Shuttles of artificial electron donors for photosystem I across the thylakoid membrane.

NADP+ reduction in isolated chloroplasts of spinach by photosystem I at the expense of various artificial donor systems is not inhibited by the plastoquinome antagonist dibromothymoquinone. The coupled ATP formation in such photoreductions is attributed to an artificial energy conserving site, i. e. a proton liberation during oxidation of the donor at the inner surface of the thylakoid membrane. Some donor systems for photosystem I are stimulated by uncouplers whereas others are not. The stimulation shows no correlation to the efficiency of the coupled photophosphorylation. Instead a correlation of the stimulation by uncouplers to the presence of an acidic OH-group in the donor molecule is seen. The uncoupler effect is therefore not explained by a release of electron transport control by the high energy state but rather by a pH-dependent distribution of the donor compound across the membrane. This is supported by the properties of donor systems in sonicated chloroplast particles with external oxidation sites of photosystem I.


Assessment of GH releasing hormone activity in sephadex-separated fractions of porcine hypothalamic extracts by hypophyseal portal vessel infusion in the rat.

Growth hormone-releasing hormone (GH-RH) activity in Sephadex G-25 fractions of porcine stalk median eminence (SME) extracts was examined in vivo by infusing these samples into a rat hypophyseal portal vessel. The increment of immunoreactive GH levels in the serum was used as the index for GH-RH activity. The GH-RH activities were found in two different locations: in the early fractions Nos. 3-4, and in somewhat retarded fraction No. 7. These GH-RH activities were not due to TRH, vasopressin, or potassium. The location of LH releasing hormone (LH-RH) and prolactin release-inhibiting hormone (PR-IH) determined in this in vivo system was in agreement with those found in other in vivo and in vitro assay systems for LH-RH and PR-IH, respectively. These results help validate this assay system.


Implications of phenothiazine side effects: a study of antiparkinsonian agents in an older population.

A study of anti-Parkinsonism agents in prolonged phenothiazine therapy seeks to clarify some anecdotal misconceptions. Antiparkinsonian agents do not appear to affect the therapeutic efficiency of phenothiazines, nor does using them on a "demand" basis increase the problems of control of parkinsonian side effects. Older patients, however, appear to require the extended usage of antiparkinsonian agents rather more than some studies would suggest. Sustained release antiparkinsonian agents may yet further simplify the management of schizophrenia.


Changes in lysosomes during ageing of parenchymal and nonparenchymal liver cells.

Intact and viable parenchymal and non-parenchymal liver cell preparations were isolated by enzyme perfusion techniques from young and old rats. The distribution of the lysosomal enzymes acid phosphatase, beta-galactosidase, cathepsin D, acid DNAse, and arylsulphatase B over parenchymal and non-parenchymal cells was determined. In addition, morphological and morphometric changes which occur in parenchymal cells with age were investigated. All lysosomal enzymes studied are present in both cell classes, but non-parenchymal cells possess much ligher activities per mg protein than do parenchymal cells. This phenomenon is most pronounced for cathepsin D with a 13-times higher specific activity in non-parenchymal cells. Electron microscopic observations demonstrated that the lysosomal activities in non-parenchymal cells can be attributed mainly to the large and numerous lysosomal structures in Kupffer cells. Parenchymal cells from old rats have higher lysosomal enzyme activities per mg protein than do hepatocytes from young rats. This observation is in agreement with the general increase with age in the cytoplasmic volume fraction occupied by lysosomal structures in parenchymal cells. In general, non-parenchymal cells show no increase in specific enzyme activities with age. The results obtained suggest an increase in the heterogeneity--in both appearance and enzyme content--of the lysosomal structures in parenchymal cells with age.


Experimental mixed infection with two tick-borne viruses and interferon-mediated interference.

Tick-borne encephalitis virus (TEV), a Flavivirus, and Lipovník virus (LV), a member of the Kemerovo group and complex and possible member of the Rioviridae, were used to infect chick embryo cell (CEC) cultures. LV reproduction was inhibited by preinfection with TEV, mainly in aged cultures. In young CEC cultures, TEV production was stimulated slightly and interferon was sometimes depressed by this superinfection; on the contrary, in aged cultures the superinfecting LV stimulated interferon also when infective LV was not produced because of interference. Double staining by the fluorescent antibody (FA) technique localized both viral antigens often in the same cells. Interference between TEV and LV (and vice versa) was observed in cerebrally infected adult mice. The pathogenicity of LV, when injected first, was increased by TEV superinfection. Examination by the FA technique confirmed interference between TEV and LV also in suckling mouse brains and demonstrated both viral antigens in the same brains when TEV and LV were injected simultaneously.


Small- and large-plaque variants of Chikungunya virus in two vertebrate and seven invertebrate cell lines.

Nine cells lines--BHK-21, Vero, Aedes albopictus, A. aegypti (monolayer and howwow vesicles), A. w-albus, A. vittatus, Anopheles stephensi and Culex quinquefasciatus--were infected with small- and large-plaque (SP, LP) variants of chikungunya virus. Ross strain, and incubated at different temperatures. In the Aedes (29 plus or minus 1 degrees C) and the vertebrate cell lines (36 degrees C), infectivity titers of extracellular virus rapidly reached a peak; cytopathic effect (CPE) occurred only in the latter. In Anopheles cells (29 plus or minus 1 degrees C), infectivity titers increased very slowly to a peak at 10 days post-inoculation (p.i.); in Culex cells (29 plus or minus 1 degrees C or room temperature), persistence of virus only or no multiplication was observed. In infected A. albopictus, A. aegypti and A. w-albus carrier cultures, the SP variant continued to resemble the original stock virus in terms of mouse pathogenicity and plaque morphology in Vero cells, but the LP variant tended to modify toward the SP variant.


Experimental latent herpesvirus infection in rabbits, mice and hamsters: ultrastructure of the virus activation in explanted gasseric ganglia.

The frequency of latent infection as established in trigeminal ganglia of rabbits, mice and hamsters with human herpesvirus type 1 (HVH) was compared using two different virus strains. Explantation proved to be effective in reisolation of HVH from ganglion tissue, which did not yield infectious virus at time of its removal. After healing of acute keratitis, the latent infection in homolateral gasseric ganglia of rabbits was detected at a relatively high frequency (60-80 per cent) up to 120 days post infection (p.i.) in case of both virus strains. The activation rate was a little lower in hamsters. After inoculation of suckling and young mice with a sublethal dose of HVH by oral and nasal routes, approximately 40-100 per cent of the animals had virus in their gasseric ganglia during the acute period; 30-60 days later only 10-25 per cent had virus in the latent form. Immunofluorescent and electron microscopic examination of the explanted ganglion tissue showed the presence of HVH in neurons, neuronal satellites and Schwann cells. The nuclei of noneural cells contained numerous crystalline arrays. The possibility that pseudounipolar neurons of the regional sensoric ganglion are not the exclusive site of HVH latency is discussed.


Inhalatory infection of mice with influenza A0/PR8 virus. I. The site of primary virus replication and its spread in the respiratory tract.

