A study of correlation of closure of the distal radial physis with performance and injury in the Standardbred.

The distal radial physes of 113 two-year-old Standardbreds were radiographed an average of 2.6 times each during an average of 4.72 +/- 1.7 months, beginning May 1, 1975. The radiograph of each physis was graded C, B-, B, B+, or A (closed), and the date of closure was determined by a method that proved to be reliable. The average age of closure of the distal radial physes was 29.8 +/- 1.6 months, with age of closure ranging from 26 to 35 months. There were no statistical differences between age of physeal closure of colts, geldings, or fillies; pacers or trotters; "well-bred" or other horses, or between horses by the same sires or from mares by the same sires. Most of the 2-year-old racing was done before physeal closure, the average date of closure being approximately Sept 25, 1975. The average birth date of the horses was March 30, 1973. Eighty of the horses raced an average of 10.5 times, and 46 of them won an average of 2.6 races. There was progressive increase of the number of horses in the more mature classifications (B-, B, B+, to A) as they; first raced, won their first race, went their fastest mile, won their fastest mile, and attained more spped. There was no correlation between age or month of closure of the physes and money won, races won, fastest mile, or fastest win mile during the 2-year-old year. Of the 113 horses, 62 (55%) had injuries--19 major and 57 minor injuries. A significant relationship was not found between physeal classification and the incidence of injury. The number of injuries was also found not to be related to the number of races, number of races won, money won, or to the fastest mile. There was a weak indication that horses with physes classified as C or B- (very immature) may not have raced quite as well as the more mature horses and may have had a slightly higher incidence of injury.


Equine degenerative myeloencephalopathy.

Five horses of mixed breeding and 1 zebra with symmetric tetraparesis and ataxia were studied. There were 2 females and 4 males. Signs of illness were first noticed from birth to 12 months of age. Clinical and pathologic studies were performed when the animals were 4 to 24 months old. Two of the horses were related. All of the subjects had diffuse degenerative myeloencephalopathy. The degeneration was most pronounced in thoracic segments of the spinal cord. Ventral and dorsolateral funiculi were most severely affected. Axonal swelling and abnormal neuronal cell bodies were seen in the gray matter of the spinal cord and in some brain-stem nuclei. These changes were most prominent in the nuclei of the dorsal spinocerebellar tract. The cause was not determined.


Selective immunoglobulin M deficiency in foals.

Selective immunoglobulin M deficiency was diagnosed in 5 foals, on the basis of reduced serum immunoglobulin M concentrations (more than 2 standard deviations below the normal mean). All 5 foals had clinical signs or lesions involving the respiratory tract. Lkebsiella sp was isolated from joint fluids, bronchial lymph nodes, or tracheal washings from the 3 foals in which such samples were available for microbiologic evaluation.


Effects of preanesthetic medication, anesthesia, and position of recumbency on central venous pressure in horses.

Central venous pressure (cvp) was recorded in horses before and after tranquilization and during halothane-maintained anesthesia in lateral or dorsal recumbency. The cvp was significantly decreased after administration of acetylpromazine, when compared with base line measurements and measurements taken after xylazine administration. After induction of anesthesia, cvp increased with time for 75 minutes, and values in lateral recumbency were significantly higher than those in dorsal recumbency.


Superoxide dismutase (SOD) activity in hypoxic mammalian systems.

Superoxide Dismutase (SOD) activity was compared in rabbit peritoneal macrophages (ambient PO2 approximately 15 Torr) and alveolar macrophages (ambient PO2 approximately 100 Torr) and in brain, lung, cardiac muscle, and skeletal muscle of chromically hypoxic mice(ambient PO2 approximately 50 Torr) and normoxic mice (ambient PO2 approximately 150 Torr). Peritoneal macrophages (PM) have significantly less SOD activity than alveolar macrophages (AM) (PM: 2.94 +/- 0.49 (mean +/- SD); AM:6.03 +/- 1.60 units-mg protein -1 (P less than 0.01)). SOD activity of lung and brain homogenates from the hypoxic mice was significantly less than from the normoxic controls. Heart and skeletal muscle SOD activities were not significantly different. These studies show that limitations of O2 supply are associated with reductions in SOD and are consistent with the thesis that SOD plays an important role in protection against oxygen toxicity in mammalian systems.


Curve-fitting analysis of pressure-volume characteristics of the lungs.

The pressure-volume relationship of the mammalian lung is markedly curvilinear and in the normal lung, can be shown to fit an exponential function of the form V = V0(1 - K 0e-K1P) in the range of lung volume from FRC to TLC. A method is presented, using a programmable calculator, of determining the parameters V0, K0, and K1, and of deriving related constants of greater physiological significance. Values calculated from pressure-volume curves of 20 normal human subjects are listed, together with coefficients of determination, to demonstrate the adequacy of fit of the method. Half-inflation pressure (h- ln 2/K1), ranged from 2.65 to 9.21 and coefficients of determination (r2) ranged from 0.851 to 0.9998.


Determination of energy metabolism from respiratory functions alone.

To monitor variations of energy metabolism on-line a system which accurately measures VO2 and VCO2 is described. The calculation of the energy metabolism usually requires knowledge of nitrogen production. This paper presents a formula that allows calculation on-line on the energy metabolism with a maximum relative error of 4% as function of VO2 and VCO2 only. Some experimental results in normal subjects are presented.


Measurement of VO2, VCO2, and evaporative water loss with a flow-through mask.

Equations for the calculation of VO2, VCO2, and evaporative water loss were derived for use of a paramagnetic gas analyzer with a flow-through respirometry system and an open or closed mask. The magnitude of errors involved in the determination of VO2 with no CO2 absorbent are small (+/-3%) but may be greater if no H2O absorbent is used, unless an appropriate correction is made for evaporative water loss. Data collected for hummingbirds (Selasphorus sasin) and monitor lizards (Varanus gouldii) indicate the validity of the technics described for the measurement of RQ and evaporative water loss and demonstrate the use of paramagnetic gas analyzers in monitoring respiratory patterns.


Pulmonary capillary permeability to HRP in dogs: a physiological and morphological study.

Pulmonary capillary permeability was studied in 10 normal anesthetized dogs using horseradish peroxidase (HRP) as tracer. Physiological studies involved measurement of left ventricular, pulmonary arterial, and wedge pressures, and of right lymphatic duct (RLD) and thoracic duct lymph flows. Plasma and interstitial oncotic pressures were calculated from protein measurements. After 1 h of lymph collection, HRP was injected intravenously at three total dose levels, low (20-30 mg/kg), medium (55 mg/kg), and high (110-120 mg/kg) as a bolus followed by smaller doses over 1.5-3 h. HRP was measured colorimetrically in plasma and lymph and appeared in the first RLD lymph sample collected at all dose levels. Electron microscopic studies to examine the passage of HRP from plasma to RLD, while less sensitive in that HRP was seen only at the high dose, showed that it crossed pulmonary capillaries under normal physiological conditions. Freeze-cleaving studies suggest this may have occurred through pathways in the interendothelial space caused by discontinuities in the junctional strands of tight junctions.


Relative contributions of hypocarbia and hyperpnea as mechanisms in postexercise asthma.

The purpose of this investigation was to assess the relative contributions of hyperpnea and hypocapnia in the induction of postexercise asthma. To achieve these ends, eight young asthmatics were exercised on a treadmill while minute ventilations (VE) and end-tidal CO2 (PET CO2) tensions were continuously recorded. The subjects were then restudied using a partial rebreathing technique that allowed separation of minute and alveolar ventilations so that independent evaluations could be made of the relative effects of bulk airflow on pulmonary mechanics as well as a systematic study of hypocapnia in a dose-response fashion. Sustained hyperpnea with VEidentical to those recorded during exercise was totally without effect when the mean PET CO2 was isocapnic or lowered to approximately 30 Torr. Reduction in PETCO2 to 21.3 +/-0.9 Torr brought about significant changes in mechanics, but in every variable measured, exercise produced the greatest alterations and did so at PETCO2 values that had no effect when studied in a controlled fashion. Consequently, neither high VE per se, nor hypocapnia can be considered as the mechanisms underlying exercise induced asthma.


Effects of respiratory equipment on endurance in hard work.

Men ran to a voluntary end point under the following conditions listed in increasing order to resistance, expressed as inspiratory/expiratory in mmH2O at a flow of 4 l-s-1: 1) bare-headed 0/0; 2) face mask 0/0; 3) face mask -6/14; 4) mouthpiece and valves -25/29; 5) face mask -96/62; and 6) face mask -148/60. Average endurance was 545, 490, 434, 392, 343, and 338 s, respectively. Despite the diverse features of the equipment in conditions 2-6, there seemed to be a rather smooth relation between resistance and endurance. There was no indication of a threshold below which endurance would be indifferent to resistance. The duration of the phases of the respiratory cycle was measured by means of a fine thermocouple in the airway. The variability in the duration of the expiratory phase at the end of the run seemed to weaken the concept that the attainment of a critically short expiratory time might be related to the subject's decision to halt running.


Depression of serotonin clearance by rat lungs during oxygen exposure.

The effect of oxygen (O2) exposure on the ability of the isolated, perfused rat lung to clear serotonin (5-hydroxytryptamine, 5-HT) from the perfusate was evaluated in normal or vitamin E-deficient Sprague-Dawley rats. Rats were exposed to 100% O2 at 1 ATA for 4-48 h. Lungs were subsequently isolated, artificially ventilated, and perfused in a recirculating system with Krebs-Ringer bicarbonate solution, pH 7.4 containing 3% bovine serum albumin and 0.25 muM [14C] 5-HT. 5HT clearance was calculated from the disappearance rate of [ 14C] 5-HT from the perfusate. In normal rats exposed to 100% O2, there was a progressive reduction in the clearance of 5-HT with increasing duration of O2 exposure. Compared to lungs from air-exposed controls, clearance was depressed 20% (P less than 0.01) after 18 h, 22% (P less than 0.01) after 24 h, and 35% (P less than 0.001) after 48 h. With vitamin E-deficient rats, the reduction in 5-HT clearance occurred after a shorter exposure time and was of greater magnitude than in rats on a normal diet. Depression of 5HT clearance by the lungs is an early alteration of lung function fue to hyperoxia and is potentiated by vitamin E deficiency. The most likely mechanism for the depression of 5-HT clearance is interference with the transport properties of lung endothelium.


Arterial lactate responses in dogs made apneic or breathing nitrogen.

O2 stores are kept more intact in apnea than in N2 breathing which removes O2stores from the body. If lactate moves readily into the circulation, arterial lactate should rise sooner with N2 breathing than with apnea because tissue O2 is lowered faster. This was tested in 10 anesthetized, paralyzed dogs made hypoxic both ways. Arterial and mixed venous blood were sampled every minute until circulation began to fail. Calculated changes in O2 stores would have supported control V O2 for 1.3 min with N2 and 2.7 min with apnea. The PVO2 at those times were 23.1 and 20.1 Torr. Although arterial lactate rose sooner with N2 than with apnea, the mean values for lactate increase for both N2 and apnea were fitted by a single curvilinear relation with PVO2. The PVO2 at which lactate first rosores were depleted. Latent period for lactate rise, therefore, was nearly the same as that for development of tissue hypoxia.


