dissociation curve analyzer is described. PO2 electrode performance was improved by electrochemical means and circuits are described for processing the PO2 and pH signals from the analyzer. A circuit for automatically correcting the curve for Bohr shifts from pH 7.40 is described, and the performance of the Bohr shift unit is verified by experiment. The analyzer produces curves under standard conditions of PCO2 40 mmHg, pH 7.40, and 37 degrees C.


Method for chronic recording of electroencephalographic activity in conscious dogs.

The difficulties involved in recording high-quality electroencephalograms chronically from conscious dogs have been resolved by devising an implantable pure silver electrode assembly that can be screwed permanently into the skull. The assembly eliminates electromyographic artifacts and provides high-fidelity tracings. Construction is simple and commercially available, inexpensive parts are used. Highly satisfactory records can be obtained over periods of months.


Identification of several unique, low-molecular-weight basic proteins in dormant spores of clastridium bifermentans and their degradation during spore germination.

Two acid-soluble, low-molecular-weight basic proteins comprise approximately 20% of the protein in dormant spores of Clostridium bifermentans. Both of these proteins are rapidly degraded during spore germination.


Bacterial characteristics determining the potential host range of bacteriophage T4.

Many members of the Enterobacteriaceae, but not other gram-negative organisms, apparently carry a specific recognition site for the T4 tail tube on their cytoplasmic membranes.


Cellular localization of uridine 5'-diphosphate-N-acetyl-D-glucosamine oxidoreductase activity in Citrobacter freundii.

The uridine 5'-diphosphate-N-acetyl-d-glucosamine oxidoreductase activity of Citrobacter freundii ATCC 10053 was found to be located in the soluble cytoplasmic fraction of lysed spheroplasts.


Hydrogenase and ribulose diphosphate carboxylase during autotrophic, heterotrophic, and mixotrophic growth of scotochromogenic mycobacteria.

Two key autotrophic enzyme systems, hydrogenase and ribulose diphosphate carboxylase, were examined in Mycobacterium gordonae and two other chemolithotrophic, scotochromogenic mycobacteria under different cultural conditions. In all three organisms both enzymes were inducible and were produced in significant levels only in the presence of the specific substrate, hydrogen or carbon dioxide. M. gordonae exhibited increased growth rates and yields, indicating mixotrophic growth, in the presence of a number of single organic substrates, including acetate, pyruvate, glucose, fructose, and glycerol. In contrast to other aerobic hydrogen autotrophs, the presence of either acetate or pyruvate did not repress ribulose diphosphate carboxylase, and mixotrophic growth was rapid with these substrates. In the absence of carbon dioxide, growth in glycerol medium under an atmosphere of hydrogen and oxygen was severely inhibited, even with cells preadapted to heterotrophic growth on glycerol. Cyclic adenosine monophosphate was not effective in inducing hydrogenase or carboxylase in heterotrophic, mixotrophic, or hydrogen-inhibited cultures.


Phosphorylation of glycerol and dihydroxyacetone in Acetobacter xylinum and its possible regulatory role.

Extracts of Acetobacter xylinum catalyze the phosphorylation of glycerol and dihydroxyacetone (DHA) by adenosine 5'-triphosphate (ATP) to form, respectively, L-alpha-glycerophosphate and DHA phosphate. The ability to promote phosphorylation of glycerol and DHA was higher in glycerol-grown cells than in glucose- or succinate-grown cells. The activity of glycerol kinase in extracts is compatible with the overall rate of glycerol oxidation in vivo. The glycerol-DHA kinase has been purified 210-fold from extracts, and its molecular weight was determined to be 50,000 by gel filtration. The glycerol kinase to DHA kinase activity ratio remained essentially constant at 1.6 at all stages of purification. The optimal pH for both reactions was 8.4 to 9.2. Reaction rates with the purified enzyme were hyperbolic functions of glycerol, DHA, and ATP. The Km for glycerol is 0.5 mM and that for DHA is 5 mM; both are independent of the ATP concentration. The Km for ATP in both kinase reactions is 0.5 mM and is independent of glycerol and DHA concentrations. Glycerol and DHA are competitive substrates with Ki values equal to their respective Km values as substrates. D-Glyceraldehyde and l-Glyceraldehyde were not phosphorylated and did not inhibit the enzyme. Among the nucleotide triphosphates tested, only ATP was active as the phosphoryl group donor. Fructose diphosphate (FDP) inhibited both kinase activities competitively with respect to ATP (Ki= 0.02 mM) and noncompetitively with respect to glycerol and DHA. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) inhibited both enzymic activities competitively with respect to ATP (Ki (ADP) = 0.4 mM; Ki (AMP) =0.25 mM). A. xylinum cells with a high FDP content did not grow on glycerol. Depletion of cellular FDP by starvation enabled rapid growth on glycerol. It is concluded that a single enzyme from A. xylinum is responsible for the phosphorylation of both glycerol and DHA. This as well as the sensitivity of the enzyme to inhibition by FDP and AMP suggest that it has a regulatory role in glycerol metabolism.


Regulation of molybdate transport by Clostridium pasteurianum.

The regulation of the molybdate (MoO42-) transport activity of Clostridium pasteurianum has been studied by observing the effects of NH3, carbamyl phosphate, MoO42-, and chloramphenicol on the ability of cells to take up MoO42-. Compared with cells fixing N2, cells grown in the presence of 1 mM NH3 are greater than 95% repressed for MoO42- transport. Uptake activity begins to increase just before NH exhaustion (under Ar or N2) and continues to increase throughout the lag period as cells shift from NH3-growing to N2-fixing conditions. When cells are shifted from N2-fixing to NH3-growing conditions the transport activity per fixed number of cells decreases by increase of bells in absence of transport synthesis. Carbamyl phosphate (greater than or equal to 15 mM) but not NH3 inhibits 58% of the in vitro uptake activity. When 1 mM carbamyl phosphate is added just before the exhaustion of NH3, the transport activity, measured 2 h later, is 100% repressed. Cells grown in the presence of high MoO42- (1mM) are 80% repressed for MoO42- transport. Synthesis of the MoO42- transport system is also completely stopped when chloramphenicol (300 mug/ml) is added just before the exhaustion oNH 3 from the medium. These findings demonstrate that the ability of cells to transport MoO42- is dependent upon new protein synthesis and can be repressed by high levels of substrate. The regulation of MoO42- uptake by NH3 or carbamyl phosphate closely parallels the regulation of nitrogenase activity. Activity of neither nitrogenase component (Fe protein or MoFe protein) was detected even 3 h after the exhaustion of the NH3 if either MoO42- was absent or if WO42- was present in place of MoO42-. The duration of the diauxic lag increases with decreasing concentration of MoO42- in the medium. If no MoO42- is present the lag continues indefinitely. If MoO42- is added late in the lag period, growth under N2-fixing conditions resumes but only after a normal induction period.


Characterization of an Acholeplasma laidlawii variant with a REP- phenotype.

An Acholeplasma laidlawii variant has been isolated that has a REP- phenotype. The properties of this variant, relative to parental cells, are: (i) it exhibits no change in cell growth kinetics; (ii) it does not propagate single-stranded deoxyribonucleic acid (DNA) mycoplasmaviruses but does propagate double-stranded DNA mycoplasmaviruses; (iii) it converts parental circular single-stranded mycoplasmavirus DNA to double-stranded replicative forms that are not replicated further; (iv) it exhibits no change in host modification and restriction; and (v) it has an increased ultraviolet light sensitivity. The REP- isolate is the first stable mycoplasma variant to which a physiological defect has been attributed.


Properties of pGC1, a lac plasmid orginating in Yersinia enterocolitica 842.

pGC1, a self-transmissible lac plasmid that originated in Yersinia enterocolitica 842, is described. This plasmid is freely transmissible between strains of Y. enterocolitica and from such strains to strains of Escherichia coli. The plasmid can also be transferref among G. coli strains and back to Y. enterocolitica, although at a greatly reduced frequency. pGC1 has a molecular weight of about 33.0 x 10(6), has a base composition of about 44 mol% guanine plus cytosine, and is fi-.


Catechol oxygenases of Pseudomonas putida mutant strains.

Investigation of a mutant strain of Pseudomonas putida NCIB 10015, strain PsU-E1, showed that it had lost the ability to produce catechol 1,2-oxygenase after growth with catechol. Additional mutants of both wild-type and mutant strains PsU-E1 have been isolated that grow on catechol, but not on benzoate, yet still form a catechol 1,2-oxygenase when exposed to benzoate. These findings indicate that either there are separately induced catechol 1,2-oxygenase enzymes, or that there are two separate inducers for the one catechol 1,2-oxygenase enzyme. Comparisons of the physical properties of the catechol 1,2-oxygenases formed in response to the two different inducers show no significant differences, so it is more probable that the two proteins are the product of the same gene. Sufficient enzymes of the ortho-fission pathway are induced in the wild-type strain by the initial substrate benzoate (or an early intermediate) to commit that substrate to metabolism by ortho fission exclusively. A mechanism exists that permits metabolism of catechol by meta fission if the ortho-fission enzymes are unable to prevent its intracellular accumulation.