The replication of influenza A0/PR8 virus started and continued simultaneously in all parts of the respiratory tract, without any preferential susceptibility of any area of the epithelial lining when the virus was administered to mice in a sublethal dose in the form of aerosol. After intranasal instillation, the initial virus replication in the lung tissue preceded by 4 to 8 hours the rise in infectious virus titre in the trachea and by 21-24 hours the rise in the virus titre in the nasal mucosa. Under conditions of aersol inhalation, the mice represent a suitable model for pathogenetic studies.


Inhalatory infection of mice with influenza A0/PR8 virus. II. Detection of the virus in the blood and extrapulmonary organs.

In the course of experimental aerosol infection of mice with influenza A virus, the latter was regularly detected in the blood, liver, salivary glands, spleen, pancreas, kidneys, heart and irregularly in cervical and mediastinal lymph nodes. The findings of extrapulmonary virus were in direct quantitative relationship to the rate of lung involvement.


The effect of species and organ-specific features of tissue systems on the reactogenic and immunogenic properties of influenza virus.

The dynamics of changes in the reactogenic and immunogenic properties of influenza A/Hong Kong/1/68 (H3N2) virus in the course of serial passages in white mice, chick embryos, chick, cow and pig embryo kidney tissue cultures and in rat, guinea pig, chick embryo, cow embryo and pig embryo trachea organ cultures was studied. The rate of virus attenuation was found to depend not only on the species origin of tissue systems but also on their organ specificity. In rapid, 24-hour passages in chick embryos attenuation of the virus developed more rapidly and its immunogenic potency was retained better than in 48-hour passages or cultivation in another tissue system.


Effect of 60amino-6-deoxy-D-glucose hydrochloride on influenza infection in mice.

A prophylactic effect of 6-amino-6-deoxy-D-glucose hydrochloride (AG) in influenza-infected mice was demonstrated. A dose of 500 mg AG per kg body-weight given intranasally 2000 mouse intranasal LD50) protected 32-40 per cent of the animals from death. The virus titres in the lungs and serum were lower in treated mice than in untreated ones. After AG administration, low interferon titres were demonstrated in the lungs and serum. The group of infected mice, untreated with AG, was examined electron microscopically. Virus was demonstrated on the surface of granular pneumocytes and in the lumen of lung alveoli.


Phagocytosis-enhancing effect of human leukocyte interferon preparation of human peripheral monocytes in vitro.

It was found that the phagocytosis of human peripheral monocytes was enhanced by human leukocyte interferon preparations. This activity showed species-specificity and a positive correlation between interferon titer and phagocytic activity. When human monocytes were treated by interferon, the pahagocytic activity was enhanced. But when interferon was added to the latex, the enhancement was not observed. The interferon preparation did not only increase the number of phagocytic cells, but also enhanced the degree of phagocytosis in individual cells. The factor responsible for the enhancing effect was inactivated by trypsin, but was not inhibited by actinomycin D.


Antiviral and cell-growth inhibitory activities of highly purified L-cell interferon. An analysis of quantitative disproportions.

Purified mouse L-cell interferon induced with Newcastle disease virus was subjected to polyacrylamide gel electrophoresis. A profile characterized by two components was obtained. The faster peak represented about 90-99 per cent of antiviral activity. Both components exerted a cell-growth inhibitory effect on L cells. A study of the quantitative relations between antiviral and cell-growth inhibitory activites of the two peaks revealed a discrepancy between these two activities in the fast peak. An analysis of this phenomenon suggests that the binding capacity of the fast moving molecular species of interferon is lower in young cells than in aged cells, i.e. those incubated for 3 days. Such differences were not observed when the slow molecular species was examined. It was concluded that both inherent properties of molecular components of interferon and the age (and species) of cells used in tests influence the outcome of interferon action on the cell.


Ribonuclease-resistant RNA of Kemerovo virus.

RNA of partially purified Kemerovo virus sedimented in a relatively broad region in sucrose density gradient with a peak at 14 S. Approximately 65% of this RNA was resistant to the action of ribonuclease which indicates the double-stranded nature of Kemerovo virus RNA.


Synergic action of distamycin A and hydroxyurea on the reproduction of DNA viruses in cell cultures.

The antiviral activity of the combination of distamycin A (DA) Pand hydroxyurea (HU) on the reproduction of vaccinia and pseudorabies viruses was investigated. The drug combination exerted a significant synergic inhibitory effect on the vaccinia virus yield and on the plaque formation in chick embryo cells. Similar experiments on pseudorabies virus showed an additive effect. The possible mechanism of the mutual enhancement of the antiviral activity is discussed.


Contamination of some avian species with viruses of the leukosis-sarcoma complex.

Contamination of 8 avian species with leukosis viruses was studied. An insignificant portion of sera from 4 and 24 months old chickens contained neutralizing antibody to 3 sero-types (A, B and D) of Rous sarcoma virus (RSV). Sera from guinea-fowl reacted positively in the neutralization test only with the RSV-RAV-1 strain. None of the Peking duck and semi-domestic Maran fowl sera was found to contain antibody to RSV strains. Twenty-five and 6.6% of embryos derived from 7-8 and 24 to 30 months old chickens respectively, 30% of liver specimens from 3-4 months old chickens 3.3% of liver specimens from adult guinea-fowl and 72% of liver specimens from Maran fowl contained the group-specific leukosis complement-fixing antigen (gs-antigen). Duck embryos and livers from 24-30 months old chickens, crows, sparrows, rooks or jackdaws contained no gs-antigen. Duck embryos did not react and ducklings reacted poorly to inoculation with RSV whereas guinea-fowl embryos and chickens proved to be highly susceptible to RSV.


Interferon production by rabies strains isolated from wild rodents.

Eight rabies strains isolated from wild rodents produced interferon in laboratory mouse brains the second day after inoculation. Low levels of interferon were also detected in the serum, kidneys and lungs of the animals. The dosage and route of inoculation had no pronounced effect on interferon production.


Comparison of the sensitivity to ultraviolet irradiation of reovirus 3 and some viruses of the Kemerovo group.

The kinetics of UV inactivation of the tick-borne Kemerovo (strain R-10) and Lipovnik (strain Lip-91) viruses which have been preliminarily classified as possible members of the Reovirus group was examined. Reovirus 3 and Sindbis virus served as reference double-stranded RNA and single-stranded RNA viruses, respectively. The parameters of UV inresembled those of Reovirus 3. This is consistent with their tentative classification as reovirus-like viruses.


Neurohumors in acupuncture.

In acupuncture an increase of some substance at the reactive site seems to be required in order to have a biochemical response. Among the substances to be considered are the neurohormones. Experimentally, the sympathetic and parasympathetic neurohormones are shown to be involved in analgesia and in the transmission of nerve impulses.


Chemical inhibition method for alkaline phosphatase isoenzymes in human serum.

The authors adapted a chemical inhibition procedure using L-phenylalanine and urea as specific inhibitors to quantitate the activities of bone, liver, and intestinal alkaline phosphatase (ALP) isoenzymes in human serum. The results of this assay were compared with electrophoretic separation, gamma-glutamyl transpeptidase (GGTP) activity, and the clinical setting in a group of patients with elevated total ALP activity. In addition, expected ranges of serum ALP isoenzymes for healthy young men and also for a geriatric population are presented.


Epidemic Venezuelan equine encephalitis in North America in 1971: vector studies.