Influence of pericardial fluid on cardiogenic gas mixing in the lung.

Respirat. Environ. Exercise Physiol. 42(1): 5-12, 1977. -In eight open-chested dogs we measured the end-inspiratory N2 concentration within 12 airways, 2.5-8.3 mm in diameter during constant flow inflations with 0.5 liter of O2 before and after the introduction of saline into the pericardial sac. The critical flow rate necessary to achieve a given end-inspiratory FN2, as well as the maximal amplitude of cardiogenic N2 fluctuations, became smaller in the presence of pericardial fluid (PF). In the presence of only 25-50 ml of saline in the pericardium the effective diffusion coefficient, incorporating cardiogenic gas mixing, decreased by as much as 50-66%, respectively. Studies in two dogs with the chest intact showed that PF caused a qualitatively similar impairment of gas mixing during breath holding. Multiple-breath N2 washouts did not reflect the decreases in gas mixing produced by PF. We believe that PF decreases cardiogenic gas mixing mainly by protecting the lung from the rotational thrust of the heart during myocardial contraction.


Dependency of hypoxic pulmonary vasoconstriction on temperature.

We studied the effect of temperature change on hypoxic pulmonary vasoconstriction. Selective hypoxia on the left lower lobe of the lung in open-chested dogs at 37 degrees C caused the electromagnetically measured blood flow to the lobe to decrease 51 +/- 5 (SE)% and its vascular resistance to increase 155 +/- 25%. Testing hypoxic response. The hypoxic response at 31.1 +/- 0.4 degrees C was only a 26 +/- 6% decrease in lobar blood flow compared to the hypoxic response at 40.0 +/- 0.5 degrees C which was a 60 +/- 5% decrease in lobar blood flow. Hypothermia itself was associated with a significant increase in pulmonary vascular resistance. We conclude that hypothermia inhibits and hyperthermia enhances hypoxic pulmonary vasoconstriction. The mechanism of inhibition may involve prehypoxic vasoconstriction.


Fluid and electrolyte shifts during bed rest with isometric and isotonic exercise.

Fluid and electrolyte shifts were measured in seven men (19-21 yr) during three 2-wk bed rest (BR) periods, each of which was separated by a 3-wk ambulatory recovery period. During two of the three BR periods they performed isometric exercise and isotonic exercise. No prescribed exercise was given during the other BR period. On day 4 of BR, plasma volume decreased (P less than 0.05) 441 ml (-12.6%) with no exercise, 396 ml (-11.3%) with isometric, and 262 ml (-7.8%) with isotonic exercise; the decreases (NS) of extracellular volume were -4.4%, -2.6%, and -2.7%, respectively. By day 13 of BR, plasma volume stabilized at the lower level with isometric and isotonic exercise and continued to decline with no exercise; but the extracellular volume returned to or above control levels due to an overshoot of the interstitial volume of +320 to +430 ml (2.0-2.7%) that was about equal to the plasma volume loss. During BR there were isocontent losses from the plasma of protein, albumin, globulin, urea N2, uric acid, creatinine, Na, Cl, osmolarity, P, and glucose that were not influenced by either exercise regimen. However, the blood, red blood cell, and plasma volumes, and the Ca and K contents were stabilized during BR by both exercise regimens. The results suggest that during BR, preservation of the extracellular volume takes precedence over maintenance of the plasma volume, and this mechanism is independent of the effects of isometric or isotonic exercise.


Fluid and electrolyte shifts in women during +Gz acceleration after 15 days' bed rest.

Twelve women (23-34 yr), comprising a bed-rest (BR) group of eight subjects and an ambulatory (AMB) group of four subjects, were centrifuged after 14 days of ambulatory control (C),after 15 days of a 17-day BR period, and on the third day of recovery (R). Venous blood was taken before and after the third +3.0 G acceleration run (1.8 G/min). Relative to (C), the +Gz tolerance after BR was reduced -49.0% (P less than 0.05) in the BR group and -38.7% (NS) in the AMB group; during (R) the BR group regained up to 89.4% and the AMB group up to 87.1% of their (C) tolerances. In each of the three test periods, the shifts in plasma Na, Cl, PO4, and osmotic contents, which accompanied +Gz, followed the outward shift of plasma volume (PV). The correlation of the shift of PV during acceleration with the +Gz tolerance was 0.72 (P less than 0.01). During acceleration, the PV and electrolyte loss for both groups after BR was about half the loss of (C) and (R). Compared with (C) and (R) values, potassium shifts were variable but the mean corpuscular volume and mean corpuscular Hb contents and concentrations were unchanged during all +Gz runs; The results indicate that: 1) the higher the (C) + Gz tolerance, the greater the tolerance decline due to BR; 2) relative confinement and reduced activity contribute as much to the reduction in tolerance as does the horizontal body position during BR; 3) bed-rest deconditioning has no effect on the erythrocyte volume during +3.0 Gz; and 4) about one-half the loss in tolerance after BR can be attributed to PV and electrolyte shifts.


Effects of acceleration on thermoregulatory responses of unanesthetized rats.

Upon exposure of rats to 2 G environments (achieved by centrifugation), there occurred a rapid decrease in colonic temperature (Tco) followed, after about 50 min, by a slow recovery toward precentrifugation levels. The initial drop in Tco was accompanied by decreases in hypothalamic and spinal cord temperatures and increases in tail temperature (Tta). In contrast to this anomalous response (i.e., increased heat loss (manifested by increased Tta) despite decreasing temperature at spinal and hypothalamic thermoreceptor areas) the return toward normal Tco appeared to involve appropriate thermoregulatory responses. The initial fall in Tco was decreased in magnitude by inverting the rat during acceleration, thereby suggesting that mechanical forces acting on the brain may underlie this temperature decrease. Exposure to cold during centrifugation allowed further examination of the thermoregulatory system. Unlike the initial acceleration-induced changes, the cold-evoked fall in Tco was not accompanied by increasing Tta and was modified by the environmental conditioning of the rats. These results are consistent with the view that exposure to 2 G adversely affects the thermoregulatory ability of rats challenged by cold.


Experimental study of convective heat transfer coefficient for the human body in water.

The steady-state convective heat transfer coefficient in water has been determined by partitional calorimetry for 17 nude subjects. Four water velocities were investigated: 0, 0.05, 0.10, and 0.25 m-s-1; and the water temperature ranged from 33.7 to 18 degrees C. In still water, hc varied from 43 W-m-2-degrees C-1 in thermoneutral conditions and a shivering rate less than 90 W-m-2 to 54 W-m-2-degrees C-1 in cold water with a shiver rate greater than 110 W-m-2. The equation, hc=0.09 (Gr-Pr)0.275, give a good approximation of this coefficient. In stirred water and for the same limits of shivering, hc can be expressed as a power function of the velocity: hc = 272.9 v0.5 and hc = 497.1 v0.65, respectively. These equations show that the flow is laminar in thermoneutral conditions and intermediate between laminar and turbulent in cold water. A study of the influence of skinfold on the magnitude of hc shows that higher values of this coefficient were obtained for thin subjects than for fat ones, concomitant with more intense shivering. The utilization of a theoretical physical model for computations of hc gave excessively high values because such methods do not embody the body shape factor and reduction of water flow adjacent to the skin.


Collaborative study of a mineral oil method for the extraction of light filth from ground paprika.

An official first action method for the extraction of light filth from spices, 44.A03--44.A05, was modified for the analyses of ground paprika. The sample is extracted with isopropanol and light filth is isolated from 40% isopropanol with mineral oil. Eight collaborators analyzed 4 paprika samples, 2 by the official method and 2 by the proposed method. Average recoveries of added elytral squares were 97 and 66%, respectively, for the proposed and official methods. Recoveries of added rodent hairs were 94 and 61%, respectively. The method has been adopted as official first action for the extraction of light filth from ground paprika.


Collaborative study of a modified method for the extraction of light filth from ground white pepper, cardamon, celery seed, coriander, and ginger.

An improved method has been developed for the extraction of light filth from 5 ground spices, ginger, white pepper, coriander, celery seed, and cardamom. The method, a modification of 44.120, utilizes a cold isopropanol defatting, followed by wet sieving and flotation of light filth from 40% isopropanol with HCl and mineral oil-heptane (85 + 15). Collaborative results show that the proposed method is more rapid to perform than the present official first action methods, 44.116 and 44.120, and yields better recoveries. The method has been adopted as official first action.


Semiautomated fluorometric analysis of riboflavin in nutrient premixes containing reduced iron.

A simple, rapid, and reproducible semiautomated method is described for routine analysis of riboflavin in premixes containing high levels of reduced iron. Reduced iron is separated from premixes by a magnet before digestion with sulfuric acid solution. The presence of ferrous sulfate, ferric pyrophosphate, and sodium ferric pyrophosphate in premixes did not affect the analytical results for riboflavin. Pretreating sample extracts with potassium permanganate and hydrogen peroxide gave variable results for riboflavin. Possible causes and mechanisms of iron interferences in fluorometric riboflavin analysis are briefly discussed. The procedure developed is capable of analyzing 40 samples/hr.


Semiautomated method for the fluorometric determination of total vitamin C in food products.

A simple method employing simultaneous extraction and oxidation has been developed for the semiautomated determination of ascorbic and dehydroascorbic acids in food products. Recovery studies were conducted on ready-to-eat breakfast cereals and both fresh and canned fruits and vegetables, with average recoveries of 101, 100, and 102%, respectively. Reproducibility data were generated showing a relative standard deviation of 3.5%. The automated method was compared with the manual AOAC fluorometric method and with indophenol titration; correlation coefficients were 0.9960 and 0.9926, respectively. The hydrolysis product of dehydroascorbic acid, 2,3-diketogulonic acid, a reported interference in this method, was prepared and shown not to form an interfering fluorescent derivative.


High performance liquid chromatographic determination of provitamin A in orange juice.

A quantitative method has been developed for the determination of alpha- and beta-carotenes and beta-cryptoxanthin, the provitamin A carotenoids in orange juice. The carotenoids were separated by high performance liquid chromatography on a single column in approximately 30 min. The procedure may also be used to measure zeta-carotene and alpha-cryptoxanthin.


High performance liquid chromatographic analysis of supplemental vitamin E in feed.

Supplemental vitamin E is extracted from feed in one step with methanol and analyzed by reverse phase partition chromatography in less than 10 min, using isocratic elution with either methanol or water-methanol as the mobile phase. Detection response (as measured by peak area) was linear between 0.5 and 3.0 mug, and the coefficients of variation for retention time and peak height on replicate analyses of the standard sample were 0.8 and 2.3%, respectively.