Involvement of 4-hydroxymandelic acid in the degradation of mandelic acid by Pseudomonas convexa.

A microorganism capable of degrading DL-mandelic acid was isolated from sewage sediment of enrichment culture and was identified as Pseudomonas convexa. It was found to metabolize mandelic acid by a new pathway involving 4-hydroxymandelic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid as aromatic intermediates. All the enzymes of the pathway were demonstrated in cell-free extracts. L-Mandelate-4-hydroxylase, a soluble enzyme, requires tetrahydropteridine, nicotinamide adenine dinucleotide phosphate, reduced form, and Fe2+ for its activity. The next enzyme, L-4-hydroxymandelate oxidase (decarboxylating), a particulate enzyme, requires flavine adenine dinucleotide and Mn2+ for its activity. A nicotinamide adenine dinucleotide-dependent, as well as a nicotinamide adenine dinucleotide phosphate-dependent, benzaldehyde dehydrogenase has been resolved and partially purified.


Genetic linkage of chromosomal tetracycline resistance and pigmentation to a purine auxotrophic marker and the isoleucine-valine-leucine structural genes in Staphylococcus aureus.

Three tetracycline resistance determinants (tmn-3106, tmn-3110, and tmn-3511) reported by Asheshov (1975) to be chromosomal in Staphylococcus aureus have been linked by transformation to a purine auxotrophic marker (pur-110), a cluster of eight genes involved in the biosynthesis of isoleucine, valine, and leucine (the ilv-leu region), a marker (ilvR10) that may be involved in the regulation of the ilv-leu region, and a gene involved in pigmentation (pig-131). The linkage group thus defined is tmn-3106-pur-110-ilvR10-(ilv-leu)-pig-131. The orientation of the ilv-leu region relative to ilvR10 and pig-131 was not determined. The tmn-3106, tmn-3110, and tmn-3511 determinants exhibit the same linkage relationships to the other markers. It is concluded that this linkage group represents a portion of the chromosome of S. aureus.


Plasmid-protein relaxation complexes in Staphylococcus aureus.

Protein-deoxyribonucleic acid relaxation complexes have been demonstrated for six Staphylococcus aureus plasmids out of sixteen examined. Four of these encode stretomycin resistence, have molecular weights of about 2.7 x 10(6), and are isolated as supercoiled molecules that are virtally 100% relaxable by treatment with sodium dodecyl sulfate. It is probable that these four isolates represent a single widely disseminated plasmid species. The other two plasmids showing relaxation complexes have molecular weights of about 3 x 10(6) and encode chloramphenicol resistance. The complexes in these cases are unstable, and it has not been possible to induce more than 50% relaxation by any of the standard treatments. Ten other plasmids do not show detectable complexes. These include three penicillinase plasmids, four tetracycline-resistance plasmids, one plasmid carrying kanamycin-neomycin resistance, and finally, two chloramphenicol-resistance plasmids.


XYL, a nonconjugative xylene-degradative plasmid in Pseudomonas Pxy.

Pseudomanas Pxy metabolizes p- or m-xylene through intermediate formation of the corresponding methylbenzyl alcohol and toluic acid via the meta pathway. The strain Pseudomonas Pxy spontaneously loses its ability to grow with xylene or toluate, and the rate of loss of this ability is greatly enhanced by treatment of the cells with mitomycin C. The assay of enzymes involved in xylene degradation in xylene-negative Pxy cells indicates the loss of the entire enzyme complement of the pathway. The genes specifying all the xylene-degradative enzymes, including those of the meta pathway, appear to be borne on a nonconjugative plasmid and can be transferred to xylene-negative Pxy or P. putida strain PpG1 cells only in the presence of a transfer plasmid termed factor K. When transferred to strain PpG1, the xylene-degradative plasmid, termed XYL, coexists stably with factor K, but transduction of XYL is not accompanied by a cotransfer of factor K. XYL appears to be compatible wit- all the other known degradative plasmids in P. putida. The xylene pathway is inducible in wild-type Pxy as well as in Pxy and PpG1 exconjugants, suggesting the cotransfer of regulatory genes along with the plasmid. The enzymes converting xylene to toluate are induced by xylene, methylbenzyl alcohol, or the aldehyde derivatives but not significantly by toluate, whereas catechol dioxygenase and other enzymes are induced by toluates and presumable by xylene as well.


Ultrastructure and elemental composition of dormant and germinating Diplodia maydis spores.

The ultrastructure of Diplodia maydis spores was studied in thin sections with a transmission electron microscope. Storage vacuoles were evenly distributed in the two cells. Some of the vacuoles that contained a dense osmiophilic sphere(s) were surrounded by a membrane, and had membranous aggregates around their periphery. The sport wall was composed of an electron-dense layer and an electron-translucent layer. An inner cytoplasmic membrane was present. Dormant and germinating spores were studied with scanning electron microscopy and also with a Si (Li) energy-dispersive X-ray analyzer. The dormant spore was ovate and usually two-celled with a central septum. Germination proceeded via a germ tube from the side of one end of the cell. Of several methods for preparation of specimens for X-ray analysis studied, freeze-dried spores mounted on carbon stubs and then further carbon coated gave the best results. X-ray analyses revealed that spore populations contained large amounts of Si, P, Cl, and K, smaller amounts of S and Ca, and trace amounts of Mg and Al. Analyses of single spores revealed high K and Cl and low P and Mg at one end of the cell with concomitant low K and Cl and high P and Mg in the central portion and other end of the cell. In two-celled germinating spores, high K and Cl occurred in the end of the nongerminating spore cell, whereas the germinating cell contained high P and Mg and low K and Cl. X-ray image maps revealed that K and Cl were located together at one end of the spore.


Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.


Motility as a morphogenic character in the genus Arthrobacter.

Motility in Arthrobacter atrocyaneus, A. citreus, and A. simplex was found to correlate with the morphogenic cycle of these organisms. The percentage of the A. atrocyaneus and A. simplex populations that were flagellated at a given time during the growth cycle differed significantly from that of the normorphogenic Pseudomonas aeruginosa population. Flagellation in A. atrocyaneus was shown to be dependent upon the morphogenic cycle rather than upon growth. The commitment to flagellar synthesis in A. atrocyaneus was found to occur only after induction to the rod morphology. Flagellar synthesis in A. atrocyaneus was shown to be restricted to only a small segment of the morphogenic cycle.


Isolation and characterization of the rodlet layer of Trichophyton mentagrophytes microconidial wall.

The rodlet layer of the microconidial wall of Trichophyton mentagrophytes was isolated and partially characterized. The purified microconidial walls were first extracted with urea (8M), mercaptoethanol (1%), and sodium dodecyl sulfate (1%) followed by enzymatic digestion with glusulase (snail intestinal enzymes) and purified (1 leads to 3)-beta-D-glucanase and chitinase. The purified rodlet layer was 15 to 30 nm thick and accounted for approximately 10% of the original wall weight. The pattern of rodlet patches, as revealed by electron microscopy of freeze-etched preparations of the isolated layer, was essentially the same as that observed on the intact microconidial wall. The rodlet layer was found to be resistant to most of the common organic solvents, cell wall lytic enzymes, mild acid treatments, and surface-active agents, but was solubilized in boiling 1 N NaOH with concomitant disorientation of the rodlet patterns. A melanin or melanin-like pigment appeared to be intimately associated with this rodlet layer and was solubilized during a hot-alkali treatment. Protein (80 to 85%) and glucomannan (7 to 10%) were the major components of the rodlet layer. The rodlet layer did not contain any appreciable amounts of lipid or phosphorus.


Electron microscope studies of conditional spore cortexless mutants of Bacillus sphaericus.

Spore cortex of conditional cortexless mutants of Bacillus sphaericus 9602 was not detectable by electron microscopy unless the medium was supplemented with meso-alpha,epsilon-diaminopimelic acid during sporulation. Other spore structures appeared normal. Spore shape was quite irregular in the absence of meso-alpha,epsilon-diaminopimelic acid.


Replication region fragments cloned from Flac+ are identical to EcoRI fragment f5 of F.

The replication region fragments from Flac(+) cloned in plasmids pSC138 and pML31 are identical with each other and with EcoRI fragment 5 of plasmid F.


Influence of secretory immunoglobulin A and purified secretory component on dextran-sucrase activity of Streptococcus mutans.

The net stimulation of dextransucrase EC 2.4.1.5) activity from Streptococcus mutans HS6 by dextran, secretory immunoglobulin A, or secretory component was investigated. Approximately equal stimulation resulted from treatment with these three components.


Studies on the subsite structure of amylases. III. Inhibition by gluconolactone of the hydrolysis of maltodextrin catalyzed by glucoamylase from Rhizopus niveus.