A major epidemic of Venezuelan equine encephalitis occurred in south Texas in the summer of 1971. More than 1500 equines died of VEE in Texas, and 110 human cases with no deaths were reported. Vector studies in south Texas and northern Tamaulipas revealed that the overall mosquito infection rates during the peak of the epidemic were about 1:100, one of the highest rates observed for a major epidemic. Mosquito infection rates of this magnitude could easily explain the intensity of VEE outbreaks in both equines and man. A total of 943 VEE virus isolations were made from mosquitoes. Eight of the 12 mosquito species found infected were implicated in the epidemic cycle of VEE for the first time. Sufficient laboratory and field evidence is available to prove that Psorophora confinnis was one of the primary vectors of VEE. The lack of laboratory evidence necessitates the use of the term "probable" primary vectors for other species apparently equally as involved on the basis of field infections; these include Aedes sollicitans, Aedes thelcter and Psorophora discolor. Eight other species from which less than 10 VEE virus isolations were made were considered auxiliary vectors. Mosquitoes of some species were tested individually; such tests showed 2-4% of the probable primary vectors to be infected. The first isolation of VEE virus of the epidemic was made from P. confinnis on June 28, 1971. Highest mosquito infection rates occurred during the week of July 5. Mosquito infection rates declined precipitously in the last 3 weeks of July 1971, signaling the end of the epidemic in the study area. One explanation for the decline was that equines, the principal epidemic hosts, were eliminated as a source of virus by death or by acquisition of natural or induced immunity. Mosquito control appeared to be effective in reducing the infected mosquito population while the immunization of equines with TC 83 VEE vaccine was accomplished. Quarantines appeared to be effective in restricting the VEE virus activity to south Texas. Undoubtedly all of the control measures contributed to stopping the epidemic. Continued VEE surveillance by various government and other agencies failed to reveal any further epidemic VEE activity in the US in 1972. Other arboviruses isolated during the VEE studies in south Texas included St. Louis encephalitis virus, and San Angelo subtype of the California Group. A virus of the Bunyamwera Group was also isolated from Palo Blanco, Tamaulipas.


Arbovirus vector ecology studies in Mexico during the 1972 Venezuelan equine encephalitis outbreak.

Virus vector studies were conducted in the States of Durango, Chihuahua, and Tamaulipas, Mexico, in June and July 1972. Apparently only a low level of Venzuelan equine encephalitis (VEE) virus transmission to equines occured at the time of the study, and the infection was restricted to areas which had not experienced overt activity during the preceding year. The low level of infection was associated with a scarcity of mosquitoes. The IB (epidemic) strain of VEE virus was isolated from two pools of Anopheles pseudopunctipennis (Theo.) and the blood of one symptomatic equine. The low mosquito population, the relatively few equine cases observed, and the absence of reports of VEE human disease from the outbreak area suggested VEE virus persistence through a low-level mosquito-equine transmission cycle. Other studies have already indicated that wild vertebrates play no more than a minor role in outbreaks of epidemic VEE. Mosquito collections made in areas of the states of Durango, Chihuahua, and Tamaulipas, where considerable epidemic activity of VEE had occurred in 1971, failed to reveal evidence of VEE virus persistence. Twenty-nine ioslations of other arboviruses were also made in these studies: including 22 of St. Louis encephalitis virus (SLE), 2 of Flanders virus, 1 of Turlock virus, 1 of Trivittatus virus of the California Group, 1 of western equine encephalitis virus (VEE), and 2 (from Santa Rose) which possibly represent a hitherto unknown virus in the Bunyamwera Group. These are the first reports of SLE virus isolations from mosquitoes in Mexico, and the first demonstration of Trivittatus, VEE Turlock and Flanders viruses in Mexico from any source.


Training and function of a women's health-care specialist, a physician's assistant, or nurse practitioner in obstetrics and gynecology.

Serious manpower shortages exist in the United States. Proposed corrective measures indicate the need for nonphysicians to provide medical care for well or worried well patients. We have tested this proposed solution in the field of obstetrics and gynecology using allied health personnel to provide health maintenance services to well women, and feel that the experiment has been a success. Using principles of pattern recognition allied health workers recognize normal and abnormal findings. Patients with abnormalities are referred to the supervising physician for diagnosis and therapy. Allied health workers can also perform technical health-maintenance tasks such as inserting intrauterine devices and obtaining Pap smears. This article describes the historical evolution of our allied health personnel workers, called women's health-care specialists (WHCS), the selection and training of students, functions and levels of performance of the specialists, and the future role of such individuals in obstetrics and gynecology. We feel that with appropriate preparation of the physician, co-workers, and patients, the integration of the WHCS into the health-care system is both desirable and appropriate and will offer significant advantages to the patient and the practicing obstetrician-gynecologist in either the clinic or private practice setting.


Development of autonomic control of fetal circulation.

Development of parasympathetic and sympathetic reflexes controlling heart rate, vascular pressures, and blood flows was investigated in fetal lambs weighing 300-5,800 g (65-165 days' gestation). Cardiovascular responses to veratridine injections, atrial stretching, bilateral cervical vagotomy, and cholinergic blockade with atropine were used to test parasympathetic activities. Responses to propranolol and phenoxybenzamine were used to test beta- and alpha-adrenergic activities. Autonomic ganglionic blockade and stimulation provided additional information on both cholinergic and adrenergic systems. Fetal responses to various tests were compared to those of the mother. Results show: a) little parasympathetic tone on resting heart rate and other circulatory functions exists prior to fetal maturity; b) despite the feeble resting tone, the parasympathetic system is capable of exerting significant control when stimulated in both premature and mature fetuses, the capability increases as fetus approaches term; c) alpha- and beta-adrenergic tone in control of resting heart rate and peripheral circulation exists in early fetal life and increases as the fetus reaches maturity, and both adrenergic receptors respond strongly to stimuli in immature, premature, and mature fetuses; d) in immature fetuses, veratridine does not elicit a vagally mediated reflex; instead, it produces a centrally mediated alpha- and beta-adrenergic stimulation; e) the fetal cardiovascular response to any given test is dampened by the existence of the various vascular shunts, the umbilicoplacental circulation and, possibly, by incomplete maturation of vasomotor tone.


Effects of alveolar and perfusion hypoxia and hypercapnia on pulmonary vascular resistance in the lamb.

The effects of ventilatory hypoxia and hypercapnia and perfusion hypoxia and hypercapnia on pulmonary vascular resistance were studied in the intact lamb using right heart techniques to isolate and perfuse the left lower lobe. Ventilatory hypoxia increased vascular resistance in the left lower lobe by constricting predominantly vessels upstream from small lobar veins, presumably small arteries. The response to hypoxia was not blocked by phentolamine and diphenhydramine in doses that markedly decreased pressor responses to norepinephrine and histamine in the lung. Perfusion hypoxia did not alter vascular resistance in the perfused lobe. Ventilatory hypercapnia increased vascular resistance in the lung by constricting mainly upstream vessels, whereas perfusion hypercapnia decreased resistance by dilating upstream vessels. These data indicate that histamine and catecholamines are not involved in the response to alveolar hypoxia. These results suggest that the sensor site for ventilatory hypoxia is close to the alveolus since the response is unrelated to lobar arterial Po2. It is concluded that systemic reflexes are not necessarily involved in the response of the pulmonary vascular bed to ventilatory hypoxia or hypercapnia and that the magnitude and rapidity of this response suggest that it may represent an important local mechanism for the control of ventilation-perfusion relationships in this species.