Determination of pirimiphos methyl in formulations by high performance liquid chromatography.

A simple and rapid method for the determination of pirimiphos methyl (O-[2-(diethylamino)-6-methyl-4-pyrimidinyl] O,O-dimethyl phosphorothioate) in formulations by high performance liquid chromatography is described. The sample is dissolved in methanol, and biphenyl is added as an internal standard. Then the sample is chromatographed by reverse phase chromatography on a Zipax octadecylsilane column. A 4 mul injection containing 0.01% pirimiphos methyl produces a 50% full scale peak at the maximum absorbance at 254 nm.


Detection of sprouted wheat by a rapid colorimetric determination of alpha-amylase.

A method is  described for the rapid, convenient detection or estimation of sprout-damaged wheat by determining its alpha-amylase content colorimetrically. A commercially available substrate in the form of dyed amylose tablets is used. The extent of sprout damage can be determined qualitatively, or quantitatively when actual alpha-amylase concentration is required. Amylase content is determined by visual comparison with prepared colored standards or by spectrophotometric analysis. The method requires only 5 min incubation time and no elaborate equipment, and is sufficiently simple for use at locations having minimal laboratory facilities.


Collaborative study of assay method for erythromycin in pelleted feeds containing binding agents.

A modified method for analysis of erythromycin in feeds containing the pelleting adjuvants bentonite and Masonex has been evaluated by collaborative study. Erythromycin is freed from the binding action of these adjuvants by an aqueous methanolic pH 8 buffer presoak before extraction with methylal. The recovery values for mash and pellets of 6 different sample types range from 86.6 to 103.0% of theory with a grand mean of 96.8%. This modification has been adopted as official first action.


Simple and rapid paper chromatographic method for the simultaneous determination of histidine and histamine in fish samples.

A simple and rapid method for the simultaneous determination of histamine and histidine in fish samples is described. The method entails paper chromatography and is sensitive from 0.5 mug to at least 30 mug. The method is reproducible to within +/- 5% and compares favorably with a biological method for histamine determination.


Electron impact and chemical ionization mass spectrometry of some butyrophenones.

The mass spectra of some butyrophenones are presented. Electron impact ionization and chemical ionization with isobutane as the reagent gas are compared. The chemical ionization spectra show the quasi molecular ion (M + H)+ to be the most abundant ion. Important fragments in the electron impact spectra are discussed for easy determination of other similar compounds.


Determination of dexamethasone in milk by high pressure liquid chromatography.

A rapid and sensitive high pressure liquid chromatographic method for the analysis of dexamethasone (9 alpha - fluoro - 11 beta, 17 alpha, 21 - trihydroxy - 16 alpha - methylpregna - 1,4-diene-3,20-dione) in milk has been developed. The corticosteroid can be quantitated at 20 ppb, with the limit of detectability estimated at 5 ppb. Recoveries ranged from 72 to 94%. After dexamethasone was injected into 5 Holstein-Friesian cows, no residues were dound in milk, even after the first milking.


Collaborative study of a semiautomated method for the analysis of prednisolone and prednisone tablets.

A semiautomated colorimetric method for the determination of prednisolone and prednisone was collaboratively studied by 6 collaborators. In the method, an alcoholic solution of the drug is extracted with chloroform and the extract is reacted with tetramethylammonium hydroxide and blue tetrazolium; the absorbance of the resulting color is read at 525 nm. Collaborators were supplied with 4 composites of tablets of different dosage levels, 2 containing prednisolone and 2 containing prednisone. Results agreed with those obtained by the author using the USP total steroid assay method. The coefficients of variation of the individual collaborator's results for prednisolone and prednisone ranged from 0.54 to 2.38 and from 0.34 to 2.19%, respectively. This method has been adopted as official first action.


Identification of 1-(1-cyanocycloalkyl) amines, intermediates in the synthesis of phencyclidine and its analogs.

Eight 1-(1-cyanocycloalkyl) amines are identified by spectroscopic techniques. Their ultraviolet and mass spectra are similar, but proton magnetic resonance and infrared spectra are distinctly different; reference spectra and data are provided. Gas-liquid and thin layer chromatographic systems for their qualitative analysis are discussed.


Separation and ultraviolet absorbance techniques for determining dihydrotachysterol in pharmaceutical preparations.

Three separation techniques and ultraviolet (UV) spectrophotometry are described for dihydrotachysterol (DHT). The absorbance procedure eliminates the necessity of establishing a regular baseline, which is advantageous in certain cases. The relative absorbance values obey Beer's law in the range of 1-10 mug DHT/ml ethanol. An absorbance ratio check ensures the applicability of the method with respect to the degree of allowable interference present. The absorbance ratio values at the indicated wave-lengths at this concentration range and in the absence of any interferences are relativiely constant. The UV absorbance could serve as the common final determinative step for the other techniques, either singly or in combination, for quantitating DHT in preparations that require varying degrees of cleanup through extraction, saponification, and thin layer chromatography. The respective mean per cent recoveries and their relative standard deviations for a 50 mug DHT standard sample treated by the 3 techniques are 99.6 -+/ 0.5, 98.5 -+/ 1.1, and 97.3 -+/ 3.3%. Content uniformity determination of DHT in tablets and in soft gelatin capsules is also presented, and precautions are discussed.


Simple and rapid gas-liquid chromatographic determination of diethylstilbestrol in biological specimens.

Diethylstilbestrol (DES) is readily extracted into toluene from biological fluids or tissue homogenates. Sodium carbonate is added to the initial mixture to eliminate potentially interfering substances. The toluene is extracted with a very small volume of aqueous trimethylphenylammonium hydroxide. This solution generates dimethyl DES in the vaporizer of a gas-liquid chromatograph. An internal standard, dienestrol (DI), is added at the beginning of the procedure and is partitioned and methylated in the same manner as DES. The DES and DI derivatives are well separated in less than 6 min on an ov-17 column. The entire analysis requires less than 15 min for a fluid specimen and less than 25 min for a solid tissue specimen. Seven samples can be analyzed each hour on a single column with a flame ionization detector. The relative standard deviations at levels from 2.5 to 100 ppm in bile are less than 5%. The lower limit of sensitivity is 8 ppb in a 1 ml bile sample.


Micro-determination of p-hydroxybenzoic esters in pharmaceuticals and cosmetics.

A rapid and specific method is described for the determination of microamounts of methyl, propyl, and butyl p-hydroxybenzoic esters (parabens) in pharmaceuticals and cosmetics. The method involves the direct extraction of parabens into benzene or chloroform followed by derivatization with phosphorochloridate. The diethyl phosphate ester derivatives are cleaned up on a Florisil minicolumn and finally measured by gas-liquid chromatography on 5% OV-210 on Gas-Chrom Q. A flame photometric detector or a KCl thermionic detector may be used. The concentration response was linear up to 40 ng parabens. The reproducibility and margin or error were tested with fortified samples. This method may be applied to the estimation of other phenol derivatives.


Identification of polychlorinated styrene compounds in heron tissues by gas-liquid chromatography-mass spectrometry.

Unknown compounds detected in Ardea herodias tissues are identified by gas-liquid chromatography-mass spectrometry as residues of octachlorostyrene. Heptachlorostyrene and hexachlorostyrene were tentatively identified.


High performance liquid chromatographic determination of sorbic acid in wine.

A method is described for the qualitative and quantitative determination of 2,4-hexadienoic acid (sorbic acid) by high performance liquid chromatography (HPLC). The results show excellent reproducibility (+/- 2.55%) and agree well with values obtained using the official AOAC ultraviolet method for both fortified wine and commercial wines. Sorbic acid is separated by HPLC, using a strong anion exchange resin, Zipax SAX, eluted with 0.01M Na2B4O7, and detected using UV (254 nm) detector. Sodium benzoate is used as an internal standard.


Gas-liquid chromatographic determination of m-phenoxybenzyl cis, trans-(+/-)-3-(2,2-dichlorovinyl)-2-2-dimethylcyclopropanecarboxylate in aqueous formulations.

A simple and rapid gas-liquid chromatographic (GLC) method was developed for the analysis of aqueous solutions of FMC 33297 (mphenoxybenzyl cis,trans-(+/-)-3-(2,2-dichlorovinyl) - 2,2 - dimethylcyclopropanecarboxylate). The compound is extracted from aqueous solution with benzene and cleaned up by liquid chromatography. The sample extracts are analyzed by GLC with a flame ionization detector and quantitated by comparing integrator counts of extracts with those of standards. Recoveries of FMC 33297 were 90-100%. The method has good precision (the standard deviation for 4 determinations of a 0.002394% formulation was 6 X 10(-6)) and is sensitive to 0.00004% active ingredient by weight,


Mammalian endonuclease, DNase V. Purification and properties of enzyme of calf thymus.

An endonuclease present in partially purified preparations of calf thymus DNA polymerase has been purified to homogeneity. It has a molecular weight of 53,000 +/- 2,500 as determined by sucrose gradient sedimentation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates the protein is composed of four subunits, each polypeptide possessing a molecular weight of 13,000. Its isoelectric point is 10.3 +/- 0.2. The endonuclease has a pH optimum at 6.6, requires Mg2+ or Mn2+ for activity, and does not attack RNA. The enzyme appears to be present in tissues other than calf thymus. The enzyme catalyzes the endonucleolytic cleavage of both denatured and native eukaryotic DNA. The enzyme introduces a limited number of single strand nicks into native DNA; hydrolysis of denatured DNA produces acid-soluble oligonucleotides. The average size of the limit product, sedimented in an alkaline sucrose gradient, is 1200 nucleotides for native DNA. The product contains 5'-phosphoryl and 3'-hydroxyl termini. While all four deoxynucleotides are found at the 5' termini, pyrimidine residues predominate. Calf thymus DNase V degrades closed circular duplex SV40 DNA and glucosylated T4DNA but not poly(dA-dT). The rate of hydrolysis of homopolymers is: poly(dT) greater than poly(dA) greater than poly(dC) greater than poly(dG) in the presence of Mg2+, and poly(dT) greater than poly(dC) greater than poly(dA) = poly(dG) in the presence of Mn2+.


Study of equilibration of the system involving two alternative, enzymically active complementing structures simultaneously formed from two overlapping fragments of staphylococcal nuclease.