Inhibition by gluconic acid-1 : 5-lactone (gluconolactone) and phenyl alpha-glucoside of the hydrolysis of maltodextrin catalyzed by glucoamylase [EC 3.2.1.3] from Rhizopus niveus was investigated in relation to the subsite structure of the enzyme. Inhibition by gluconolactone was of the mixed type, whereas that by phenyl alpha-glucoside was purely competitive. These inhibition types were consistent with a theoretical prediction based on the assumption that gluconolactone and phenyl alpha-glucoside bind mainly to Subsites 1 and 2, respectively. The inhibitor constant of gluconolatone was determined to be 1.5 mM, which is in good agreement with the dissociation constant estimated by difference spectrophotometry (1.5 mM) (Ohnishi, M, et al. (1975) J. Biochem, 77, 695-703).


Separation of myosin subfragment-1 into fractions containing g1 chani and g2 chain by Sepharose-adipic acid hydrazide-ATP column chromatography.

Subfragment-1 prepared by chymotryptic digestion of myosin was applied to a column of Sepharose-adipic acid hydrazide-ATP in 1 mM EDTA, 10 mM Tris-HCL (PH 7.6), and 40 mM KCL. Ninety-nine per cent of subfragment-1 was adsorbed on the column in this medium. Fourty-three per cent of the applied protein was eluted with 6 mM ADP in the above buffer and then 52% was eluted with 1 mM EDTA, 10 mM Tris-HCL (pH 7.6), AND 0.7 M KCL. The former fraction contained g3 chain and the latter g1 chain. These fractions were apparently the same as the components, p2 and p1, respectively, isolated by ion-exchange chromatography using DEAE-cellulose (Yagi & Otani (1974) J. Biochem. 76, 365-373). No significant difference of ADP binding was found between the two fractions, both could bind about 0.5 mole per 10(5) g of protein. The preparation of the two subfragment-1 fractions is described.


Spectral intermediates during the reduction of hepatic microsomal cytochrome P-450.

The slow reduction of microsomal cytochrome P-450 by dithionite consists of an initial fast and then a slow phase. During reduction of aniline and azide complexes with cytochrome P-450, an intermediate spectrum developed in the fast phase and changed to that of the reduced form in the slow phase. Only the spectra in the slow phase had an isosbestic point. No intermediate spectrum detectable during reduction of the cyanide complex and native-cytochrome P-450. Carbon monoxide accelerated the reaction, causing complete reduction in the initial phase. The electron spin resonance spectrum of cytochrome P450 was greatly reduced in the initial phase of reduction with dithionite. These results indicate that reduction of the aniline and azide complexes of cytochrome P-450 involves two steps: first reduction of cytochrome P-450 and then some changes in reduced state. The aniline and cyanide difference spectra of reduced cytochrome P-450 showed peaks at 423 nm and 429 nm, respectively, while that of azide had a peak at 445 nm and a trough at 404 nm. An essay method to obtain the difference spectrum of reduced minus oxidized cytochrome P-450 using a spectral data processor is reported. The effects of other NADH-nonreducible pigments on the spectrum is eliminated by this procedure, provided these pigments are rapidly reduced by dithionite. Therefore, the spectrum obtained was slightly differed from that measured by the usual method, especially in the region of 425 nm.


The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venon and characterization of the new active intermediates.

Bovine prothrombin was activated, in both the absence and presence of dissopropyphosphofluoridate (DEP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecysulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28,000 and 57,000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Intermediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong alpha-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35,000 of 27,000 daltons. The former was indentified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocaralytically yielded a fragment (inner fragment) of 14,000 daltons with N-terminal serine and the clotting enzyme alpha-thrombin [EC 3.4.21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture. On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin III, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51,000 and 34,000 daltons, of which the former consisted of N-terminal fragment, the inner fragment, and thrombin A chain, and the latter was thrombin B chain. This new prothrombin derivative, named prothrombin ECV, formed a high-molecular-weight complex, associating with antithrombin III. The complex was not dissociable even in the presence of SDS. Moreover, prothrombin ECV reacted with p-nitrophenyl p'-guanidinobenzoate. On the basis of the results described above, the mechanism of activaton of prothrombin by Echis carinatus venom activator can be summarized as follows: The venom activator first cleaves an Arg-Ile bond liniking thrombin A and B chains in the zymogen molecule, forming an active derivative, prothrombin ECV. This active derivative converts autocatalytically to intermediate ECV, liberating the N-terminal fragment, and active intermediate ECV generates alpha-thrombin, releasing the inner fragment. Thus, only a single peptide bond cleavage along the polypeptide chain of prothrombin is associated with activation by the venom activator...


Hybrid enzyme of liver phosphorylase and phosphorylase I.

A hybrid enzyme (LI) of liver phosphorylase [EC 2.4.1.1.] (L) and phosphorylase I, which is mainly located in brain, was isolated and its enzymatic and immunological properties were examined. The following results were obtained: (1) AMP stimulated the b forms of the hybrid (LIb), I(Ib), and L(Lb); (2) in the presence of AMP, SO42- stimulated Lb more than LIb and inhibited Ib; (3) in the absence of AMP, SO42- stimulated all three isozymes in the order: Ibeta less than LI less than Lbeta; (4) on conversion to the a forms, the activities of L, LI, and I increased 35.5-fold, 3-fold, and 1.2-fold, respectively; (5) the relative inhibition potencies of anti-Lb antibody with LIa and LIb were 63% and 4%, respectively of that with La, and those of anti-Ib antibody with LIa and LIb were 42% and 88%, respectively of that with Ia. Since the ratios of the specific activities of purified La and Ia and of Lbeta and Ibeta are 70: 82 and 2 : 70, respectively (Schliselfeld, 1973), the present findings suggest a 1 : 1 association of I and L subunits in the hybrid molecule.


Preparation of monovalent succinyl-concanavalin A and its mitogenic activity.

Monovalent succinyl-concanavalin A was prepared by photoaffinity labeling of succinyl-concanavalin A with p-azidophenyl a-D-mannopyranoside. This concanavalin A derivate showed a weak but definite mitogenic acitivity against mouse splenic lymphocytes and human peripheral lymphocytes, suggesting that direct receptor cross-linkage on the cell surface is not an obligatory prerequisite for the activation of lymphocytes.


Ovalbumin synthesis in a homologous cell-free system prepared from hen's oviduct.

Hen's oviduct polysomes are present in the precipitates prepared from the oviduct homogenates by low-speed centrifugation, in constrast other eukaryotic polysomes. The polysomes possessed synthesizing activity for ovalbumin. The amount of the released form of ovalbumin (soluble ovalbumin) synthesized in cell-free system A or B, consisting of the cell sap and the total ribosomal fraction or the polysomes, respectively, was about a half of that bound to the polysomes (nascent ovalbumin). The amount of soluble ovalbumin synthesized in cell-free system C, consisting of the pH 5 fraction and the polysomes, was only about 5% of that of nascnet ovalbumin. These results indicate that factors required to release nascent ovalbumin from polysomes are present in the pH 5 supernatant fraction. The soluble and nascent ovalbumins, which were purified by chromatography on a CM-cellulose column and by the use of antiovalbumin antiserum, respectively, seemed to be elongation products the initiations of which were supposed to occur in the oviducts before preparation of the cell-free system. The initiated chains in vitro were found to exist as nascent peptides bound to polysomes. Thus, the cell-free systems prepared in the present study lacked the ability to complete initiated peptide chains. The soluble ovalbumin synthesized in the cell-free systems was indentical with ovalbumin A1 containing two residues of phosphates, which was crystallized from hen's egg-white and was different soluble ovalbumin devoid of the prosthetic group (ovalbumin A3) prepared in the oviduct minces. This result suggests that an enzyme necessary for incorporation of the phosphate is present in the cell sap.


Biological process of carbohydrate attachment to ovalbumin.

Dithiothreitol or glutathione, essential for amino acid incorporation into polypeptides in cell-free systems, inhibited glucosamine incorporation into ovalbumin, a glycoprotein containing about three residues of N-acetylucosamine and about five residues of mannose. However, the thiol was necessary for mannose incorporation into ovalbumin in cell-free system prepared from hen's oviducts. The two monosaccharides were incorporated into soluble ovalbumin (intracellular ovalbumin released from the polysomes, corresponding to ovalbumin A3 which contains no phosphate and probably no carbohydrate) present in cell sap which was prepared from homogenates of minced oviducts by ultracentrifugation. It was difficult to study the incorporation of the carbohydrates into ovalbumin in connection with synthesis of the protein using the crude cell-free system. The incorporation of glucosamine and mannose into various forms of ovalbumin was therefore studied using minced oviducts. It was concluded from the incorporation of the two radioactive monsaccharides and of amino acids into the extracellular, soluble and nascent protein fractions that all the carbohydrate moieties were not incorporated into the growing polypeptides bound to polysomes, but were incorporated into ovalbumin molecules released from polysomes after synthesis had been completed.


Synthesis of ribosomal structural proteins by postmitochondrial supernatant from regenerating rat liver.