Inhibition of angiotensin conversion and prevention of renal hypertension.

Renal artery constriction in the unilaterally nephrectomized, trained dog, with maintained renal arterial hypotension, produces a prompt increase in systemic renin activity and blood pressure. The hypertension normally induced by renal artery stenosis is prevented by prior treatment with the nonapeptide Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro (SQ 20, 881), which blocks conversion of angiotensin I to angiotensin II. Constant intravenous infusion of the inhibitor over several days of renal artery constriction prevents the development of chronic renovascular hypertension. Furthermore, a single injection of the nonapeptide restores blood pressure to normal in the early phase of renovascular hypertension, but becomes progressively less effective as salt and water retention occurs in the chronic stage when plasma renin activity returns to control levels. These data provide strong evidence that the renin-angiotensin system is responsible for the initiation of renovascular hypertension in the one-kidney Goldblatt dog, but that other factors become increasingly important in chronic renovascular hypertension.


Sites of organic acid production and absorption in gastrointestinal tract of the pig.

The relationship between diet, pH, and microbial digestion of carbohydrate was examined in 24 pigs fed either a conventional or a low-protein, high-cellulose experimental diet and sacrificed 2, 4, 8, or 12 h after a meal. In animals fed the control diet contents of the cranial half of the stomach demonstrated marked, cyclic fluctuations in pH and high concentrations of organic acids. Contents of the caudal (glandular) half were lower in both pH and organic acid concentration. Despite concentrations of volatile fatty acids (VFAs) as high as 250 meq/liter in the large intestine, the pH remained relatively neutral. The VFA levels remained relatively constant throughout the length of the colon. The VFA transport across isolated gastric and large intestinal mucosa also was examined. All four types of gastric mucosa absorbed and transported VFA at substantial rates. Mucosa of pig cecum and colon transported VFA at much greater rates than gastric mucosa and greater rates than previously determined in equine large intestinal mucosa or even bovine rumen epithelium. Comparison with results of earlier studies in the pony suggested that the higher concentration of VFA in the large intestinal contents of pigs was due to the more rapid rate of digesta passage rather than to less efficient absorption of fatty acids.


Cardiovascular responsiveness to beta-adrenergic stimulation and blockade in chronic hypoxia.

Previous studies have shown that exposure to high altitude results in an initial increase in heart rate, followed by a return to sea-level values within several days; circulating catecholamines rise progressively during this time. Nine conscious dogs were studied in normoxia (N) and after 10 days' exposure to 445 torr (CH). The mean (plus or minus SE) hematocrit was higher in CH (50 plus or minus 2 vs. 42 plus or minus 1%) while Pa-o2 (53 plus or minus 1 vs. 97 plus or minus 2 torr) and PaCO2 (27 plus or minus 1 vs. 35 plus or minus 1 torr) were lower than in N. A 3.5-fold increase in plasma norepinephrine above the N value was found in CH. Arterial pH, heart rate (HR), and mean femoral arterial pressure (MAP) did not differ significantly in N and CH. Isoproterenol (ISO), 0.5 mug/kg iv, produced an average increase in HR of 92 plus or minus 9 beats/min in N, but only 66 plus or minus 8 beats/min in CH (P smaller than .02). Reduction in MAP after ISO were similar. Pretreatment with propranolol, 0.15 mg/kg iv, reduced HR equally in N and CH without affecting MAP, but diminished the HR response to ISO significantly more in CH than in N. The attenuated chronotropic response to beta-adrenoceptor stimulation following chronic hypobaric hypoxia suggests a relative cardiac refractoriness secondary to an increased level of sympathetic activity.


Relations of weak-electrolyte transport and acid-base metabolism in rat small intestine in vitro.

The jejunal and ileal regions of rat small intestine in vitro exhibit different patterns of weak-electrolyte transport and acid-base metabolism. The jejunal pattern of weak-electrolyte transport is net transport of weak acids M yields S and of weak bases S yields M (M, mucosal; S, serosal), and the pattern of acid-base metabolism is luminal acidification and serosal alkalinization. In the absence of sodium or in the presence of metabolic inhibitors, weak-electrolyte transport and serosal alkalinization are inhibited, but luminal acidification is not inhibited. The ileal pattern of weak-electrolyte transport is the opposite of that of the jejunum and may be associated with the presence of a luminal alkalinization process observed in this region. In the absence of chloride the ileal patterns of weak-electrolyte transport and acid-base metabolism become similar to those of the jejunum. It is suggested that these observations support a previously proposed model for weak-electrolyte transport in the intestine consisting of a series three-compartment system in which the pH of the intermediate compartment is greater than that of the bulk phases.


Effect of methylprednisolone upon arterial pressure and the renin angiotensin system in the rat.

The effect of methylprednisolone or deoxycorticosterone upon systemic arterial blood pressure and components of the renin-angiotensin system was studied in the rat. Rats maintained on regular diets given methylprednisolone suspension 20 mg/kg body wt demonstrated a significant increase in arterial pressure of + 37 plus or minus 5 mmHg, mean plus or minus SE, over a 2-wk period, whereas those treated with DOC and untreated controls showed no significant change. On normal diets, plasma renin concentration (PRC) of methylprednisolone-treated rats was significantly higher than that of DOC-treated rats. Methylprednisolone treatment also resulted in a significant elevation of plasma renin substrate concentration (PRS). Calculated plasma renin activity (PRA) was highest in methylprednisolone-treated rats, significantly above that of the DOC and no-steroid groups. NaCl supplementation resulted in a significant fall in PRC and PRA in all three groups; however, PRS remained significantly above normal in the methylprednisolone-treated rats. The pressor effect of angiotensin II was slightly increased in methylprednisolone-treated rats. Infusion of [Sar1,Ala8]angiotensin II (P-113) in methylprednisolone-treated rats resulted in a significant fall in diastolic arterial pressure. The results imply that methylprednisolone hypertension in the rat may be in part angiotensin dependent.


Induction of a myogenic response in tonic airway smooth muscle by tetraethylammonium.

In multi-unit tracheal smooth muscle (TSM), quick stretches applied at a velocity of 5 times the measured maximum velocity of isotonic shortening of the muscle, of a magnitude 3 times the measured extension of the series-elastic component when the muscle contracts maximally, and at optimal muscle length (L-o) were unable to elicit any myogenic response (MR). Experimental conditions such as hypoxia (P-O2 smaller than 60 mmHg) and acidosis (pH equals 6.8) or the presence of Ba2+ (2 mM), acetylcholine (10-6 M), or high (K+)-o (59 mM) were also unable to elicit the MR. However, tetraethylammonium chloride (TEA, 0.4-67 mM) produces 1) spontaneous phasic contractions and 2) a MR to quick stretch. The ionic basis for these changes was then investigated by studying the Ca and Mg dependence of the response to TEA. The dose-response relationship to TEA was shifted to the left by decreasing external Mg2+ from 2.5 to 0.5 mM. The ability of TSM to produce a MR was absolutely dependent on external Ca, but the threshold concentration required shifted from 2.5 times 10-5 M at normal external Mg (2.5 mM) to 5 times 10-4 M at the reduced external Mg (0.5 mM). The effects of TEA on spontaneity and the MR were abolished by D-600. These results suggest that 1) TEA functionally converts multiunit smooth muscle into a single unit one and leads to the development of a MR and 2) the MR results from a depolarization-activated mobilization of Ca and is inhibited by ionic conditions known to increase membrane permeability.