Quantitative complementation of two overlapping fragments of staphylococcal nuclease, Nuclease-(1-126) (residues 1 to 126) and Nuclease-T-(50-149) (residues 50 to 149), simultaneously forms in 1 min, two alternative, enzymically active ordered structures (types I and II) resembling nuclease (149 residues) (Taniuchi, H., and Anfinsen, C.B. (1971) J. Biol. Chem. 246, 2291-2301). We determined the ratio of type I to type II complex formed from the two fragments as a function of time, temperature, and the presence or absence of the ligands thymidine 3',5'-diphosphate and calcium ion. The ratio of type I to type II complex was determined on the basis of the quantities of their derived complexes obtained after each experiment by removing the redundant amino acid sequences by limited digestion with trypsin in the presence of ligands. The quantity of the derived complexes was estimated by quantitative determination of the component fragments separated by gel filtration. The ratio of type I to type II complex formed in 2 min after mixing the two fragments was approximately 0.3 and appears to be independent of temperature and the presence or absence of ligands. The equilibrium of the system of type I and II complexes is attained through unfolding and folding. The ratios of type I to type II complex at the apparent equilibrium state of the system at 6 and 23 degrees were approximately 1.1 and 2.4, respectively. The observations indicate that the rate of unfolding of type II complex is greater than that of type I complex at 6 degrees and increases more than that of type I complex with increasing temperature. Thus, the change of the complementing structure from type I complex with increasing temperature. Thus, the change of the complementing structure from type I to type II causes a decrease in the activation free energy, an increase in the activation enthalpy, and thereby an increase in the activation entropy of unfolding. Since the unfolded states with which type I and II complexes are in equilibrium are the same, the distribution of the population of type I and II complexes at the equilibrium state will be determined on the basis of the respective decreases in Gibbs standard free energy from the unfolded state to type I and II complexes. On this basis type I complex has a lower energy by deltaG0 = -0.05 and -0.51 kcal mol-1 at 6 and 23 degrees, respectively, than type II complex. Nevertheless, at the initial complementation the population of type I complex formed is approximately one-third that of type II complex at both 6 and 23 degrees. That is, the probability (rate) of folding is not related to the decrease in energy from the unfolded to the folded state. Using van't Hoff's equation deltaH = 7.5 kcal mol-1 and then deltaS degrees = 27 cal deg-1 mol-1 from type II to type I complex.


Deoxyribonucleic acid structure in human diploid fibroblasts stimulated to proliferate.

When contact-inhibited human diploid fibroblasts that have been maintained on medium containing 0.5% serum are stimulated by 10% serum they leave the G0 state and enter the cell cycle at G1. The onset of DNA synthesis occurs at 12 to 13 h. DNA was isolated from cultures that had been stimulated for 0, 2, 4, 6, 12, 16, 20, 24, and 30 h. These DNA preparations were characterized by sensitivity to a single strand specific nuclease, CsSO4/AgClO4 buoyant density, sedimentation in neutral and alkaline sucrose gradients, and sedimentation in neutral sucrose gradients after digestion with S1-nuclease. The DNA from 4-h stimulated cultures has an increased amount of single strandedness as indicated by sensitivity to S1-nuclease and buoyant density. The amount of single strandedness increases as the cells enter S-phase and decreases as they enter the G2 period. The DNA isolated from G0 cells behaves as native, duplex DNA with virtually no breaks. The DNA from 2-h and 4-h stimulated cultures contained a significant number of breaks. The number of breaks increases to a maximum in S-phase and declines as the cells enter the G2 period. There are no nuclease-sensitive sites (gaps) in G0 or 2-h cultures. The number of sites increases 4 h after stimulation, reaches a maximum during S-phase, and decreases as the cells enter G2. These results lead us to suggest a chromosomal cycle at the level of DNA itself, whereby structural changes are occurring throughout the entire G1 period, aimed toward the initiation of DNA synthesis. These changes reach a maximum degree during replication and decline as replication is terminated.


Neutral amino acid transport. Characterization of the A and L systems in isolated rat hepatocytes.

Hepatocytes isolated from adult rat liver by enzymatic dispersion were used to investigate amino acid transport. Steady state and influx experiments were carried out with alpha-amino[1-14C]isobutyric acid and [1-14C]cycloleucine in the presence and absence of sodium under various experimental conditions. Hepatocytes concentrated alpha-aminoisobutyric acid to a 3-fold higher degree than cycloleucine. At low external alpha-aminoisobutyric acid levels (2 to 5 mM), about 25% and 75% of entry were accounted for by nonsaturable and saturable processes, respectively. The nonsaturable component was sodium-independent, and had the properties of passive diffusion. The saturable transport was dependent on external sodium; the rate of transport reached its maximal value with sodium greater than or equal to 75 mM. Sodium increased the apparent Vmax of transport without changing the apparent Km. This component was largely dependent on energy supplies and was strongly reduced at pH less than or equal to 6.5. The value for activation energy (Ea approximately equal to 15 kcal/mol, calculated from the Arrhenius plot) favors a mediated active transport. The Na+-dependent influx of alpha-aminoisobutyric acid was competitively inhibited by N-methyl-alpha-aminoisobutyric acid (Ki approximately equal to 9.3 mM) and alanine (Ki approximately equal to 2 mM) to the extent of 70% and 100%, respectively. The N-methyl-alpha-aminoisobutyric acid-sensitive part of alpha-aminoisobutyric acid influx represents transport through the "A" system, whereas the N-methyl-alpha-aminoisobutyric acid-insensitive part of transport is believed to occur through the "ASC" system. No evidence was obtained to suggest that alpha-aminoisobutyric acid is transported by the "L" system. Cycloleucine transport was a composite phenomenon involving at least two saturable processes, one of which was sodium-dependent and inhibited by alpha-aminoisobutyric acid, and probably represents entry through the A and ASC systems. The sodium-independent component was completely and competitively inhibited by 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (Ki approximately equal to 2 mM). This component exhibited accelerative exchange-diffusion and was pH-insensitive, properties which suggest a facilitated diffusion process. However, the weak inhibition exerted by oligomycin and cyanide along with the concentrative effect observed indicated that uphill transport was also operative. These data are in good agreement with those reported for the L system. We conclude that, as in Ehrlich ascites tumor cells and in embryonic heart cells, the A, ASC, and L systems are operative in isolated hepatocytes for the transport of amino acids.


Genetic differences in induction of cytosol reduced-NAD(P):menadione oxidoreductase and microsomal aryl hydrocarbon hydroxylase in the mouse.

The stimulation of reduced-NAD(P):menadione oxidoreductase (EC 1.6.99.2) activity in liver cytosol is highly correlated with the stimulation of hepatic microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2) activity in 3-methylcholanthrene-, beta-naphthoflavone-, phenobarbital-, or pregnenolone-16alpha-carbonitrile-treated inbred C57BL/6N and DBA/2N mice and in eight other inbred strains treated with 3-methylcholanthrene. No oxidoreductase activity is detectable in mouse liver microsomes. Cytochrome c and 2,6-dichlorophenolindophenol are equally good electron acceptors for the oxidoreductase. There is no preferential in vitro inhibition of induced versus control oxidoreductase activities by either alpha-naphthoflavone or metyrapone. In 3-methylcholanthrene-treated F1 and F2 progeny and offspring from backcrosses between the F1 and either C57BL/6N or DBA/2N parent, however, there is not a strict correlation between induced or noninducible aryl hydrocarbon hydroxylase and oxidoreductase activities. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, at doses (80 mug kg-1) sufficiently high to induce the hydroxylase almost as well in DBA/2N as in C57BL/6N mice, induces the oxidoreductase about 3-fold in C57BL/6N and less than 50% in DBA/2N mice. All the data are consistent with an hypothesis that two loci (Ox-1 and Ox-2) regulate oxidoreductase induction by 3-methylcholanthrene, that one of the genes is linked to the Ah locus (with an estimated recombination frequency between 2% and 23%), and that the other gene is not linked to the Ah locus. These experimental data might be useful in the protein activator hypothesis of the Britten-Davidson model for gene regulation.


Studies of human histone messenger RNA. I. Methods for the isolation and partial characterization of RNA fractions containing human histone message from HeLa S3 polyribosomes.

Large quantities of nonpolyadenylated [poly(A(-))] 4 to 18 S RNA were isolated from the polyribosomes of S phase HeLa S3 cells and were fractionated into multiple discrete RNA components by continuous elution preparative electrophoresis. Previous studies have shown that treatment os S phase HeLa cells with cytosine arabinoside inhibits DNA replication and causes translatable histone messenger RNA (mRNA) species to disappear from cytoplasmic polyribosomes (Borun, T. W., Scharff, M.D., and Robbins, E. (1967) Proc. Natl. Acad. Sci. U.S.A. 58, 1977-1983; Gallwitz, D., and Mueller, G. C. (1969) J. Biol. Chem. 244, 5948-5952; Borun, T. W., Gabrielli, F., Ajiro, K., Zweidler, A., and Baglioni, C. (1975) Cell 4, 59-67; Gallwitz, D. (1975) Nature 257, 247-248). In the present study it was found that cytosine arabinoside treatment does not appreciably affect major 7.5 to 8 S RNA species but does cause the disappearance of 8.6 to 13 S RNA components from preparative electrophoresis elution profiles of S phase polyribosomal 4 to 18 S RNA. Base ratio analysis of the 8.6 to 13 S putative histone mRNA species indicates that they are GC-rich but not like the HeLa 18 or 28 S rRNA in base composition.


Studies of human histone messenger RNA. II. The resolution of fractions containing individual human histone messenger RNA species.

Polyribosomal 4 to 18 S RNA from S phase HeLa S-3 cells has been fractionated by chromatography on oligo(dT)-cellulose and resolved into multiple discrete components by continuous elution preparation electrophoresis. The human histone messenger RNA (mRNA) species associated with various polyadenylated [poly(A(+))] and nonpolyadenylated [poly(A(-))] components of 4 to 18 S RNA were determined by translation of these RNA fractions in vitro using a Krebs II ascites cell-free system followed by resolution of histones synthesized in vitro on polyacrylamide gels containing Triton X-100. The results of these studies indicate that poly(A(-)) 4 to 18 S RNA from S phase HeLa polyribosomes contains: (a) large quantities of discrete 7.4 and 8 S RNA species which are not functional histone mRNA; (b) a discrete 8.6 S RNA fraction which contains the templates of human histone H4; (c) 9.2 to 10.7 S RNA which contains mixtures of incompletely resolved histone H2B, H2A, and H3 mRNA (These mRNA species do not closely correspond to discrete RNA subfractions resolvable by our techniques.); (d) discrete 12 and 13 S RNA fractions which contain templates of human histone H1 polypeptides. The present studies also indicate that the mRNA templates of histone variants H3.2 and H3.3 have a slightly lower electrophoretic mobility than H3.1 mRNA and that H2A.2 mRNA has a slightly lower electrophoretic mobility than H2A.1 mRNA. In addition, appreciable quantities of H3.2, H3.3, and H2A.2 mRNA are bound to oligo(dT)-cellulose in 0.5 M KCl. These results indicate that mRNA species of the same histone class differ slightly in primary structure and are consistent with the hypothesis that some histone mRNA species contain short tracts of poly(A).


Photocatalyzed labeling of adipocyte plasma membranes with an aryl azide derivative of glucose.