1. When the postmitochondrial supernatant (PM-supernatant) from regenerating rat liver was incubated with [3H]methionine, the incorporation of [3H]methinoine into the N-terminal residues of nascent peptides on ribosomes was observed. This incorporation was sensitive to a low cocentration (2X10(-6) M) of pactamycin. The results suggest that PM-supernatant has low but definite activity for the initiation of nascent protein synthesis. Polysomes and cell sap from regenerating rat liver showed negligible pactamycin-sensitive incorporation of [3H]methionine into N-terminal, residues of nascent peptides. 2. PM-supernatant from regenerating rat liver was incubated with [35S]methionine in the complete reaction mixture. After addition of ribosomal proteins labelled with [3H]methionine in vivo, ribosomal structural proteins were prepared from the incubation mixture by acetic acid extraction, CM-cellulose column chromatography, Sephadex G-200 gel filtration and finally by two-dimensional acrylamide gel electrophoresis. Incorporation was observed in the greater part of ribosomal proteins on the two dimensional gel. From the 35S-to-3H ratios of ribsomal protein fractions during the purification procedures, it appeared that the incorporation of labelled methionine into the ribosomal proteins by PM-supernatant was about 3% of that into the total proteins. When [3H]leucine was used, the values were about 4% in the same cell-free system and 5 to 6% in in vivo labelling. The results indicate that ribosomal proteins are synthesized with high efficiency by PM-supernatant from regenerating rat liver.


Effects of polyamines on in vitro dna synthesis by DNA polymerases from calf thymus.

DNA synthesizing reactions catalyzed both large and small species of calf thymus DNA polymerase (DNA polymerase-alpha and -beta) [EC 2.7.7.7] were stimulated to comparable extents by the presence of spermidine or spermine, and it was not possible to differentiate these two species in terms of their sensitivities to polyamines. Optimal concentrations for stimulation were 0.5-1.0 mM for spermidine and 2-10 mjM for spermine. Excess polyamines strongly inhibited the reactions. The modes of stimulation were as follows: 1) Stimulation was observed with templates bearing long single-stranded sections when either natural DNA or synthetic homopolymer-oligomer duplex was used. 2) The nautral DNA-dependent reaction was stimulated by polyamines at suboptimal concentrations of Mg2+; the apparent Km value for Mg2+ was lowered on adding polyamines, while the Vmax value was unchanged. When synthetic homopolymer-oligomer duplex was used as a template, the reaction was stimulated spermidine even at the optimal concentration of Mn2+. 3) Polyamines markedly influenced the salt requirements of the reactions of DNA polymerase. Spermidine could replace salts such as KC1 or NaC1 at concentrations less than 1/100 of those of salts.


Some enzymatic properties of 3beta-hydroxysteroid oxidase produced by Streptomyces violascens.

The 3beta-hydroxysteroid oxidase produced by Streptomyces violascens was purified from the culture broth by procedures including batch-wise treatment on DEAE-cellulose, ammonium sulfate fraction, gel filtration on Sephadex G-75, and column chromatography on DEAE-cellulose. The highly purified enzyme preparation exhibited no significant absorption maxima in the visible region other than a maximum at 280 nm. Optimum pH and temperature for the enzyme activity were approximately pH 7.5 and 50 degree, respectively. The Michaelis constant (Km) for cholesterol determined under two different experimental conditions were 4.5 and 6.7 X 10(-4) M. The enzymatic activity was remarkably inhibited by various metal salts such as FeC1(3), FeSO4, AgNO3, etc. On the other hand, neither EDTA nor Fe-chelating agents had any inhibitory effect on the enzymatic activity, while other metal-binding agents, KCN and NaN3, caused significant inhibition. The enzyme activity was inhibited almost completely by N-bromosuccinimide and iodine but not p-chloromercuribenzoate. The highly purified enzyme did not require any external electron acceptors other than oxygen. In addition, the activity was not influenced by the addition of external electron donors. The enzyme showed a high substrate specificity for 3beta-hydroxysteroids and the relative oxidation rates were 100 for cholesterol, 91 for 5alpha-cholestan-3beta-ol, 83 for pregn-5-en3beta-ol-one, 80 for androst-5-en-3beta-ol-17-one, 64 for 5alpha-androstan-3beta-ol-17-one, ect. The oxidation of cholesterol by the enzyme was remarkably inhibited by the addition of 5alpha-cholestan-3beta-ol, 5alpha-cholestan-3-one, 5beta-cholestan-3beta-ol, 5alpha-cholestane3beta, 5alpha-doil or 5alpha-lanosta-8, 24-den 3beta-ol. These findings indicate the present enzyme belongs to the class of 3beta-hydroxysteroid oxidase but some of its physical and enzymatic properties obviously differ from those of 3beta-hyroxysteroid oxidase of Brevibacterium.


Purification and properties of a four iron-four sulfur protein from a Pseudomonas species.

We have isolated an iron-sulfur proteins from a Pseudomonas species grown on glucose. This protein has different properties from the two known iron-sulfur proteins isolated from other Pseudomonas species: rubredoxin and putidaredoxin. The iron-sulfur protein was purified to homogeneity by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The absorption spectrum of the oxidized iron-sulfur protein shows a peak at 283 nm with shoulders at about 290, 320, and 410 nm. The protein contains 4 g atoms of iron and 4 moles of labile sulfur per mole of protein, and has a molecular weight of approximately 14,000. The amino acid composition of the protein shows a predominance of acidic amino acids. The Pseudomonas protein was found to be active for both photosynthetic nicotinamide nucleotide reduction by chloroplasts and cytochrome c reduction by spinach ferredoxin-NADP+ reductase [EC 1.6.7.1]. On the basis of these results, this protein appears to be unique among all known ferredoxins. From an evolutionary point of view, it appears to be more closely related to Azotobacter ferredoxin than to Desulfovibrio ferredoxin.


Interaction of hydrophobic probes with the apoenzyme of pig heart lipoamide dehydrogenase.

The interaction of hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2, 2'-disulfonate (MBAS), with pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3] was investigated. When ANS or MBAS was mixed with the apoenzyme of lipoamide dehydrogenase, the fluorescence quantum yield, of each dye was enhancedd markedly and the emission maxima concurrently shifted to the blue. The quantum yield, 0.038, of ANS bound to the apoenzyme, calculated from the corrected emission spectrum, was eight times higher than that in buffer solution, and the value, 0.0090, for bound MBAS was eighteen times higher than that in buffer solution. Moreover, the absortion bands of both ANS and MBAS shifted to the red upon binding with the apoenzyme. A general feature of the absorption spectra of these dyes observed on changing the solvent from polar to apolar was a red shift of the absorption bands. These results indicate that ANS or MBAS bound to the apoenzyme of lipoamide dehydrogenase is situated in a hydrophobic region of the apoenzyme molecule. It was found that 2 moles of each dye was bound per mole of the apoenzyme, which contains two polypeptide chains. The dissociation constants for the ANS- and MBAS-apoenzyme complexes were estimated to be 1.03X10(-5) and 1.54X10(-5) M, respectively. The enhanced fluorescence of both dyes bound to the apoenzyme decreased linearly upon adding FAD and disappeared at about 2 moles of FAD per mole of the apoenzyme. This suggests that both ANS and MBAS were displaced from their binding sites on the apoenzyme by FAD. The protein fluorescence spectrum of the apoenzyme had a maximum at 352 nm, which was blue-shifted by 6 nm from that of tryptophan in the buffer. Upon binding ANS or MBAS, the maximum of the protein fluorescence of the apoenzyme returned to 350 nm for the holoenzyme, and the fluorescence intensity decreased. Thus, the conformation around some tryptophan residues was affected by the binding of the dyes. When guanidine hydrochloride (GuHCl) was added to the ANS-apoenzyme complex solution, the enhanced fluorescence due to the bound ANS decreased and the emission maximum concurrently shifted to the red. Further, the maximum of the protein fluorescence of the apoenzyme shifted to the red, indicating the exposure of some tryptophan residues buried in an apolar region of the apoenzyme. Thus the binding of ANS to the apoenzyme was inhibited by protein denaturation due to GuHCL. In contrast, the holoenzyme of lipoamide dehydrogenase did not bind ANS or MBAS at all.


Synergistic action of two different types of endo-cellulase components from Irpex lacteus (Polyporus tulipiferae) in the hydrolysis of some insoluble celluloses.

The substrate specificities of three endo-cellulase [EC 3.2.1.4] components, F-1, F-2, and S-1, obtained from the culture filtrate of Irpex lacteus (Polyporus tulipiferae), were investigated in detail. It was confirmed that the former is of a more random type, belonging to the carboxymethyl-cellulase (CMCcase) group, and the latter two are of a less random type, beloning to the Avicelase group. It was found that a mixture of CMCase and Avicelase shows a remarkable synergistic action in the degradation of cotton and Avicel and that CMCase lowers the degree of polymerization of both cotton and CM-cellulose faster than Avicelases, relative to the production of reducing sugar. Thus, it was assumed that cotton and similar cellulosic substrates were degraded mainly by the synergistic action of these cellulase components produced by this celluloytic fungus.