Pacui virus, phlebotomine flies, and small mammals in Brazil: an epidemiological study.

Pacui virus, originally obtained from forest rodents, was isolated 100 times from 61,437 specimens (658 pools) of the phlebotomine fly Lutzomyia flaviscutellata, collected from rodent-baited traps in the forests of Belem, Para, Brazil in the period October 1968 through September 1970. Isolations were made from engorged and unengorged females and from males (3 strains), and occurred in all 24 months. Pacui virus also was isolated from the blood of two wild rodents (Oryzomys), but not from 424 L. infraspinosa, 12,000 mosquitoes, or sentinel mice. Pacui virus neutralizing antibodies were detected in serum of six bait animals after exposure to biting flies in the forest, in 30% of wild rodents surveyed (including two from Amapa Territory), and in 10% of marsupials, but were absent in human survey sera and in bats. Low-passage Pacui virus produced viremia in and was lethal to infant mice by the subcutaneous route. L. flaviscutellata was most abundant in the dry season, in which period Pacui virus isolations increased. This fly is strongly attracted to rodents close to the ground. L. flaviscutellata also yielded single strains of Guama, Icoaraci, and BeAr 177325 viruses.


The effect of acid-base balance on neostigmine antagonism of d-tubocurarine-induced neuromuscular blockade.

d-Tubocurarine (dTc) was infused intravenously into 35 cats anesthetized with chloralose and urethane at a constant continuous rate to produce and maintain 90 per cent depression of twitch height of the anterior tibial muscle following supramaximal stimulation of the peroneal nerve. The mean infusion rates that produced 90 per cent depression were not significantly altered by respiratory acid-base changes. Metabolic alkalosis decreased (32.5 per cent) and metabolic acidosis increased (27.7 per cent) the required infusion rate of dTc. When pH and Paco2 were maintained at 7.37 and 38 torr, respectively, the addition of a bolus of neostigmine, 10.5 mug/kg, intravenously, to the continuing infusion of dTc produced 50 per cent antagonism of the dTc-depressed twitch. Respiratory alkalosis and metabolic acidosis did not alter the dose of neostigmine needed to produce 50 per cent antagonism. However, during respiratory acidosis (pH 7.13, Paco2 66 torr) and metabolic alkalosis (pH 7.59, Paco2 36 torr) 20.0 and 18.0 mug/kg neostigmine, respectively, were needed to produce 50 per cent antagonism. Still larger doses of neostigmine (75 mug/kg) could not completely antagonize the block unless pH and Paco2 were returned to 7.30-7.50 and 35-45 torr, respectively. It is concluded that respiratory acidosis and metabolic alkalosis limit and oppose antagonism of dTc by neostigmine.


MAC expanded: AD50 and AD95 values of common inhalation anesthetics in man.

Two important measures may be derived from patient responses to a range of anesthetic doses. The AD50, corresponding to MAC, estimates the median anesthetic concentration--that dose where half the patients are anesthetized and half are not. The AD95 approaches the theoretical "minimum" anesthetic concentration by estimating the dose that anesthetizes 95 per cent of a patient population. The AD50 and AD95 are logical extensions of the MAC concept and can be evaluated with current experimental methodology. Recomputed from available data , the AD50's of nine inhaled anesthetics proved to be numerically identical to their MAC values. The AD95's of nine inhaled anesthetics proved to be numerically identical to their MAC values. The AD95's were 5 to 40 per cent greater than the AD50's.)Key workd: Potency, anesthetic, MAC, AD50, AD95; Pharmacology, dose-response curves.)


Effects of peridural block: V. Properties, circulatory effects, and blood levels of etidocaine and lidocaine.

Ten healthy, unpremedicated, male volunteers, aged 21-33 years, were given 20 ml 1 per cent etidocaine with 5 mug/ml epinephrine for peridural analgesia via a catheter placed L2. On a different occasion they were given 20 ml 2 per cent lidocaine with 5 mug/ml epinephrine in the same manner. Initial onset of sensory analgesia to pin prick was faster for etidocaine (7 min) than for lidocaine (9 min). Analgesia lasted significantly longer after etidocaine with respect to both two-segment regression (177 plus or minus SE min vs. 114 plus or minus 8 min) and total duration (379 plus or minus 22 min vs. 190 plus or minus 8 min). Onset of maximal motor blockade was significantly faster with etidocaine (15.4 plus or minus 2.5 min) than with lidocaine (31.7 plus or minus 3.3 min); blockade lasted longer with etidocaine (331 plus or minus 25 min vs. 167 plus or minus 13 min). Changes in mean arterial pressure cardiac output, central venous pressure, limb blood flows, total peripheral resistance, and stroke volume were similar with the two drugs, although those after etidocaine were more prolonged as a result of the longer blockade. Mean maximum arterial concentrations of etidocaine were 0.96 plus or minus 0.05 SE mug/ml (plasma) and 0.55 plus or minus 0.03 mug/ml (whold blood), achieved at 17 plus or minus 2 min. Mean maximum arterial concentrations of lidocaine were 2.22 plus or minus 0.09 mug/ml (plasma) and 1.85 plus or minus mug/ml (whold blood), achieved at 24 plus or minus 2 min. No sign of central toxicity was observed with either drug, although subjects receiving lidocaine tended to sleep, which was not the case with etidocaine. Hematologic screening, blood chemistries, and urinalyses performed 24 hours before and after each study showed no abnormality.


Pulmonary shunting during alveolar hypoventilation in the dog.

Pulmonary shunting (Qs/Qt) was measured in 16 anesthetized and paralyzed healthy beagle puppies during a control period when the end-expired CO2 concentration was 5 per cent and during alveolar hypoventilation, when the PaCO2 increased to 100 plus or minus 13 torr (mean plus or minus SE) and the ;Ha vecame 7.04 plus or minus 0.04. A constant tidal volume was maintained throughout each study, and hypoventilation was achieved by slowing only the respiratory rate. During the control period Qs/Qt was 2.9 plus or minus 0.3 per cent; during hypoventilation Qs/Qt was 3.2 plus or minus 0.3 per cent (an insignificant difference). The puppies, therefore, responded to anesthesia and alveolar hypoventilation without development of increased pulmonary shunts.


Smoking as a cause of erythrocytosis.

Five smokers had erythrocyte masses sufficiently larger than normal to pose a problem in the differential diagnosis of polycythemia. Evaluation excluded lung disease, shunt physiology, hemoglobin with increased oxygen affinity, erythropoietin-producing tumor, renal disease, or polycythemia rubra vera as the primary cause of erythrocytosis in these patients. All were found to have levels of carboxyhemoglobin sufficient to cause clinically significant hypoxemia and to account for the increased erythrocyte masses. In two patients the erythrocytosis improved when they stopped smoking. Heavy smoking is a reversible cause of polycythemia and should be considered in the differential diagnosis of this problem.