The photoreactive glucose derivative, N-(4-azido-2-nitrophenyl)-2-amino-2-deoxy-D-glucose, has been synthesized and used as a site-directed probe to label adipocyte plasma membranes. In the absence of photoactivation, the glucose derivative was shown to inhibit D-glucose and 3-O-methyl-glucose uptake in the isolated adipocyte system. Glucose was also shown to partially inhibit the uptake of the photoreactive probe. Photolysis of this reagent with light of wavelength greater than 300 nm in the presence of intact adipose cells resulted in the covalent incorporation of radioactivity into the plasma membrane fraction. Analysis of this material by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that four membrane components were significantly labeled and that two of these components corresponded in electrophoretic mobility to the major glycoproteins with apparent molecular weights of 100,000 and 81,000. Photolysis of the glucose analog in the presence of the particulate pellet derived from homogenized adipocytes resulted in the incorporation of the radioactive label into most of the membrane proteins. Although the cells appear to be permeable to the photoreactive probe, only a few membrane components are labeled when the cells are intact, suggesting that the probe has a high affinity for these membrane components which may be functionally involved in the glucose transport system.


20beta-Hydroxysteroid oxidoreductase. Kinetics and binding of corticosteroids and corticosteroid-21-aldehydes.

Corticosteroid-21-aldehydes were reduced only at C-20 by 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53) of Streptomyces hydrogenans, and the reduction occurred by transfer of hydrogen from the B-side of NADH. A kinetic investigation of cortisol, cortisone, cortexolone, and the 21-aldehydes of each indicated: (a) the magnitude of the Michaelis constant for any substrate was independent of the second substrate concentration; (b) the 21-aldehydes had larger Michaelis constants (5- to 8-fold) and larger maximum velocities (16- to 40-fold) than the steroids from which they were synthesized; (c) the Michaelis constant for NADH, 29 muM, was independent of the steroid substrate. With cortisol and cortisol-21-aldehyde, product inhibition patterns showed only slope effects with steroid product and NAD+, suggesting a "random" mechanism. Inhibition studies with the "poor" substrate cortisol indicated that cortisol and cortisol-21-aldehyde were reduced at the same site. The inhibition constant (180 muM) agreed with the Michaelis constant of cortisol (140 muM). The steroid product, 20beta-hydroxyprogesterone, gives noncompetitive inhibition patterns with respect to NADH and cortisol-21-aldehyde, indicating a separate binding site exists on the enzyme for this inhibitor. The intrinsic protein fluorescence of 20beta-hydroxysteroid dehydrogenase was quenched by NADH (56%) with a dissociation constant of 16 muM. NAD" quenched the protein fluorescence somewhat less (31%) with a dissociation constant of 104 muM. The fluorescence of 2-p-toluidine-6-naphthalene sulfonate is enhanced in the presence of enzyme, and there is a blue shift in the emission wavelength maximum. The enzyme-enhanced 2-p-toluidine-6-naphthalene sulfonate fluorescence is quenched by NAD+ (32%) with a dissociation constant of 128 muM. Corticosteroids and their corresponding 21-aldehydes completely quench the enhanced 2-p-toluidine-6-naphthalene sulfonate fluorescence and this feature can be used to determine enzyme-steroid dissociation constants. Corticosteroid-21-aldehydes and NAD+ dissociation constants determined in this manner agree with values obtained in kinetic measurements. The dissociation constants determined for cortisol, cortisone, cortexolone, progesterone, and 20beta-hydroxyprogesterone were at least 1 order of magnitude greater than the corresponding kinetic constants, and these findings suggest the presence of a kinetically insignificant binding site.


Association-dissociation behavior of sesame alpha-globulin in electrolyte solutions.

The major protein fraction, alpha-globulin, of sesame seed (Sesamum indicum L.) contains subunits which are associated predominantly by hydrophobic interactions. Effects of various salts show the following effectiveness of anions in dissociating the proteins, SO4(2-) less than Cl- less than Br- less than ClO4- less than SCN- less than or equal to I- less than CCl3COO-, the first two members being association-inducing ions. CCl3COONa is found to be the most effective among the series in causing dissociation. The cations Li+, Na+, K+, and Cs+ induce association, the order of effectiveness being Cs+ approximately K+ greater than or equal to Na+ greater than Li+. The low concentration of salts (anions) necessary to induce dissociation does not involve a detectable change in protein conformation. The discrepancy between the effectiveness of the anions in dissociating the protein and the Hofmeister pattern of these ions has been discussed.


Nucleotide polymerases in the developing avian erythrocyte.

Avian erythroid cells were separated into five developmental stages by sedimentation on discontinuous isotonic albumin gradients. Solubilized enzyme activities from whole cells were partially purified and characterized by ion exchange and ion filtration chromatography and velocity sedimenttation analysis. Three nucleotide polymerase types were investigated: (a) DNA-dependent RNA polymerases; (b) RNA-dependent terminal ribonucleotidyltransferases, and (c) DNA-dependent DNA polymerases. The two characteristic forms of eucaryotic DNA-dependent RNA polymerases, polymerase I (nucleolar) and polymerase II (nucleoplasmic), were identified. Polymerase III was only marginally detectable even in the earliest developmental populations. At least two species of RNA-dependent terminal ribosyltransferases were present. One apparently was the poly(A) polymerase observed in other systems. The other terminal transferase was present in two chromatographic forms, required an RNA primer, and used UTP and/or CTP as particularly efficient substrates. Three DNA polymerase activities were resolved, two of which were characteristic of the alpha and beta DNA polymerases described in other eucaryotic systems. The third polymerase was not the gamma polymerase but a separate entity. Poly(dC)-dependent RNA polymerase activity, associated with the alpha polymerase, was relatively enriched in the third DNA polymerase species. The activity levels of the nucleotide polymerases were monitored as a function of red cell maturation. Characteristic declining patterns of activity were obtained for each enzyme which correlate well with the synthetic rates of their in vivo products where these are known. These results correlate well with the synthetic rates of their in vivo products where these are known. These results are consistent with the postulate that the general transcriptive and replicative control processes operating during development may involve changes in the level of the requisite polymerases.


Interactions between solubilized cytochrome P-450 and hepatic microsomes.

Solubilized cytochromes P-450 and P-448 have been prepared from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rats, respectively. These hemoproteins can bind to microsomes and increase the microsomal monoxygenase activities. The binding of cytochrome P-450 enhances the microsomal benzphetamine demethylase activity, whereas cytochrome P-448 enhances the ethoxycoumarin dealkylase and benzo[a]pyrene hydroxylase activities. The added cytochrome P-450 is believed to be incorporated into the microsomal membrane, and the enriched microsomes can be separated from the free hemoprotein by gel filtration. A correlation between the increased cytochrome P-450 content and the enhanced catalytic activity of the microsomes is shown. Several lines of evidence suggest that the exogenous cytochrome P-450 molecules become catalytically active only when they are incorporated into the membrane. By measuring the enhanced ethoxycoumarin dealkylase activity, the rate of the proposed incorporation of cytochrome P-448 into microsomes can be measured, and the temperature dependence of the rate is reported. The addition of cytochromes P-448 and P-450 causes a great increase in the monoxygenase activities of microsomes which have been treated with linoleic acid hydroperoxide. The hydroperoxide treatment denatures almost all the cytochrome P-450 molecules in the microsomes but retains most of the NADPH-cytochrome P-450 reductase activity. Experiments with such microsomes indicate that the added cytochrome P-450 molecules, after incorporation into the membrane, have a direct access to the reductase molecules and are able to receive electrons directly from the latter. The present results are consistent with a nonrigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.


Progesterone-binding components of chick oviduct. Receptor B subunit protein purified to apparent homogeneity from laying hen oviducts.

The progesterone receptor B subunit has been purified to apparent homogeneity from hen oviduct cytosol using ammonium sulfate precipitation and chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite, and gel filtration on Agarose A-1.5m. The material is obtained (1 mug of purified protein/g of tissue) in about 17% yield. The protein is highly purified; a single band is seen on gel electrophoresis in acid-urea and sodium dodecyl sulfate gels as well as gel electrophoresis under nondenaturing conditions. In the latter system, labeled progesterone co-migrates with the protein band, demonstrating that the isolated protein is a progesterone-binding species. Labeled progesterone bound to the protein is displaced by progestational steroids but not by estradiol or hydrocortisone. The protein has a molecular weight of 116,000 g/mol as determined by gel electrophoresis and has a single polypeptide chain. The isolated protein contains 98% endogenous nonradioactive progesterone and is 2% labeled by incubation in vitro with radiolabeled progesterone. It behaves identically on all of the chromatographic steps to chick receptor B subunits described in earlier publications.


Progesterone-binding components of chick oviduct. Biochemical characterization of purified oviduct progesterone receptor B subunit.

A number of physical and chemical properties of pure hen oviduct progesterone receptor B subunit have been determined. The molecule consists of a single polypeptide chain with a molecular weight of 115,000 g/mol as determined by gel filtration in the presence of 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate. The labeled subunit has retained the biologically important properties which it displayed in cruder preparations: it binds to nuclei (Kd = 1 X 10(-9) M) and chromatin (Kd = 1.5 X 10(-9) M) but does not bind to DNA. Reaction of the purified subunit with dansyl (5-dimethylaminonaphthalene-1-sulfonyl) chloride revealed a single NH2-terminal lysine. The amino acid composition has been determined and has been shown to be distinct from that of other steroid-binding proteins and consistent with the known properties of the molecule. In addition, no evidence for carbohydrate or phosphorylated amino acids was observed. The protein contains about 12% alpha helix as determined by circular dichroism. The ultraviolet spectrum of intact steroid receptor complexes revealed that the purified subunit had no pyridine nucleotide cofactor or nucleic acid, and that each receptor molecule contains a single hormone binding site. Electron microscopic analysis confirms the prolate-ellipsoid shape of the protein, with a long axis of 114 A. The purified protein isolated as described in a companion paper is shown here to have the characteristics of the crude receptor subunit B. Due to the apparent role in the hormone response, this protein has been named progestophilin B.


Subunit structure and chromophore composition of rhodophytan phycoerythrins. Porphyridium cruentum B-phycoerythrin and b-phycoerythrin.