Effect of albumin and methylated albumin on the glucose permeability of lipid membranes.

The diffusion of glucose from phospholipid membranes has been measured in the presence of serum albumin or methylated serum albumin. At neutral pH, serum albumin enhanced the rate at which glucose diffused from liposomes containing more than a certain amount of lysolecithin. Net charge of the membrane is not important for the reaction, since positively charged membranes containing stearylamine showed almost the same reactivity as negatively charged liposomes containing dicetyl phosphate. Carboxylmethylated albumin showed enhancement of the diffusion rate of glucose from negatively but not from positively charged liposomes. The amount of methylated albumin required to affect liposomes was much smaller than the amount of albumin required to damage liposomes containing lysolecithin. Cholesterol incorporation suppressed the sensitivity of liposomes to both proteins, albumin and methylated albumin. The effect of temperature and fatty acid composition of phospholipids on the sensitivity of liposomes to proteins suggests the importance of the fluidity of the membrane, especially in the case of methylated albumin.


Properties of liposomal membranes composed of short-chain lecithins.

Permeability properties of multilamellar liposomes prepared from synthetic, saturated short-chain (C8, C10, C12) lecithins (3-sn-phosphatidyl choline) were studied. Dioctanoyllecithin, didecanoyllecithin, and dilauroyllecithin form "stable" bilayers which are practically impermeable to glucose when prepared with proportions of more than 1.2, 0.75, and 0.6 of cholesterol (molar ratio to phospholipid), respectively. Dioctanoyllecithin liposomes were rather leaky above 30 degrees, even when a proportion of 1.5 of cholesterol (molar ratio to lecithin) was incorporated. Judging from their sensitivities to temperature, Triton X-100, serum albumin, and other reagents, short-chain lecithin bilayers show characteristic properties, different from those of both saturated long-chain lecithins (dipalmitoyllecithin and dimyristoyllecithin) and lecithins having unsaturated fatty acid residues (egg lecithin and dioleoyllecithin).


Fragmentation of rat serum albumin by cyanogen bromide cleavage and the amino acid sequences of four fragments.

Rat serum albumin was cleaved into seven peptides by cyanogen bromide treatment followed by reduction and carboxymethylation. The amino acid composition and the N-terminus of each peptide and the amino acid sequences of four peptides were determined by conventional methods. An alignment of these peptides in the original protein was proposed in the light of the established sequences of bovine and human plasma albumin.


Fluorometric method for assay of kallikrein-like arginine esterases.

The fluorometric method of Nash for determination of methanol was applied in assay of kallikrein-like arginine esterases using N-a-tosyl-L-arginine methyl ester (TAME) as substrate. The results obtained using this method corresponded well with those obtained in the usual colorimetric methods using hydroxamate, chromotropic acid, and 3-methyl-2-benzothiazolone hydrazone and the present method was more sensitive than these colorimetric methods.


Primary structure of bovine plasma high-molecular-weight kininogen. The amino acid sequence of a glycopeptide portion (fragment 1) following the C-terminus ot the bradykinin moiety.

On incubation of bovine plasma high-molecular-weight (HMW) kininogen with purified plasma kallikrein [EC 3.4.21.8], a large glycopeptide fragment and the vasoactive peptide, bradykinin, were initially liberated; the former, named fragment 1-2, was subsequently cleaved into fragment 1 (glycopeptide) and the previously established fragment 2 (histidine-rich peptide). The isolated fragment 1-2 contained a total of 108 to 110 amino acid residues and carbohydrates, and the amino-terminal sequence Ser-Val-Gln was established. The other fragment, fragment 1, consisted of a total of 69 amino acid residues with serine and arginine (and lysine) at the amino and carboxyl termini, respectively. It contained eleven residues each of histidine and glycine, together with an oligosaccharide chain consisting of galactosamine, hexose and sialic acid. The complete amino acid sequence of fragment 1 was determined by Edman degradation and standard enzymatic and chemical techniques. These results established the following sequence: H-Ser-Val-Gln-Val-Met-Lys-Thr-Glu-Gly-Ser-Thr-Pro/Thr-Val-Ser(CHO)-Val/Leu-Pro-His-Ser-Ala-Met-Ser-Pro-Val-Gln-Asp-Glu-Glu-Arg-Asp-Ser-Gly-Lys-Glu-Gln-Gly-Pro-Thr-His-Gly-His-Gly-Trp-Asp-His-Gly-Lys-Gln-Ile-Lys-Leu-His-Gly-Leu-Gly-Leu-Gly-His-Lys-His-Lys-His-Asp-Gln-Gly-His-Gly-His-His-Lys/ArgOH.


Purification and characterization of a proteinase from pineapple fruit, fruit bromelain FA2.

Fruit bromelain FA2, the main proteinase component of the juice of pineapple fruit, has been purified and characterized. 1. Efficient extraction of this enzyme from the crude material was possible using "Cellulosin AP," a microbial polysaccharidase preparation containing cellulase, hemicellulase, and pectinase. The enzyme was purified mainly by successive applications of anion-exchange chromatography, yielding an apparently homogeneous protein as judged by several physical, chemical, and immunochemical criteria. Properties of FA2 include: molecular weight, 31,000; isoelectric point, pH 4.6; absorbance at 280 nm of a 1% solution at pH 7.0 per cm, 19.2. 2. FA2 gave only alanine phenylthiohydantoin upon amino-terminal group analysis by the Edman procedure. Stepwise degradation yielded the amino-terminal sequence Ala-Val-Pro-Gln-Ser-Ile-Asp-Trp-Arg-Asp-Tyr-Gly-Ala. The amino acid composition of FA2 was not markedly different from that of stem bromelain, except for a much smaller lysine content and a smaller alanine content relative to glycine in FA2. FA2 contained neither amino sugars nor neutral carbohydrates as determined by several methods, so FA2 is not a glycoprotein. 3. By labeling the reactive cysteine residue (CYS) with [14C]iodoacetate, the following partial amino acid sequence has been determined. Asn-Glx-Asn-Pro-Cys-Gly-Ala-CYS.


Protein synthesis in a cell-free system from an extreme thermophile. Effects of preincubation in the cold on polyuridylic acid-dependent polyphenylalanine synthesis at high temperature.

1. It was found that preincubation of the reaction mixture in the cold enhanced polyuridylic acid-directed polyphenylalanine synthesis by a cell-free extract of Thermus thermophilus HB8 at high temperature. 2. The effect of preincubation was most marked at 10-25 degrees in the presence of 20 mM Mg2+. Preincubation at 65 degrees failed to stimulate the incorporation. 3. The presence of phenylalanyl-tRNA, polyuridylic acid, and ribosomes was essential for preincubation in the cold to be effective. 4. A ternary complex of amino acyl-tRNA, polyuridylic acid, and a ribosome formed at low temperature was isolated by CPG-10 column chromatography; the isolated complex initiated polyphenylalanine synthesis effectively at high temperature. 5. The amount of the ternary complex formed depends on the preincubation time and the concentration of Mg2+. Since the amount of the complex correlated positively to the rate of polyphenylalanine synthesis at high temperature, the effectiveness of preincubation in the cold is presumably due to the formation of the ternary complex of phenylalanyl-tRNA, polyuridylic acid, and a ribosome.


Properties and subunit structure of pig heart pyruvate dehydrogenase.

Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.


Hybridization of glyceraldehyde-3-phosphate dehydrogenase in borate.

Conditions for the hybridization of glyceraldehyde-3-phosphate dehydrogenase (GPD) [EC 1.2.1.12] in the presence of dilute borate were examined with horseshoe crab and rabbit GPDs. Hybridization was strongly dependent upon pH, borate concentration, and temperature. The optimum medium for hybridization was 10mM Tris-HCl-1 mM 2-mercaptoethanol-1mM EDTA containing 10-20 mM borate, pH 8.5-9.0. Hybridization was performed by incubation of two electrophoretically distinct GPDs at 30 degrees for 3-15 hr in the above medium at a protein concentration of 1-2 mg/ml. The time course of hybridization was analyzed under the optimized conditions. Symmetrical A2B2-type hybrid appeared only 5 min after incubation for 1 hr. Hybridization of GPDs from 7 different species was examined under the optimal conditions. Hybridization was detected with rabbit-horseshoe crab, yeast-rabbit, yeast-chicken, and chicken-horseshoe crab combinations. Subunit-subunit interaction, the mechanism of hybridization, and the structure of GPD are discussed based on the results obtained.


A simple fluorometric assay for type B monoamine oxidase activity in rat tissues.