Drug spotlight program: antacids and anticholinergic drugs.

Controlled clinical trials have provided some useful evidence of the effectiveness or lack of effectiveness of antacids and anticholinergic drugs. However, few such trials are free of serious methodologic or clinical criticism, and the use of these agents cannot be defined with any degree of scientific certainty. Their principal uses still focus on reduction of gastric acid content, and their best use requires individualization for each patient. With anticholinergic drugs there is little if any pharmacologic effect without some side-effects, and dose adjustment may be more important than the choice among the many drugs considered effective. Some new antacids offer better neutralization than old ones, but are at least as likely to affect colonic function. Although toxicity, side-effects, and interactions with other drugs are minimal, they should be watched for.


Cholecystectomy and gallstone dyspepsia. Clinical and physiological study of a symptom complex.

The symptom complex of gallstone dyspepsia is defined and then analysed before and after cholecystectomy in 108 patients. Only 46% of patients were symptom-free after operation and 30% were no better. When pyloric function was studied patients with these symptoms before or after cholecystectomy and those with normal radiographs showed duodenogastric reflux, often precipitated by intraduodenal fat. Symptomless matched control subjects showed no reflux. Synchronous radiology and pressure recordings demonstrated that the pylorus in these patients failed to contract in response to a duodenal contraction, whereas the normal pylorus could prevent the reflux produced by an isolated duodenal contraction. The effect of metoclopramide on gastroduodenal contractions and in treating the symptoms was assessed. Gallstone dyspepsia is essentially a functional disease--a disorder of gastroduodenal motility.


[Carbon metabolism under conditions of regulated penicillin biosynthesis].

Carbon metabolism of P. chrysogenum under conditions of periodical addition of the nutrients was studied. It was found that a proper rate of the carbon source addition to the culture was of significant importance for intensive biosynthesis. The use of carbon for the energetic and constructive needs was not the same at different fermentation periods.


[Rate of benyzylpenicillin inactivation in native solutions during extraction].

The constants of benzylpenicillin inactivation in acidified fermentation broth filtrates were different in various filtrate batches. Marked inactivation of the antibiotic in the filtrates immediately after their acidification was observed. Possible losses of the antibiotic due to inactivation under conditions of extraction were estimated.


[Quantitative method for determining variamycin].

A quantitative spectrophotmetric method for the assay of variamycin in commercial samples was developed. It was based on the measurement of the optical density of variamycin solutions in 0.01 N hydrochloric acid at a UV spectrum wave length of 412 nm. A method of thin-layer chromatography for a semi-quantitative estimation of tetraside, the main admixture of variamycin was devised. On storage for 18 months the content of variamycin in the preparations did not change as compared to that at the moment of the drug manufacturing and the amount of tetraside in 18 months did not exceed 2 per cent.


Chromosome mutations and pH disturbances.

Chromosomal anomalies, namely hypodiploidies, hyperdiploidies, and endoreduplications, developed in nearly 25% of metaphase plates of replicating human lymphocytes exposed in vitro to either acid (6.5 to 6.9) or alkaline (8.4 to 8.8) pH equilibriums. The G group was most susceptible to aneuploid formation. Mammalian mutations may develop as the result of a pH disturbance during cleavage of the fertilized zygote.


CO2 fixation and its regulation in Anacystis nidulans (Synechococcus).

Anacystis nidulans (Synechococcus) had a minimal doubling time of 5 hrs at 30 degrees C at saturating light intensity and carbon dioxide concentration. Half maximal growth rates in saturating CO2 occured at a light intensity of 0.54 mW per cm2, and there was an apparent threshold intensity of 0.13 mW per cm2 below which no growth occurred. Growth rate in saturating light was dependent on the concentration of CO2+H2CO3 in the medium, rather than on total dissolved CO2; half maximal rates were estimated at 0.1 mM CO2+H2CO3. Under saturating conditions of light and CO2, 14CO2 was fixed primarily into 3-PGA, and subsequently moved into sugar phosphates and amino acids. Incorporation into aspartate was relatively slow. CO2 fixation was strictly light-dependent. The changes in adenylate and pyridine nucleotide pools were followed in light/dark and dark/light transitions. Whereas adenylates relaxed slowly over 15-20 min to the concentrations characteristic of illuminated cells following the abrupt changes induced by darkening, the sharp drop in intracellular NADPH showed little dark recovery although rapid restoration occurred on reillumination. Other pyridine nucleotides showed no changes during these transitions. The nucleotide specificity and Km of partially purfied GAP dehydrogenase suggest a role for this enzyme in the regulation of CO2 fixation.


Differential fluorescence labelling with 5-dimethyl-aminonaphthalene-1-sulfonyl chloride of intact cells and isolated membranes in Salmonella typhimurium and Acholeplasma laidlawii.

For fluorescence labelling intact cells and isolated cell envelopes (membranes) from Salmonella typhimurium and Acholeplasma laidlawii were treated with mixed dansylchloride-lecithin-cholesterol vesicles. This kind of dansylation, which has been supposed to be specific for cell surface proteins, produced fluorescent protein pattern after SDS-polyacrylamide gel electrophoresis only when isolated envelopes were dansylated. Acid hydrolysis of fluorescent cell envelopes of Salmonella typhimurium yielded O-dansyltryosine and epsilon-N-dansyl-lysine besides the free sulfonic acid and unidentified compounds. However, no fluorescent proteins were detectable in cell envelopes isolated from dansylated intact bacteria from Salmonella typhimurium. In accord Acholeplasma laidlawii showed only fluorescence from proteins with a molecular weight higher than 100000.


The metabolism of pyrimidines by proteolytic clostridia.

Uracil was used by growing cultures of Clostridium sporogenes, and by proteolytic strains of C. botulinum types A and B. Uracil was not used by C. bifermentans; C. botulinum, type B (non-proteolytic); C. botulinum, type F (non-proteolytic); C. botulinum, type E; C. butyricum; C. cochlearium; C. difficile; C. histolyticum; C. oedematiens, type A; C. paraputrificum; C. scatologenes; C. specticum; C. sordellii; C. sticklandii; C. tertium; C. tetani; C. tetanomorphum; C. welchii, types A, B, C, E and 4 untyped strains. The growth of C. sporogenes was not increased by uracil; it was reduced to dihydrouracil. Experiments with washed cells of C. sporogenes showed that the uracil-reducing system was inducible. Washed cell suspensions incubated under hydrogen with uracil, thymine, iso-barbituric acid, 5-amino uracil and cytosine consumed 1 mole H2/mole pyrimidine. The reduction product of cytosine was dihydrouracil indicating that it was deaminated before reduction. The reduction products of the  remaining pyrimidines were the corresponding dihydro derivatives. Extracts of C. sporogenes reduced uracil in the presence of NADPH2 but not NADH2.


Psychotropic drugs on general medical and surgical wards of a teaching hospital.

We examined the prescribing habits for psychotropic drugs of internists, surgeons, and gynecologists on their inpatient wards in a teaching hospital. Data were gathered from patients' charts and pharmacy records. In a six-week period, 9% of all admissions received such a drug. The male:female ratio and black:white ratios were studied; the maximum incidence of receiving these medications was in the 50- to 60-year age group. Minor tranquilizers were prescribed most frequently, followed, respectively, by major tranquilizers, barbiturates, and antidepressants. Less than half the available drugs were used, but drugs of differernt groups were often used interchangeably in an unsystematic fashion, and there was little evidence as to how effective a drug had been. It also seemed that depression was often overlooked or insufficiently treated.