A comparative study is presented of the two phycoerythrins of the unicellular red alga Porphyridium cruentum. Native B-phycoerythrin has a molecular weight of 236,000 +/- 18,000 in 0.05 M potassium phosphate at pH 7.0, and an absorption spectrum with maxima at 545 nm (epsilonM = 2.41 X 10(6) M-1 cm-1) and 563 nm, and a shoulder at 498 nm. The protein carries 38 phycoerythrobilin and at least two phycourobilin prosthetic groups per 240,000 daltons. B-Phycoerythrin is composed of three dissimilar subunits, alpha and beta, each of 17,500 daltons, and gamma of 30,200 daltons. Physical, chemical, and spectroscopic data are consistent with a subunit structure (alphabeta)6gamma for B-phycoerythrin. The alpha and beta subunits carry solely phycoerythrobilin chromophores, while the gamma subunit carries both phycoerythrobilin and phycourobilin groups. The NH2-terminal sequences of the alpha and beta subunits determined by sequential Edman degradation, are shown below: alpha subunit: Met-Lys-Ser-Val-Ile-(Gly-Arg-Phe: beta subunit: Met-Leu-Asp-Ala-Phe-(Thr)-Arg-Val-Val-Val-Asn-Ala-Asx-Ala-(  )-Ala-Ala-Tyr-Val. The NH2 terminus of the gamma subunit is blocked. b-Phycoerythrin is polydisperse and exhibits native molecular weights ranging from approximately 40,000 to approximately 260,000, depending on pH, ionic strength, and protein concentration. The absorption spectrum is characterized by maxima at 543 nm (epsilonM = 3.41 X 10(5) M-1 cm-1/35,000 daltons) and 563 nm. The protein carries six phycoerythrobilin groups per 35,000 daltons. b-Phycoerythrin is made up of two dissimilar types of subunits, alpha and beta, of 17,500 daltons each. The alpha and beta subunits derived from b-phycoerythrin appeared equivalent to the corresponding subunits of B-phycoerythrin on the basis of the following criteria: (a) identical chromatographic behavior on Bio-Rex 70 in acid urea; (b) similar amino acid compositions; (c) identical mobilities on polyacrylamide gels in the presence of sodium dodecyl sulfate; (d) similar phycoerythrobilin contents; (e) identical NH2-terminal sequences. These data support, but do not establish unambiguously, the conclusion that b-phycoerythrin may be a component of B-phycoerythrin. The absence or presence, and relative height, of an absorption peak (or shoulder) at 498 nm represents the major difference among the absorption spectra of different classes of phycoerythrins. The present study shows that this spectral feature is dependent on the presence and amount of phycourobilin chromophores in the native protein and is correlated with the presence of the gamma subunit.


Removal of the projections from cytoplasmic microtubules in vitro by digestion with trypsin.

Trypsin was found to selectively destroy the high molecular weight (HMW) proteins associated with microtubules. Both major groups of high molecular weight bands observed by sodium dodecylsulfate-polyacrylamide gel electrophoresis were destroyed at the same rate (t 1/2 = 1 min), while tubulin was little affected under the conditions employed. Destruction of the high molecular weight bands was correlated with the disappearance of the lateral projections observed on the surface of microtubules. The trypsin-treated protein was capable of reassembly to form microtubules and showed characteristic ring-shaped structures at low temperature. Analysis of fragments formed from the high molecular weight proteins indicated the initial appearance of a fragment of M = 255,000 which failed to bind to microtubules. These results suggest that a small fragment of the high molecular weight protein molecule may remain bound to the microtubule after removal of the lateral projection, and that this fragment may retain the ability to promote microtubule assembly.


Comparison of mRNA binding by Met-tRNAf binding protein and mRNA-associated proteins.

One of the heterogeneous mRNA binding activities in the 0.5 M KCl eluate of rabbit reticulocyte polyribosomes co-purified to apparent homogeneity through phosphocellulose and DEAE-cellulose chromatography and isoelectric focusing with the GTP-dependent Met-tRNAf binding protein. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following iodination revealed putative subunits of 51,000 and 39,000 apparent molecular weights. Specificity of mRNA binding by this protein was suggested since the ability of poly(A)-rich mRNA to compete for binding of [3H]poly(A)-rich mRNA exceeded by 10- to 100-fold that of most natural or synthetic RNAs tested, except for the hybrid poly(G) - poly(C) which was almost as effective, and poly(G), which was more effective, at competing for protein-dependent binding. The mRNA binding activity exhibited complete GTP independence and no apparent divalent cation requirement. GDP inhibited Met-tRNAf binding but neither GDP, GMP, nor 7-methylguanosine 5'-monophosphate inhibited mRNA binding by this protein. Similar data were obtained with respect to the ability of natural or synthetic RNAs to compete for binding of [3H]poly(A)-rich mRNA by proteins associated with purified rabbit reticulocyte polyribosomal mRNA-protein particles; while poly(A) was an ineffective competitor, poly(G) was more effective than even mRNA at competing for protein-dependent binding. No significant binding of Met-tRNAf by mRNA-protein particles was detected. Polyacrylamide gel electrophoresis following reduction of mRNA-protein particles revealed apparent co-migration of a major protein with one subunit of the GTP-dependent Met-tRNAf binding protein, but no protein comparable to the 39,000 dalton subunit protein.


Binding of acetyl-CoA to chicken liver pyruvate carboxylase.

Pyruvate carboxylase from chicken liver is a tetramer whose catalytic activity is completely dependent on the presence of acetyl-CoA. However, no direct evidence concerning the nature of the binding of the activator to the enzyme has been available. This is due in part to the instability of the enzyme and its ability to hydrolyze acetyl-CoA at an appreciable rate. The present studies on binding of the activator with the rapid flow dialysis method of Colowick and Womack ((1969) J. Biol. Chem. 244, 774-777) as modified by Klapper ((1970) Biochem. Biophys. Res. Commun. 38, 172-179) show four binding sites for acetyl-CoA. The binding dissociation constant at pH 7.2 is 13.9 muM as compared with an activation constant of 13.3 muM for the catalytic reaction at this pH. The relationship between acetyl-CoA concentration and catalytic activity is highly cooperative (nH = 2.9). The binding process also exhibits positive cooperativity but to a lower degree (nH = 1.9). Pyruvate carboxylase from chicken liver is rapidly inactivated and dissociated in the cold (0 degrees). The inactive protomeric form of the enzyme has been shown to be unable to bind acetyl-CoA at 0 degrees although the tetrameric species can do so. These results provide a plausible explanation for the catalytic inactivity of the protomer. The presence of acetyl-CoA results in an ultraviolet difference spectrum for the enzyme with a maximum at 280 nm. Half-maximal optical density difference is observed at an acetyl-CoA concentration of 9 muM, in reasonable agreement with the binding and activation constants.


Pancreatic elastase in human serum. Determination by radioimmunoassay.

This study demonstrates that a serine endopeptidase of pancreatic origin (elastase 2) circulates in human blood. A specific and highly sensitive radioimmunoassay has been developed for pancreatic elastase 2 in human serum. The inactivation of elastase 2 employed as radioiodinated tracer with an active site-specific reagent (phenylmethanesulfonyl fluoride) was necessary to prevent its binding by serum alpha1-antitrypsin and alpha2-macroglobulin while maintaining its immunoreactivity. The assay is based upon competition of standard human pancreatic elastase 2 with 125I-labeled phenylmethanesulfonyl elastase 2 for specific antibody binding sites, after which a second antibody precipitation step is used to separate bound from free 125I-labeled phenylmethanesulfonyl elastase 2. The minimum detectable concentration of elastase 2 was 0.9 ng/ml. The average normal fasting serum level determined was 71 ng/ml, approximately 80-fold greater than the minimum detectable amount. The form of radioimmunoassayable elastase 2 in normal human serum has been investigated by gel filtration of serum samples on Sephadex G-200 followed by radioimmunoassay of column fractions. The majority of the immunoreactive elastase 2 is eluted from G-200 in the void volume. While a minor amount of elastase 2 is eluted in a position consistent with alpha1-antitrypsin-elastase 2 complex, no free elastase or free proelastase is detectable. Addition of exogenous elastase 2 to normal serum prior to gel filtration on G-200 produced an increase only in the peak of radioimmunoassayable elastase bound to alpha1-antitrypsin. In vitro experiments have demonstrated that while elastase 2 bound to alpha1-antitrypsin is immunologically reactive, alpha2-macroglobulin-bound elastase 2 cross-reacts less than 2% in this radioimmunoassay. The assay has been shown to be specific for elastase 2. Human pancreatic elastase 1, anionic trypsin, chymotrypsin I, and chymotrypsin II do not cross-react in this assay system. The major advantages of this radioimmunoassay over enzymatic assays are its high sensitivity and ability to measure the enzyme in terms of its total protein concentration.


Direct desaturation of eicosatrienoyl lecithin to arachidonoyl lecithin by rat liver microsomes.

A microsomal enzyme system from rat liver was shown to catalyze desaturation, in presence of reduced pyridine nucleotides and oxygen, of 1-acyl-2-[14C]eicosatrienoyl-sn-glycero-3-phosphorylcholine to 1-acyl-2-[14C]arachidonoyl-sn-glycerophosphorylcholine. This desaturation was linear with time and proportional to microsomal protein concentration, and proceeded with no significant breakdown of the lecithin substrate. The microsomal enzyme system will also desaturate 1,2-di-[14C]eicosatrienoyl-sn-glycero-3-phosphorylcholine and [14C]eicosatrienoyl-CoA, but not free [1-14C]eicosatrienoic acid in the absence of ATP, Mg2+, and CoA. Desaturation of 1-acyl-2-[14C]eicosatrienoyl-glycerophosphorylcholine as well as [14C]eicosatrienoyl-CoA was dependent on oxygen and either NADH or NADPH, and was inhibited by cyanide but not by carbon monoxide, indicating the involvement of cytochrome b5 and not P450. The activity of both eicosatrienoyl-glycerophosphorylcholine desaturase and the eicosatrienoyl-CoA desaturase was increased in rats that had been starved for 48 h and refed a fat-free diet. These data indicate the existence of a new route to synthesis of arachidonate, namely, by desaturation of eicosatrienoyl lecithin to arachidonoyl lecithin.


Thermodynamic studies on subunit assembly in human hemoglobin. Self-association of oxygenated chains (alphaSH and betaSH): determination of stoichiometries and equilibrium constants as a function of temperature.