A simple fluorometric assay for monoamine oxidase (MAO) [EC 1.4.3.4] activity towards beta-phenylethylamine (PEA) was devised. The procedure consists in measuring the disappearance of PEA fluorometrically. The disappearance of PEA was completely inhibited by pargyline, a potent inhibitor of MAO. MAO activity for PEA was linear with 10 mg to 100 mg of liver tissue in 3 ml of reaction mixture for up to 90 min of incubation. Using this method, the V max values and the apparent Km values of MAO for PEA in several rat tissues were determined, and compared with those for benzylamine and 5-hydroxytryptamine (5-HT).


Effect of priming doses of chemically modified antigen on helper activity.

Mice primed with chemically modified bacterial alpha-amylase (BalphaA) [EC 3.2.1.1 alpha-amylase, B. subtilis], which was neither cross-reactive with anti-BalphaA antibody nor able to induce a humoral anti-BalphaA antibody response, developed enhanced responses to a subsequent challenge with native BalphaA (Nakashima et al. (1974) J. Biochem. 76, 349-357). The present studies were designed to examine the relationship of priming doses of BalphaA derivatives to the level of enhancement of the helper activity. Increasing the priming dose of modified antigens resulted in a greater degree of helper cell response until the maximal level of enhancement was reached. When injections for priming and challenge were given intraperitoneally, priming doses of D-BalphaA, M-BalphaA, and RM-BalphaA required for the maximal enhancement of helper activity were about 15, 50, and 15 mug, respectively. Further increase in the priming dose, conversely, resulted in suppressin of the enhanced helper activity, irrespective of whether the time interval between priming and challenge was 10 or 28 days. Suppression of the enhanced helper activity upon excessive dose priming with modified BalphaA derivatives was not specific for the anti-BalphaA antibody response. On the basis of these results it is suggested that this phenomenon of suppression might be partly accounted for by the regulatory mechanism functioning in antigenic competition.


The effect of temperature on ribose-5-phosphate isomerase from a mesophile, Thiobacillus thioparus, and a thermophile, Bacillus caldolyticus.

The enzyme ribose-5-phosphate isomerase [EC 5.3.1.6] was partially purified from a mesophilic organism, Thiobacillus thioparus, and from an extreme thermophile, Bacillus caldolyticus. The stability and kinetics of the two enzymes were compared with regard to temperature in the presence of a series of neutral salts and alcohols. The thermal stability of both enzymes was altered such that the salts (NH4)2SO4, NaCl, KCl, and LiCl increased stability, while LiBr, CaCl2, methanol, ethanol, and 1-propanol decreased stability. Ethylene glycol had little effect on the mesophilic enzyme, but increased the stability of the thermophilic protein. The kinetics of both enzymes were also affected by the salts and alcohols, and Arrhenius plots of two kinetic parameters, Km and Vmax, displayed discontinuities, or sharp changes in slope, at characteristic temperatures, TD. Neutral salts and alcohols altered the temperature of discontinuity in a sequence similar to that observed in studies of thermal stability. It is suggested that the slope change is due to temperature-dependent alterations in the enzymes at specific, but undefined, loci at the active site, although no evidence is offered for the absence of a larger conformation change in the entire enzyme.


Macromolecular components of the vitelline membrane of hen's egg. II. Physicochemical properties of glycoprotein I.

Of the three major macromolecular components of the vitelline membrane of hen's egg, the lowest molecular weight component (previously designated component I) has been studied and its physicochemical properties clarified. The molecular weight of this component is 27,000 and its chemical composition is typical of a glycoprotein, consisting of protein (91%), total hexose (4.4%), hexosamine (glucosamine 2.3%; galactosamine 0.7%), and sialic acid (1.7%). Uronic acid was not found. The molar ratios of the constituent neutral sugars of this glycoprotein (GP-1) are as follows: fucose 3, mannose 5, galactose 5, glucose 1, and xylose 1. The amino acid profile shows a relatively high proportion of hydrophobic amino acids (39%), which may partly account for the insolubility of GP-I in water.


Roles of nucleoside triphosphates in microtubule assembly.

Depolymerization of microtubules in the ATP-reassembly buffer permitted the preparation of GDPETNGTP. Incubation of this tubulin fraction at 35 degrees with ATP induced the phosphorylation of E-site GDP into GTP, which was then dephosphorylated during microtubule assembly. Incubation of GDPETNGTP with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40] also induced polymerization. Depolymerization of microtubules in the GTP-reassembly buffer yielded GTPETNGTP, which was capable of polymerizing into microtubules even in the absence of free GTP. In the presence of 4 M glycerol, GDPETNGTP assembled into microtubules with no change in the bound nucleotides.


Biostereometrics and the communication of biological form.

Biostereometrics-the spatial and spatio-temporal analysis of biological form and function based on principles geometry-is a modern approach to the measurement and analysis of biological form which recognizes that organic structures are irregular and three-dimensional (or four-dimensional, as in movement or growth). Stereometric sensors of various types are used to determine the coordinates of points distributed over the surface (internal or external). The resulting data provide a more comprehensive, parsimonious and unambiguous spatial quantification than heretofore possible using traditional lengths, breadths, and circumferences. Recent and prospective biomedical applications are described.


Steroidogenesis in the zona glomerulosa of the adrenal coretx. I. Isolation and some properties of mitochondria from the zona glomerulosa of the bovine adrenal cortex.

Isolation procedures for mitochondria from the zona glomerulosa of the bovine adrenal cortex are described and the properties of the mitochondria thus prepared are compared with those isolated from the zona fasciculoreticularis. The cristal membranes of mitochondria in the zona glomerulosa in situ are tubular or tubulovesicular, whereas those of mitochondria in the zona fasciculoreticularis in situ are vesicular. When mitochondria are isolated from the former zone, they invariably showed the condensed configuration regardless of isolation media, whereas those isolated from the latter zone in an ST medium showed the orthodox configuration. When Ca2+ was added to mitochondria isolated either from the zona glomerulosa or the zona fasciculoreticularis in an STE medium in the condensed configuration, a transition from the condensed to the orthodox configuration tool place; the cristal membranes of mitochondria from the zona glomerulosa became tubular or tubulovesicular and those of mitochondria from the zona fasciculoreticularis became vesicular. Contaminations of mitrochondria of the zona glomerulosa with other cellular organelles were examined using various marker enzymes. There was no difference in cytochrome content between mitochondria of the two zones specified above. The coupling efficiency of mitochondria of the zona glomerulosa was found to be remarkably effected by temperature during the isolation procedures. Effects of various substrates, isolation media, and bovine serum albumin on the coupling efficiency of mitochondria of the zona glomerulosa are also described.


Mechanism of activation of bovine factor VII. Products of cleavage by factor Xa.

Coagulation Factor VII from bovine plasma is a glycoprotein containing a single peptide chain. The NH2-terminal sequence of Ala-Asx-Gly-Phe-Leu- is homologous with the NH2 termini of prothrombin, Factor IX, and the light chain of Factor X. Factor Xa in the presence of calcium ions and phospholipid cleaves Factor VII at an Arg-Ile bond in the sequence Arg-Ile-Val-Gly-Gly-, producing a two-chain molecule with at least 85 times the coagulant activity of single-chain Factor VII and a new NH2-terminal sequence homologous with the corresponding chains of thrombin, Factor IXa and Factor Xa. A second slower cleavage at an Arg-Gly bond destroys Factor VII activity. Bovine Factor VII, unlike prothrombin, Factor IX, and Factor X, is rapidly inhibited by diisopropylphosphorofluoridate (iPr2PF). [3H]iPr2PF is readily incorporated into one-chain, two-chain, and three-chain forms of Factor VII up to ratios of approximately 0.9 moles of [3H]diisopropylphosphate per mole of protein. The radioactive peptides generated from each form of [32P]iPr2PF-inhibited Factor VII by tryptic and thermolytic digestion were found to migrate together on paper electrophoresis. This indicates that the iPr2PF is incorporated stoichiometrically into the same specific site in each form.


Effect of estradiol on rat uterus DNA-dependent RNA polymerases. Studies on solubilized enzymes.

DNA-dependent RNA polymerases of immature and castrated rat uteri were studied after estradiol administration. The enzymes were solubilized from either whole uterus homogenate or nuclei and their activities were measured on an exogenous DNA template. alpha-Amanitin was used to distinguish alpha-amanitin-resistant from alpha-amanitin-senitive forms of the enzyme. The number of alpha-amanitin-sensitive RNA polymerase molecules was measured by a binding assay using labeled amanitin. In the first series of experiments RNA polymerases were solubilized from whole uterus homogenate. alpha-Amanitin-sensitive and -resistant activities were constant during the first 6 hours after estradiol treatment, followed by a late and moderate increase in their activities (50% at 12 hours for the resistant form and 40% at 24 hours for the sensitive form). The number of sensitive polymerase molecules evolved in an identical manner to its activity (+40% at 24 hours), suggesting that the increase in activity is due to the synthesis of new enzyme molecules. For both forms, no diffusible stimulatory factor was detected in the uterus of hormone-treated animals. In the second series of experiments, disrupted nuclei were washed with 0.15 M (NH4)2SO4 in order to release only enzyme molecules which were not firmly bound to DNA in a transcription complex. The amount of the sensitive form of polymerase which remains firmly bound to chromatin, was constant for 6 hours after estradiol administration and was doubled by 24 hours. The firmly bound alpha-amanitin-resistant activity was solubilized and was measured in the presence of an exogenous template. There was a progressive increase in activity first detectable in 1 to 2 hours, amounting to 50% at 6 hours and 100% at 24 hours. The reported results show that during the first 6 hours of hormone treatment: (a) the total content of RNA polymerases remains unchanged in the uterus; (b) the number of alpha-amanitin resistant molecules tightly bound to DNA increases progressively while the alpha-amanitin sensitive remains constant. At a later time (24 hours), an increase is observed both for the total amount of enzymes and for their fraction engaged in a transcription complex.