Psychotropic drug use in the Boston area. A report from the Boston Collaborative Drug Surveillance Program.

During 1972, adults admitted to general medical and surgical wards in Boston area hospitals were interviewed to determine their use of prescribed psychotropic drugs prior to hospitalization. Patients hospitalized for psychiatric disorders or psychogenic ("functional") disease were excluded. Patients who had taken prescribed drugs that could not be identified were considered nonusers. Of the total sample, 20% gave a histroy of psychotropic drug use. Antianxiety drugs were taken by 15% of the patients and accounted for two thirds of total psychotropic drug use. Hypnotics were taken by another 4% of the sample. Slightly over half of antianxiety and hypnotic drug use was for a year or more. Use was more frequent in patients with potentially chronic medical disorders. The findings, in general, are consistent with other studies employing other methods to investigate the use of psychotropic drugs. Simple studies of use, however, do not provide a sole or adequate definition of treatment option, need, or efficacy.


Cyclic adenosine monophosphate in cerebral cortex. Alterations following trauma.

Stab-wound injury produced a seven-fold elevation in cyclic adenosine monophosphate (AMP) in mouse brain within one minute. The increase in cyclic AMP in the brain was blocked by prior treatment of the animal by theophylline, chlorpromazine, trifluoperazine hydrochloride, and diphenhydramine hydrochloride. Neither dichloroisoproterenol, pronetalol, nor reserpine blocked in the rise in cyclic AMP concentration due to injury. The results following administration of drugs suggest that the increases in cyclic AMP due to injury may be mediated by adenosine. Theophylline and phenothiazine derivatives have been shown previously to decrease adenosine-mediated increases in cyclic AMP in brain slices. The absence of any effect after administration of dichloroisoproterenol or pronetalol suggests that the increase in cyclic AMP after injury is not through catecholamine release. Hypothermia reduced the increase in cyclic AMP in injured brain after one minute.


A psychological study of patients undergoing cosmetic surgery.

This is an investigative study of the personality of patients requesting cosmetic rhinoplasty. Analysis of data obtained from objective projective tests and psychological interviews indicated that patients seeking cosmetic surgery are not as psychologically disturbed as often as described. Comparative studies showed certain personality characteristics to be associated with patients who seek cosmetic surgery. While evaluations 18 months after surgery showed no major personality change, self-concept was improved. Certain disturbing personality patterns indicative of psychological risk were identified. These fell more in the range personality disorders, exemplified by the infantile-narcissistic and the manipulative controlling personalities rather than in somatic ranges. We recommend a simple interview question method of counseling designed to identify underlying psychological manifestations and to control the potential problem patient.


Characteristics of three strains of avian adenoviruses isolated in Queensland. II. Biochemical, biophysical, and electron-microscope studies.

Three strains of adenovirus were relatively stable to the effects of chloroform, extreme pH, trypsin, heat, lyophilization, and ultrasonication. The structure of the viruses and their mode of replication in cell cultures confirmed these viruses to be members of the avian adenoviruses.


Coccidiosis and intestinal pH in chickens.

The intestinal contents of white Leghorn cockerels were significantly lower in pH in birds infected with Eimeria mivati, E. maxima, or E. necatrix than in uninoculated control birds. The particular Eimeria species used affected the region of the intestine involved, the days (5-9 postinoculation) on which pH decreased, and the magnitude of the decrease. The effects on the magnitude and frequency of pH decrease were as follows: E. mivati, marked in the duodenum and jejunum, variable in the ileum; E. maxima, slight in the duodenum, variable in the jejunum and ileum; E. necatrix, no change in the duodenum, variable in the jejunum and ileum. Infection with E. tenella did not change the intestinal pH. The pH of the gizzard was occasionally higher in birds infected with E. mivati or E. necatrix than in uninoculated control birds. With all four species, on some days the pH in the ceca was higher than in the ceca of controls. These results and previous reports indicate that infection with one of the above four species or with E. acervulina or E. brunetti produces the greatest and most consistent decrease in pH in the region of the intestine where the particular species characteristically produces the severest infection.


[The effect of a dimenhydrinate combination of the cardiovascular system and cerebral blood circulation in the anaesthetized dog].

The influence of Vertigo-Vomex (VV) and Vertigo-Vomex Retard (VVR) on the perfusion rate of the a. carotis interna and on the peripheral circulation was tested in 30 anaesthetized dogs. These drugs represent beta-dimethylamino-ethyl-benzhydrylether-1,3-dimethyl-8-chloroxanthine (dimenhydrinate, Vomex A) with the addition of pyridine-3-carbonic acid (nicotinic acid, niacin) and pyridoxine-HCl (vitamin B6) and its retard preparation. A) Angiographic researches of the brain vessels after i.v. injection of VV. B) Perfusion rate in the a. carotis int. by electromagnetic flowmeter; measurements of blood pressure, heart rate, and acid-base balance; reactions after injection of niacin, dimenhydrinate, and pyridoxine-HCl as well as after a combination of both. C) Same measurements as in B but after oral application. In one out of four cases there was a dilation of the brain vessels, in two cases there was a small one, in one case there was none. Niacin induces a short-lasting augmentation of the perfusion rate in the a. carotis int. simultaneously with rising aortic blood pressure. Dimenhydrinate with pyridoxine-HCl slightly increases the heart rate. Combined with niacin the perfusion rate shortly increases immediately after injection of niacin without any rise of blood pressure but combined with a fall in blood pH and standard bicarbonate. After oral application of VVR there was a slight but not significant augmentation of the perfusion rate in the a. carotis int. combined with rising blood pressure but with no change of the blood pH. Therefore the certain intivertigineous effect of VV and VVR cannot be explained by a rise of the perfusion rate in the inner ear or in the brain. In view of our results the central effect of dimenhydrinate combined with niacin and pyridoxine-HCl hypothetically may be improved by metabolic influences on the cells and their membranes.


The treatment of threatened premature labor by tocolytics, Ca++-Antagonists and anti-inflammatory drugs.

Prevention of premature labor is of considerable interest in research and clinics.  Three groups of pharmacological agents can inhibit uterine activity: tocolytics, spasmolytics, and anti-inflammatory drugs.  Tocolvtics like isoprenaline and fenoterol stimulate the beta-adrenergic receptors in decreasing the amplitude of contractions but with little effort on the muscle tone.  Spasmolytic uterine inhibitors produce primarily a decrease in contraction frequency and tonus, and finally in high concentrations a decrease of the amplitude of contraction.  An alteration of the electro-mechnaical coupling mechanism of the muscle cell via calcium antagonism is discussed.  A combined application of both drugs, tocolytics and spasmolvtics, potentiates the inhibitory effect and may simultaneously reduce undesirable systemic and cardiovascular side effects.  Anti-inflammatory agents of the acetylsalicylic acid or indometacin type reduce uterine activity by inhibition of prostaglandin synetheis and release.  They are clinically used as complementary agents in case of unsuccessful tocolytic or spasmolytic inhibition of uterine activity. In our study only 21.4 per cent out of 603 cases of threatened premature labor justified a tocolytic treatment.  Before attempting an inhibition one must decide on certain clinical parameters.