The homogeneous self-association of isolated alphaSH chains and betaSH chains from human hemoglobin has been studied by analytical molecular sieve chromatography over the concentration range 0.004 to 15.2 mg/ml. Detailed studies were carried out as a function of temperature at pH 7.4 in 0.1 M Tris/HCl, 0.1 M NaCl, 1 mM Na2EDTA in order to establish stoichiometries, equilibrium constants, and enthalpies for the self-association reactions. The dissociation data best describe the alphaSH system as being a monomer-dimer equilibrium (2alpha1  in equilibrium alpha2). Under the same conditions the betaSH system is best described by a monomer-tetramer equilibrium (4beta1 in equilibrium beta4). van't Hoff enthalpies were determined from the temperature dependence of the equilibrium constants. For the 2alpha in equilibrium alpha2 reaction the molar enthalpy delta H = 4.3 +/- 0.5 kcal, and for the reaction 4beta1 in equilibrium beta4, deltaH = 23.5 +/- 1.0 kcal. Unitary entropies were determined to be: deltaSu = 40.6 e.u., deltaSu = 177.5 e.u., respectively. Thermodynamic parameters for association of the two types of chains are roughly comparable in magnitude if four bonding interactions are assumed in the beta4 tetramer. Both reactions are entropy-driven, and the overall results (including salt effects) are consistent with a dominant role of hydrophobic interactions. Increasing the NaCl concentration to 2 M at 21.5 degrees under the same buffer conditions increases the association constant for both the alphaSH and betatsh chains. This increase in the association constants with increasing salt concentration is attributable to the increased binding of salt, or the release of bound water upon formation of the association complexes, or both. The present results do not distinguish between these possibilities. The introduction of inositol hexaphosphate (IHP) was found to have no effect upon subunit association in betaSH chains. This result and the previously observed effect of IHP upon oxygenation of beta chains imply that oxygenation and self-association are completely unlinked in this system. Accurate determinations of (a) the enthalpy changes for homogeneous reactions of isolated chains, carried out in this study and of (b) the enthalpy of forming alpha2beta2 tetramers from alphabeta dimers provide a basis for the interpretation of (c) calorimetric studies on reconstitution of hemoglobin from the isolated chains, described in accompanying papers.


Thermodynamic studies on subunit assembly in human hemoglobin. Temperature dependence of the dimer-tetramer association constants for oxygenated and unliganded hemoglobins.

Equilibrium constants for dimer-tetramer association of oxygenated human hemoglobin have been determined as a function of temperature, by analytical molecular sieve chromatography, yielding a van't Hoff enthalpy of 3.8 +/- 1.6 kcal/mol, and a unitary entropy of 48.4 +/- 5.7 e.u. Under the same conditions (0.1 M Tris/HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4) the equilibrium constants for dimer-tetramer association of unliganded hemoglobin have been determined as a function of temperature from kinetic studies of the forward and reverse rate constants. The derived enthalpy and unitary entropy for association of unliganded hemoglobin are 0deltaH2 = 28.9 +/- 0.5 kcal/mol and 0deltaS2 = -41.8 +/- 1.7 e.u., respectively. Thus the oxygenation-linked enthalpy of subunit association is 32.7 +/- 1.7 kcal. The corresponding entropic coupling is 90.2 +/- 5.9 e.u. The thermodynamic parameters are consistent with an increased role of hydrophobic interactions within the dimer-dimer contact region upon oxygenation, or a decreased role of hydrogen bonds and ion pair interactions. The opposite signs for association entropies in unliganded and oxygenated states suggest the existence of two distinct subunit interaction processes during the course of oxygenation with a reversal in sign at some particular binding step. The results provide a necessary basis for interpretation of calorimetric results on reconstitution of hemoglobin, described in an accompanying paper.


Thermodynamic studies on subunit assembly in human hemoglobin. Calorimetric measurements on the reconstitution of oxyhemoglobin from isolated chains.

Calorimetric heats generated upon mixing solutions of alphaSH and betaSH chains of human hemoglobin have been studied by isothermal heatburst microcalorimetry as a function of mixture composition. Based upon studies described in accompanying papers, the contributions to the measured heats arising from (a) alpha chain self-association, (b) beta chain self-association, (c) association of dimers to form tetramers, have been evaluated. Taking these processes into account, the calorimetric data have been used to determine the enthalpy of formation for alphabeta dimers, yielding a value of -15.71 +/- 0.96 kcal in the fully oxygenated state at 21.5 degrees in 0.1 M Tris/HCl, 0.1 M NaCl, 1 mM Na2EDTA, pH 7.4. The total enthalpy for assembly of a mole of hemoglobin tetramers from oxygenated chains is -27.6 +/- 2.1 kcal. Combining results of this study with independently determined information, limits can be placed upon the magnitude of the enthalpy for dimer formation in unliganded hemoglobin. The total enthalpy for assembly of a mole of unliganded hemoglobin tetramers from unliganded chains is -61.6 +/- 3.5 kcal, or approximately twice the value for oxygenated hemoglobin. This difference lies entirely in the dimer-tetramer stage of assembly. There are essentially no oxygenation-linked thermodynamic quantities (deltaG, deltaH, deltaS) associated with alphabeta dimer formation from isolated chains.


Consequences of blocked sterol synthesis in cultured cells. DNA synthesis and membrane composition.

When cell cultures were incubated with certain oxygenated derivatives of cholesterol which specifically depress 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity (i.e. 25-hydroxycholesterol, 7-ketocholesterol, or 20 alpha-hydroxycholesterol), a number of consequences followed the decline in enzyme activity. DNA synthesis, expressed in terms of (methyl-3H)thymidine incorporation/mg of cellular protein, declined progressively after the addition of an inhibitor to L-cell cultures and had essentially ceased by 80 h. A decline in the rate of cell growth, as determined by the measurement of cellular protein, became apparent about 24h after the addition of the inhibitor. These effects of the inhibitory sterols were reversed when mevalonate or cholesterol was added to the medium within 48 h after the addition of the inhibitor. Specific rates of uridine incorporation into RNA and of leucine into protein were not significantly altered during 80 h of incubation with the inhibitor. Sterol synthesis was suppressed; and the concentration of desmosterol in L-cells declined to about one-half of the control level within 36 h. Since phospholipid concentrations were not altered significantly by the treatment, the molar ratio of sterol to phospholipids declined to levels lower than any previously reported for mammalian cells. Changes in the molar ratio of sterol to phospholipids in plasma membranes isolated from L-cells that had been incubated with 25-hydroxycholesterol or cholesterol were in agreement with those found in cells under similar conditions. The inhibitory sterols caused a similar but slower decline in the concentrations of unesterified and total cholesterol in fetal mouse liver cell cultures and rat hepatoma cell cultures.


Isolation and physical characterization of hyaluronic acid prepared from bovine nasal septum by cetylpyridinium chloride precipitation.

Raw extract in 2 m CaCl2 of bovine nasal septum cartilage was eluted from 4 per cent agarose gel to give a "void volume" Fraction v-4, which was indistinguishable in composition and behavior on viscometric and sedimentation analysis from the densest fraction obtained by associative centrifugation in a cesium chloride density gradient. The sulfated proteoglycan was precipitated (Fraction A) by cetylpyridinium chloride from acidic solutions of Fraction v-4 or of dialyzed raw ectract. Neutralization under conditions of low ionic strength precipitated a further small fraction (B), which contained from 0.5 to 1 per cent of the uronic acid in the original extract. Analysis by associative and dissociative density gradient centrifugation demonstrated that Fraction B resembled in effective density known samples of hyaluronic acid from other sources. Gel chromatography of proteolytic digests of Fractions A and B on 6 per cent agarose indicated that cetylpyridinium chloride precipitation essentially separated sulfated proteoglycan (A) from hyaluronic acid (B). A viscosity-average molecular weight of about 5 x 10(5) was estimated for a sample of Fraction B purified in a dissociative (4 M guanidine hydrochloride + CsCl) density gradient. Sedimentation velocity data were consistent with this result. Analysis of hexosamines showed that the sample contained 96 per cent glucosamine, confirming the identification of hyaluronic acid. The proteoglycan fraction (A) resembled "subunits" in its sedimentation behavior.


Studies on the kinetic mechanism of oxidative phosphorylation.

The kinetics of the synthesis of ATP from ADP and Pi by beef heart submitochondrial particles were examined. When Pi was the variable substrate positive cooperativity was observed, whereas if ADP was varied, linear double reciprocal plots were obtained. The analog of Pi, thiophosphate, was a noncompetitive inhibitor of ATP synthesis with respect to ADP, while the analog of ADP, AMP (CH2)P, was an uncompetitive Pi leads to ATP exchange inhibitor. The kinetics of the initial velocity isotopic exchanges of oxidative phosphorylation were also examined. When the Pi leads to ATP exchange was examined, it was found that if ADP concentration was held constant while ATP and Pi concentrations were varied at a constant ratio, linear double reciprocal plots were obtained. However, if Pi concentration was held constant and ADP and ATP concentrations were varied at constant ratio, apparent substrate inhibition was observed. The 2, 4-dinitrophenol-sensitive ADP leads to ATP exchange showed linear double reciprocal plots regardless of which components were varied. These results are interpreted to indicate that in the direction of ATP synthesis, the reaction is ordered, with Pi adding to the enzyme before ADP addition.


Purification of biologically active globin mRNA using cDNA-cellulose affinity chromatography.

A complementary DNA (cDNA) copy of mouse globin mRNA was synthesized using the RNA-dependent DNA polymerase from avian myeloblastosis virus and the oligo(dT) covalently attached to cellulose as primer. All four deoxyribonucleotide triphosphates, NaCl, the globin mRNA template, and an oligo(dT) primer were required for optimal synthesis of cDNA. By saturating the primer sites using a 3-fold excess of mRNA, sufficient concentrations of immobilized cDNA could be synthesized to allow the hybridization reactions to be performed using an excess of globin cDNA. Conditions which permitted the annealing of globin mRNA to cDNA-cellulose were selected and the sequence specificity for hybridization to cDNA-cellulose was determined using 28 S ribosomal RNA, polyadenylic acid, and mouse L-cell RNA. Both analytical and preparative applications of this chromatographic medium were explored. When radioactively labeled poly(A)-containing 9 S RNA isolated from nucleated erythroid cells was analyzed by affinity chromatography on globin cDNA-cellulose, 46 per cent of the applied radioactivity hybridized to the cDNA-cellulose column. Only 1 per cent of the labeled RNA was retained by the column during reapplication of the unbound fraction, while 96 per cent of the bound RNA reannealed to cDNA-cellulose. Hybridizations utilizing unfractionated RNA extracts from either mouse reticulocytes or nucleated erythroid cells provided a one-step purification method for globin mRNA sequences. The relative purity of the RNA isolated by cDNA-cellulose affinity chromatography was determined by hybridization kinetic analysis. The cDNA-bound fraction obtained from the unfractionated RNA of either cell type was shown to have a Crt1/2 of 2.7 x 10-3. This represents a 60-fold purification of the globin sequences present in reticulocyte polysomal RNA and a 280-fold enrichment of the globin mRNA in nucleated erythroid cells. Hybridization to cDNA-cellulose did not result in any change in the sedimentation rate of globin mRNA. Furthermore, experiments were performed which demonstrated that the globin mRNA isolated by hybridization to cDNA-cellulose retained its biological activity when assayed in a wheat germ cell-free lysate.


A pregnenolone-binding protein in soluble fraction of guinea pig adrenal cortex.

A pregnenolone-binding component has been detected in the soluble fraction of the guinea pig adrenal cortex. Enzymatic degradation studies revealed that the binding component was a protein. The binding was destroyed at 60 degrees but was not inhibited by sulfhydryl reactants. Pregnenolone was bound optimally at pH 7 to 7.5 The equilibrium association constant at 0 degrees was 10(7) M-1. The pregnenolone-binding protein had an apparent molecular weight of 58,000, as determined by gel filtration. With the exception of pregnenolone sulfate, structurally similar steroids did not interfere with pregnenolone binding. No such binding activity was detected in the guinea pig liver and kidney. Serum contained pregnenolone-binding activity which was distinguishable from the adrenal cytosol factor by a variet of physicochemical means. The physiological importance of this finding remains to be determined.