Free ribosomes in physiologically nondividing cells. Human peripheral lymphocytes.

Ribosomes of physiologically nondividing human peripheral blood lymphocytes were studied by ultraviolet absorbance measurements of cytoplasmic extracts subjected to ultracentrifugation in sucrose gradients at high and low ionic strength. At least 70% of the total cytoplasmic ribosomes were free ribosomes which sedimented at 80 S in low salt and dissociated to 40 S and 60 S subunits in high salt. These particles were not involved in protein synthesis. The remaining ribosomes were equally divided among native subunits, active monosomes, and larger polysomes. Free ribosomes were shown to exist as 80 S particles in the intact cell, and labeling studies indicated that they did not freely return to the pools of protein-synthesizing components. New ribosomes appeared first as native subunits and in polysomes. After a lag of 15 to 20 min, the particles began to enter the free ribosome pool. Thus, free ribosomes arise in the resting cell by a unidirectional flow which continuously removes particles from the protein-synthesizing pool and sequesters them as an accumulation of inactive 80 S particles. The transition from native subunits to free ribosomes is accompanied by functional changes in association behavior of subunits and by alteration of sedimentation behavior of the subunits. These changes may be due to absence of a protein or proteins from the free ribosomes which is required to permit effective dissociation of subunits prior to initiation of translation. Deficiency of this dissociation factor may be responsible for the continuous formation of free ribosomes in resting cells. Our data also suggest a limitation of the rate of initiation of protein synthesis which may result from deficiency of an initiation factor.


Characteristics of acetaldehyde oxidation in rat liver mitochondria.

Rat liver mitochondria oxidized acetaldehyde (180 muM) at the rate of approximately 12 nmol/min/mg of protein at 37 degrees. This was stimulated by 88% with the addition of ADP. The ADP/O ratio (2.6) was similar to that with glutamate as substrate. 2,4-Dinitrophenol and phenazine methosulfate also stimulated the rate of acetaldehyde oxidation in the mitochondria. By contrast, acetaldehyde metabolism was virtually abolished by rotenone and antimycin A. These results indicate that acetaldehyde oxidation is linked to the mitochondrial respiratory chain and coupled with mitochondrial oxidative phosphorylation. Indeed, little acetaldehyde was metabolized when mitochondrial membranes were disrupted by sodium deoxycholate. In the disrupted mitochondria, however, acetaldehyde oxidation was fully recovered by addition of NAD+, suggesting that the ability of mitochondria to supply NAD+ controls the rate of acetaldehyde oxidation in intact mitochondria. The stimulatory effect of ADP on mitochondrial acetaldehyde oxidation was diminished by increasing the acetaldehyde concentration. Concomitantly, the ADP/O ratio decreased, suggesting an inhibitory effect of high concentrations of acetaldehyde on mitochondrial respiration. Chronic feeding of ethanol significantly reduced the capacity of intact liver mitochondria to oxidize acetaldehyde. This was associated with a significant reduction of the mitochondrial respiration. By contrast, the activity of aldehyde dehydrogenase in disrupted mitochondria remained unchanged.


Stereospecific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. Evidence that the binding species is receptor for induction of aryl hydrocarbon hydroxylase.

We previously hypothesized that the genetic trait of aromatic hydrocarbon nonresponsiveness (the failure in certain inbred strains of mice of polycyclic hydrocarbons to induce aryl hydrocarbon hydroxylase activity, and the diminished sensitivity to the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is due to mutation which results in an induction receptor with a diminished affinity for the inducing compound. Following the intraperitoneal administration of [14C]TCDD (6 nmol/kg), hepatic accumulation of the radiolabel was greatest in C57BL/6J mice, intermediate in the hybrid B6D2F1/J mice, and least in DBA/2J mice, a pattern which mirrors the strain sensitivity to hydroxylase induction by TCDD (C57BL/6J greater than B6D2F1/J greater than DBA/2J). These data are compatible with receptor mutation theory and suggested that the hepatic uptake of TCDD is determined by the affinity of the receptor. In vitro experiments on the binding of [3H]TCDD to hepatic cytosol from C57BL/6J mice revealed a small pool of high affinity sites which stereospecifically and reversibly bind TCDD. The specific binding of [3H]TCDD to hepatic cytosol had an equilibrium dissociation constant KD of 0.27 nM and a maximum binding capacity of 84 fmol/mg of cytosol protein. Much less high affinity specific binding of [3H]TCDD was observed in hepatic cytosol from DBA/2J mice, but the KD was not estimated because of the limited aqueous solubility of the ligand. The binding affinity of 23 halogenated dibenzo-p-dioxins and dibenzofurans for this hepatic cytosol-binding species closely correlated with the potencies of these compounds as inducers of hepatic aryl hydrocarbon hydroxylase activity. The polycyclic hydrocarbons that induce hepatic hydroxylase activity competed with [3H]TCDD for hepatic cytosol binding, but phenobarbital, pregnenolone-16alpha-carbonitrile, and the steroid hormones had no specific binding. The data suggest that the hepatic cytosol species which binds TCDD is the receptor for the induction of hepatic aryl hydrocarbon hydroxylase activity, and that the mutation in nonresponsive mice results in an altered receptor with a diminished affinity for inducing compounds.


Conformational relevance of the beta6Glu replaced by Val mutation in the beta subunits and in the beta(1-55) and beta(1-30) peptides of hemoglobin S.

The beta subunits of hemoglobin S showed a higher ellipticity than the beta subunits of the hemoglobin A, both in the Soret and near-ultraviolet regions. The apoderivatives of the beta subunits of hemoglobin S showed a lower helical content and a larger amount of beta conformation than the apoderivatives of the beta subunits of hemoglobin A. The beta(1-55) peptides and beta(1-30) peptides from the beta subunits of hemoglobin A and S have been separated and analyzed. The betaS(1-55) peptide showed a higher content of beta conformation and lower amount of alpha helix when compared to the betaA(1-55) peptide. This difference was present also in different concentrations of methanol. The apoderivative from the beta subunits of hemoglobin S and the betaS(1-55) peptide aggregated with increasing ionic strength. The CD measurement showed that their secondary structure did not change upon a 10-fold dilution of the sample. Very little secondary structure was present in the betaS(1-30) peptide, and the CD spectrum was very similar to that of the betaA(1-30) peptide. No significant difference in aggregation was found between the betaS(1-30) and betaA(1-30) peptides.


Lipid peroxidation and alteration of membrane lipids in isolated hepatocytes exposed to carbon tetrachloride.

Lipid peroxidation, determined by malondialdehyde formation, occurs at a low, but detectable, rate in parenchymal cells isolated from livers of fasted rats. Pretreatment of rats with phenobarbital increased malondialdehyde formation about 2-fold, probably because of the increased amount of endoplasmic reticulum. Lipid peroxidation was increased in the cells by the addition of either NADPH or CCl4, and the effect of the two agents together was more than additive. Phenobarbital pretreatment increased peroxidation due to exposure of the cells to CCl4 but not that associated with NADPH addition. The amount of CCl4 producing a 50% increase in malondialdehyde formation was about 3-fold less for cells from phenobarbital-treated rats than for those from control rats. Decreased cytochrome P450 levels in isolated hepatocytes produced by prior treatment of rats with allylisopropylacetamide enhanced lipid peroxidation from endogenous substrates but did not affect lipid peroxidation caused by the addition of NADPH. Allylisopropylacetamide markedly lowered lipid peroxidation caused by CCl4 in proportion to the loss of cytochrome P450. Malondialdehyde production associated with metabolism of endogenous substrates in the cells, or resulting from incubation of the cells with NADPH was not accompanied by changes in the fatty acid or protein content of three membranous fractions (microsomal mitochondrial, and cell debris) isolated from homogenates of these cells. Exposure of liver cells to CCl4, however, caused major losses in all fatty acids and of protein from the microsomal fraction, but only polyunsaturated fatty acids were decreased in the cellular debris fraction. Incubation with NADPH and CCl4 together enhanced malondialdehyde formation, but caused no further decrease in fatty acid content in these two fractions. Mitochondrial fatty acids were not decreased by any treatments described.


Delta-Aminolevulinic acid synthetase in the heart.