Specificity of fructose-1, 6-P2 aldolase (muscle) and partition of the enzyme among catalytic intermediates in the steady state.

Others have concluded that beta-fructose 1, 6-bisphosphate is a substrate for muscle aldolase on the basis of rapid kinetic measurements. In view of new data showing excellent aldol cleavage of an analog of the keto form and a very high rate of spontaneous ring opening, Midelfort et al. (Midelfort, C. F., Gupta, R., and Rose, I.A. (1976) Biochemistry 15, 2178-2185) have suggested that the beta form may be used only after spontaneous conversion to the keto form in solution, followed by reaction of the keto form with the aldolase. In order to determine whether beta-fructose-1, 6-P2 is itself a substrate a steady state approach was devised in which the beta form is specifically produced by phosphofructokinase for use by the aldolase present. Since aldolase binds very tightly to bisphosphates similar in size to beta-fructose-1, 6-P2, it was expected that if a spontaneous ring opening were essential for catalysis, it would be possible to decrease the rate of production of triose-Ps by using very high concentrations of aldolase. If the beta form were itself a substrate, the rate would reach a constant value with increasing aldolase, limited by the phosphofructokinase rate. It was found that under conditions where only approximately 2 per cent of the total fructose-1, 6-P2 present in the steady state would be free, the turnover of the complexed fructose-1, 6-P2 was about 20-fold greater than that in which spontaneous ring opening is a required step. Using similar methods, the turnover of enzyme-bound glyceraldehyde-P and dihydroxyacetone-P were determined. It was concluded that at saturation both the beta and acyclic forms have about the same rates as substrates.


Isolation and purification of double-stranded ribonuclease from calf thymus.

A RNase from calf thymus, which specifically cleaves native or synthetic double-stranded RNA molecules endonucleolytically, has been isolated and purified from calf thymus. For optimal activity, the enzyme requires a sulfhydryl reagent and divalent cations; over 95 per cent of the activity is inhibited by 0.5 mm ethidium bromide. The degradation of [3H]poly(C)-poly(I) by purified enzyme preparations yields labeled dinucleotides and octanucleotides; the latter oligonucleotide contained 5'-phosphate and 3'-hydroxyl termini. The enzyme cleaves high molecular weight RNAs such as RNA products formed in vitro by T3 phage-induced RNA polymerase from T3 phage DNA, heterogeneous RNA isolated from duck reticulocyte nuclei, and 45 S RNA isolated from rat liver nucleoli. The mode of degradation of RNA in vitro with the double-stranded RNase is similar to that of Escherichia coli RNase III and appears to act endonucleolytically. The degradation of 45 S RNA with the enzyme results in the production of 29 S and 19 S RNA fragments. These findings suggest that the enzyme may be involved in the processing of high molecular weight precursor RNAs to mRNA or rRNAs in a manner analogous to that reported for RNase III of E. coli.


Native heparin from rat peritoneal mast cells.

[35S]Heparin was produced in vitro by incubation of rat peritoneal mast cells with [35S]sulfate and in vivo by injection of [35S]sulfate into rats. The [35S]heparin together with nonlabeled heparin in the mast cells was isolated in native form by mild methods that avoided the use of proteolytic enzymes or high alkali concentrations. The heparin had low anticoagulant activity. Incubations of mast cells with [35S]sulfate for less than several hours in vitro resulted in [35S]heparin of approximately Mr=200,000 to 400,000 based on gel filtration, while longer incubations yielded [35S]heparin of approximately Mr=750,000 that was similar to the nonlabeled heparin in the mast cells. When [3H]serine was included in the in vitro incubations, 3H-labeled material was found to co-chromatograph with the [35S]heparin. None of the heparin could be degraded by any of several proteolytic enzymes, but incubation for 14 h at 25 degrees with 0.5m NaOH degraded all samples to a size of approximately Mr=40,000. One-third of the [3H]serine label continued to co-chromatograph with the [35S]heparin after alkali treatment, while the remaining two-thirds appeared as smaller molecules completely separated from the [35S]heparin. Thus, native heparin of the mast cell may be an unusual proteoglycan that is resistant to proteolytic enzymes.


Purification and properties of pig heart crotonase and the presence of short chain and long chain enoyl coenzyme A hydratases in pig and guinea pig tissues.

A short chain enoyl-CoA hydratase (crotonase) from pig heart has been purified to apparent homogeneity. The enzyme has an estimated native molecular weight of 155,000 and appears to be composed of six subunits of molecular weight 27,300. A study of the kinetic properties of the enzyme revealed that the maximal velocity decreases nearly linearly with increasing chain length of the substrates from 1,670 units/mg with crotonyl-CoA to 40 units/mg with hexadecenoyl-CoA. However, the same Km values of 30 muM were obtained for all substrates except for crotonyl-CoA for which a value of 13 muM was determined. Since the presence of both crotonase and long chain enoyl-CoA hydratase in pig heart has been reported earlier, the presence of the same two enoyl-CoA hydratases in various tissues of several animals was investigated by sequential extraction and chromatography on hydroxylapatite of tissue homogenates. The simultaneous occurrence of both types of enoyl-CoA hydratase in tissues of pig and guinea pig has thus been established. It is proposed that the complementary actions of the two enoyl-CoA hydratases assures a high rate of hydration of enoyl-CoA intermediates of all chain lengths in fatty acid oxidation.


The quantitative role of the kidneys in the in vivo metabolism of mevalonate.

The roles of the sterol and nonsterol pathways in the metabolism of circulating mevalonate have been estimated in the intact rat. On an average, the sterol pathway accounts for 74 per cent of the mevalonate metabolized, while the nonsterol, or shunt, pathway is responsible for 26 per cent of the mevalonate metabolized in the whole animal. The contribution of the kidneys to each of these processes was evaluated by two approaches. First, the localization of labeled sterols and sterol precursors derived from [14C]mevalonate was determined in each of the major tissues of the body and, second, the effect of nephrectomy upon mevalonate metabolism by the sterol and shunt mechanisms was examined. The results confirm our earlier conclusion that the kidneys represent the primary tissue site of conversion of circulating mevalonate to sterols and sterol precursors. In the present study, it was shown that by 6 h after administration of [14C]mevalonate, the major end product of mevalonate metabolism in the kidneys is cholesterol and that, moreover, the kidneys are responsible for most of the cholestreol synthesized in the intact animal from injected mevalonate. Following nephrectomy, the extrarenal tissues can readily assume the dominant role normally played by the kidneys in synthesizing cholesterol and other sterols from circulating mevalonate. The major observation of the present study is that the kidneys represent the primary site of mevalonate metabolism by the shunt pathway, in that nephrectomy results in approximately a 60 per cent decrease in the mevalonate metabolized by the shunt pathway. These studies, therefore, reinforce and expand the evidence that the kidneys represent the most important single tissue site for the metabolism of circulating mevalonate.


Insulin-like effects of polyamines in fat cells. Mediation by H2O2 formation.

Significant amounts of H2O2 were produced when the polyamines, spermine, or spermidine were incubated with a Krebs-Ringer phosphate buffer that contained bovine serum albumin (Fraction V). This effect was specific for certain amines and could be prevented by treatment of the albumin fraction with isoniazid or aminoguanidine. These features suggest that H2O2 is formed during oxidative deamination of the polyamines catalized by spermine oxidase, a known contaminant of Fraction V bovine serum albumin. The insulin-like effects elicited by polyamines in fat cells (e.g. enhancement of glucose transport and inhibition of cAMP-mediated lipolysis) were dependent on H2O2 production. Incubation of cells with catalase or treatment of the albumin fraction with isoniazid abolished the stimulation of glucose uptake by polyamines but did not alter the stimulatory effects of insulin or vitamin K5. The H2O2 generating activity was partially separated from the albumin by gel filtration; only those fractions which formed H2O2 provided support for the activation of glucose transport by polyamines. Also, the time needed to activate glucose uptake was markedly shortened by incubation of the albumin buffer with the polyamines before addition of the cells. These findings indicate that the polyamines do not themselves mimic the actions of insulin but that the insulin-like effects result from the formation of H2O2 which has been shown to stimulate glucose transport.


Transport of amino acids in renal brush border membrane vesicles. Uptake of the neutral amino acid L-alanine.

The transport of L-alanine was studied using membrane vesicles derived from the brush borders of the rabbit renal proximal tubule. Preincubation of the renal membranes with L-alanine, but not D-alanine, accelerated exchange diffusion of L-alanine, i.e. stereospecific counter transport. The equilibrium uptake of L-alanine decreased with increasing medium osmolarity. Extrapolation to infinite medium osmolarity, i.e. zero intravesicular space, indicates no uptake. These findings demonstrate that the uptake of L-alanine represents transport into membrane vesicles and not surface binding to the membrane. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium stimulated L-alanine uptake. Accumulation of the amino acid in the vesicles was maximal at 5 min and then decreased, indicating efflux. The final level of uptake in the presence of the Na+ gradient was identical with that obtained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the "overshoot" the uptake of L-alanine was slmost twice the final equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of L-alanine into renal brush border membrane vesicles against its concentration gradient. Stimulation of L-alanine uptake was specific for Na+. When the intravesicular medium contained no Na+, the rate of uptake enhanced with increases in the concentration of Na+ in the external medium. Increasing the Na+ gradient lowered the apparent Km for L-alanine. In the absence of the Na+ gradient, the rates of uptake of L- and D-alanine were identical. In the presence of the Na+ gradient, the rate of D-alanine uptake was stimulated, but significantly less than that of L-alanine. The uptake of L-alanine, at a given concentration of amino acid reflected the sum of the contributions from Na+ gradient-dependent and -independent transport systems. The dependent system was saturated at about 2 mM L-alanine. The independent system exhibited minimal saturability and may itself represent the sum of passive diffusion and a "carrier"-mediated system. At physiological concentrations of L-alanine, the rate of the Na+ gradient-dependent uptake was 5-fold that in the absence of the gradient. Valinomycin enhanced the Na+ gradient-dependent uptake of L-alanine, provided a K+ gradient (vesicle greater than medium) was present. This finding indicates that the Na+ gradient-dependent transport of L-alanine into renal brush border membrane vesicles is an electrogenic process and suggests that the membrane potential is a determinant of L-alanine transport. In the presence of a Na+ gradient, the uptake of L-alanine was strongly inhibited by other neutral L-amino-acids. Imino acids and glycine also inhibited, but acidic and basic amino acids were without effect. In the absence of a Na+ gradient, little selective competition was found...