The regultion of cardiac delta-aminolevulinic acid synthetase activity was studied in rat heart homogenates. Optimal conditions were determined for the measurement of delta-aminolevulinic acid and an appropriate assay was established for the heart. The activity of cardiac delta-aminolevulinic acid synthetase was determined in rats either fed ad libitum or starved for 24 or 48 h. Marked decreases in delta-aminolevulinic acid synthetase activity were observed in homogenates or mitochondrial fractions prepared from hearts of fasted animals and an explanation for previous findings that the enzyme is undetectable in heart tissue is provided. Dexamethasone treatment was effective in reversing the decreases brought about by fasting but had no effect on the delta-aminolevulinic acid synthetase activity in heart homogenates from fed rats. ACTH treatment had no effect in fed or starved rats. Decreases in delta-aminolevulinic acid synthetase activity induced by fasting were not reversed in homogenates or mitochondrial preparations by succinyl-CoA-generating systems or when alpha-ketoglutarate was substituted for succinate in homogenate preparations. Cardiac delta-aminolevulinic acid dehydratase levels are not altered by fasting. Agents such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine, which produce marked increases in hepatic delta-aminolevulinic acid synthetase activity, have no effects on the activity of this enzyme in the heart.


Effect of delta9-tetrahydrocannabinol on mitochondrial NADH-oxidase activity.

Delta9-Tetrahydrocannabinol (delta9-THC), the active ingredient of marihuana was found to be a highly effective inhibitor in vitro of the NADH-oxidase activity of rat brain and heart mitochondria. The degree of inhibition of the enzyme system obtained from rat brain tissue varied with the region from which it was derived as follows, in the presence of 10(-5) M delta9-THC: hypothalamus plus thalamus plus midbrain, 73 +/- 4%; cerebellum, 66 +/- 4%; medulla oblongata plus pons, 63 +/- 6%; cerebral cortex, 50 +/- 8%. The same concentration inhibited rat heart NADH-oxidase activity 69 +/- 9%. Inhibition of NADH-oxidase activity by a corresponding concentration of deoxycorticosterone was significantly less in all tissue preparations tested, ranging from 11% to 26%. The inhibition of delta9-THC appeared to be competitive and near the amytal-sensitive site of the electron transport system. Suggestive evidence was also obtained for a second site of action, above the cytochrome c site.


Negative cooperativity among beta-adrenergic receptors in frog erythrocyte membranes.

(-)-[3H]Dihydroalprenolol, a potent competitive beta-adrenergic antagonist, has been previously documented to bind to the adenylate cyclase-coupled beta-adrenergic receptor sites in mammalian and non-mammalian tissues. Steady state binding of (-)-[3H]dihydroalprenolol to sites in frog erythrocyte membranes, a model system for adenylate cyclase-coupled beta-adrenergic receptors, displays characteristics consistent with negative cooperativity among the beta-adrenergic receptors: Scatchard plots are curvilinear with upward concavity and slopes of Hill plots are consistently less than 1.0. The existence of site-site interactions of the negatively cooperative type were demonstrated directly by the ability of unlabeled (-)-alprenolol to accelerate the dissociation of (-)-[3H]dihydroalprenolol under conditions were no rebinding of radioligand occurred. The dissociation rate of (-)-[3H]dihydroalprenolol alone is directly related to temperature and increases with increases in temperature from 4-37 degrees. (-)-[3H]Dihydroalprenolol dissociation is enhanced by unlabeled (-)-alprenolol at all temperatures studied; however, at 4 degrees, the time required to observe an enhancement of radioligand dissociated is greater than the time required for unlabeled (-)-alprenolol to occupy the empty receptor sites, suggesting that increased rigidity of the biomembrane at 4 degrees may be responsible for the absence of readily observable site-site interactions. The ability of a number of beta-adrenergic agonists and antagonists to induce negative cooperativity among the beta-adrenergic receptors was directly related to their affinity for the receptor sites rather than their intrinsic activity in the adenylate cyclase-coupled beta-adrenergic system. The ability to induce site-site interactions among the beta-adrenergic receptors occurs at physiological concentrations of beta-adrenergic agents, since occupancy of less than 10% of the receptor sites is sufficient to reduce receptor affinity. Changes in pH from 6.5 to 9.0 did not significantly alter the negatively cooperative site-site interactions among the receptor sites. The negatively cooperative phenomenon was also independent of Mg2+, Ca2+, and NaF concentrations in the buffer medium. The presence of guanyl-5'-yl imidodiphosphate, a nonhydrolyzable nucleotide analog which enhances adenylate cyclase stimulation (Vmax) by beta-adrenergic agonists and decreases the concentration of agonist required to half-maximally stimulate adenylate cyclase, did not alter the ability of either agonists or antagonists to induce negatively cooperative site-site interactions among the beta-adrenergic receptors.


Isolation and characterization of subcellular membranes with altered phospholipid composition from cultured fibroblasts.

Plasma membranes, microsomes, and mitochondria were isolated from mouse fibroblast (LM) suspension cells by modification of several established procedures. Choline analogues such as N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine were incorporated in vivo into phospholipids of all three cell fractions studied, but to varying degrees depending on the type of analogue used. The in vivo incorporation of these bases into membrane phospholipids produced no significant effect on the activities of seven membrane-bound enzymes: (Na+, K+)-ATPase, 5'-nucleotidase (plasma membranes); TPNH-cytochrome c reductase, glucose-6-phosphatase, inosine diphosphatase (microsomes); and succinate cytochrome c reductase (mitochondria). The incorporation of base analogues into phospholipids was accompanied by several compensatory mechanisms. (a) The quantity of both phosphatidylcholine and phosphatidylethanolamine decreased up to 75% and 50% respectively in 3 days. (b) The molar ratio of desmosterol/phospholipid in the plasma membranes of LM cells grown in suspension culture in the presence of choline analogues decreased from 0.65 to 0.45. (c) The percentage of lysophosphatidylcholine increased over 2-fold in the phospholipid of all subcellular fractions studied. The quantity of lysophosphatidylcholine was directly proportional to the number of methyl groups on the nitrogen atom of the base analogue supplemented to the cells. This was a specific effect since the quantity of lysophosphatidylethanolamine, the other major lysophospholipid, remained unchanged. (d) The ratio of zwitterionic phospholipids to acidic phospholipids remained relatively constant in all isolated membrane fractions regardless of analogue supplementation. Neither increase in the degree of unsaturation nor shortening of fatty acid chain length was noted in response to analogue supplementation.


Synthesis in vitro of intrinsic membrane proteins by free, membrane-bound, and Golgi apparatus-associated polyribosomes from rat liver.

A fraction of intrinsic membrane proteins was prepared from the major membranous cell components of rat liver by extraction of the membranes with KCl and deoxycholate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the compositions of the intrinsic protein fractions from rough and endoplasmic reticulum, smooth endoplasmic reticulum. Golgi apparatus, plasma membrane, and nuclear envelope were similar to each other but distinct from that of mitochondria. Among endomembranes, differences were in the ratios of protein constituents plus a few protein bands of Golgi apparatus and plasma membranes not found in endoplasmic reticulum or nuclear envelope. The abilities of total rough endoplasmic reticulum, polysomes released from rough endoplasmic reticulum, and free polysomes to incorporate amino acids into the intrinsic protein fraction were tested in vitro. Polysomes bound to endoplasmic reticulum has the greatest capacity to synthesize proteins of this fraction as shown by co-purification of radioactive products and by immunoprecipitation. Although the majority of the radioactive products synthesized by bound polysomes were distinct from those synthesized by free polysomes, certain radioactive products synthesized by free polysomes also co-purified with intrinsic membrane proteins. The results show no absolute segregation between free and bound polysomes in the synthesis of intrinsic membrane proteins. However, the majority of these proteins appear to be synthesized by polysomes bound to the endoplasmic reticulum. Several intrinsic proteins found in plasma membranes do not appear in rough endoplasmic reticulum. To determine where these proteins were synthesized, the ability of other endomembrane components to support in vitro incorporation of [14C]leucine into protein was examined. In contrast to plasma membranes, isolated Golgi apparatus fractions did incorporate [14C]leucine to an extent greater than could be explained by contamination with rough endoplasmic reticulum. Golgi apparatus in situ and isolated from rat liver have polyribosomes associated with a zone of cytoplasm at the Golgi apparatus periphery occupied by tubules and vesicles. The polysomes are not directly attached to membranes as with rough endoplasmic reticulum and may represent a special class of "Golgi apparatus-associated" polysomes. The polysomes, when associated with Golgi apparatus membranes, incorporated amino acids in vitro. The products synthesized in vitro were analyzed by treatment with KCl and deoxycholate and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Certain proteins synthesized by the Golgi apparatus-associated polysomes remained insoluble after the treatment with KCl and deoxycholate. The proteins synthesized by the Golgi apparatus fraction had mobilities similar to proteins in plasma membranes which were absent from endoplasmic reticulum, and which were relatively minor components of Golgi apparatus...