Calorimetric analysis of aspartate transcarbamylase from Escherichia coli: binding of cytosine 5'-triphosphate and adenosine 5'-triphosphate.

The binding of CTP and ATP to aspartate transcarbamylase at pH 7.8 and 8.5 at 25 degrees has been investigated by equilibrium dialysis and flow microcalorimetry. The binding isotherms for CTP at both pH 7.8 and 8.5 and ATP AT PH 8.5 can be fit by a model which assumes three tight, three moderately tight, and six weak binding sites. The binding isotherms for ATP at pH 7.8 are best fit by a model which assumes six tight and six weaker sites. Both finite differenceH binding and finite differenceS binding are negative for both nucleotides at both pH values, so that the binding is enthalpy driven. For both nucleotides, finite differenceH is the same for the first two classes of binding sites, implying that the difference in the dissociation constants of these two classes of sites is the result of entropic effects. Direct pH measurements and calorimetric measurements in two buffers with very different heats of ionization (Tris and Hepes) indicate that the binding of both nucleotides is accompanied by the binding of protons. In the pH range 6.7-8.4, the number of moles of protons bound per mole of nucleotide increases as the pH decreases.


Temperature and requency dependence of solvent proton relaxation rates in solutions of manganese(II) carbonic anhydrase.

Longitudinal and transverse proton relaxation rates of water in solutions of manganese(II) bovine carbonic anhydrase have been measured by pulsed nuclear magnetic resonance spectrometry as a function of temperature (2-35 degrees), frequently (5-100 MHz) and pH. The pH dependence of the longitudinal relaxation rate was fitted to a sigmoidal curve with a pK value at 7.8, while the esterase activity of the manganese(II) enzyme in the hydrolysis of p-nitrophenyl acetate revealed an inflection point at pK = 8.2. The hydration number of manganese(II) carbonic anhydrase could be derived using either the frequency dependence of T1p or the T1p/T2p ratio at only one (high) frequency. Both treatments are in agreement with a model in which one water molecule is bound to the metal at high pH. At low pH the relaxation data imply that no-H20 exists in the first coordination sphere of the manganese ion. The various parameters which are responsible for the proton relaxation mechanisms have been evaluated and are compared to other manganese(II) enzyme systems. The pH dependence of the binding constant of manganese to apocarbonic anhydrase is also reported.


A study of subtilisin types Novo and Carlsberg by circular polarization of fluorescence.

The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.


Equilibrium of Bowman-Birk inhibitor association with trypsin and alpha-chymotrypsin.

Association constants, enthalpies, and stoichiometries of Bowman-Birk soybean inhibitor for trypsin and alpha-chymotrypsin were measured in the pH range 4-8 at 25 degrees, 0.01 M Ca2+. The results are quoted in terms of moles of protease active sites, from active site titration. Enthalpies were obtained from calorimetry. The inhibitor was modified by carboxyl group modification, and by tryptic and chymotryptic attack. Association thermodynamics and stoichiometries of the modified inhibitors with both proteases were also determined. There is one independent site for each protease on the inhibitor protein. Modification decreases association to some extent, but does not appear to change stoichiometry or protease binding site independency. In the pH 4 region the association enthalpies are endothermic, of the order 6 kcal/mol for both trypsin and chymotrypsin. With increasing pH, the enthalpies decrease and become exothermic at pH 8 for chymotrypsin. Positive entropies, 50 cal mol-1 deg-1, occur at pH 4-5. They decrease as pH increases, but are always positive in sign. The observed to accompany the overall reaction, such as H+ transfer steps. The enthalpies and entropies probably compensate over the pH range 4-8, with a characteristic temperature of 390 plus or minus 30 degrees K. Estimates were made of the macromolecular Coulomb charge products in inhibitor-protease interaction. These range from about +5 to -60, over pH range 4-8, depending on the protease. Although intermolecular Coulombic forces cannot be easily delineated at the specific side chain level, they may operate at the macromolecule level.


Hydrogen exchange in nucleosides and nucleotides. Measurement of hydrogen exchange by stopped-flow and ultraviolet difference spectroscopy.

Time-dependent changes in the ultraviolet absorbance of the adenine chromophore are observed in the stopped-flow spectrophotometer when adenosine and its analogs are rapidly transferred from protium oxide to deuterium oxide. These absorbance changes are shown to result from hydrogen exchange in the exocyclic amino groups of the purine ribonucleosides by using derivatives of adenosine in which methyl groups replace exchangeable hydrogens and by showing that the general characteristics of hydrogen exchange in adenosine analogs agree with those found here. A study of the dependence of hydrogen-exchange rate constants on adenosine, AMP, and phosphate concentration showed there is a second-order dependence on AMP concentration which is primarily due to intermolecular catalysis by the phosphate group of the nucleotide. The deuterium oxide perturbation difference spectrum, obtained at equilibrium, was found to contain two components that result from blue shifts of the adenine chromophore absorbance: (1) a shift cause by the substitution of deuterium for protium in the ring (N1) nitrogen and exocyclic nitrogens, and (2) a shift associated with a change in the polarizability of the medium. Since the theory of solvent perturbation, which is used to measure the relative "exposure" of chromophores in macromolecules, assumes that the spectral shifts observed are solely due to (2) above, the use of deuterium oxide as a measure of chromophore exposure to perturbants the size of water must be reexamined.


A multienzyme complex for CO2 fixation.

Acetyl-coenzyme A carboxylase from Euglena gracilis strain Z was isolated as a component of a multienzyme complex which includes phosphoenolpyruvate carboxylase and malate dehydrogenase. The multienzyme complex was shown to exist in crude extracts and was purified to a homogeneous protein with a molecular weight of 360,000 by gel filtration. The ratio of the activities of the constituent enzymes was acetyl-CoA carboxylase:phosphoenolpyruvate carboxylase:malate dehydrogenase, 1:25:500. The complex is proposed to operate in conjunction with malic enzyme, which is present in Euglena, to facilitate the formation of substrates, malonyl-CoA, and NADPH, for fatty acid biosynthesis. The interaction of the enzymes may represent a means of control of acetyl-CoA carboxylase activity in organisms which do not possess an enzyme subject to allosteric regulation. The acetyl-CoA carboxylase activity from Euglena is unaffected by citrate and isocitrate.


Dissociation and characterization of enzymes from a multienzyme complex involved in CO2 fixation.

A multienzyme complex from Euglena, molecular weight about 360,000, containing phosphoenolpyruvate carboxylase, malate dehydrogenase, and acetyl-coenzyme A carboxylase has been dissociated into active constituent enzymes. The respective molecular weights are 183,000, 67,000, and 127,000. The malate dehydrogenase contained in the complex is electrophoretically distinct from other malate dehydrogenase isozymes found in Euglena. The K-m for HCO3minus of the free and complexed acetyl-CoA carboxylase is 4.2-5.4 mM, and the substrate dependency for acetyl-CoA describes a sigmoidal relationship. The HCO3minus K-m for the free phosphoenolpyruvate carboxylase is 7.3-5.4 mM while that for the same enzyme contained in the complex is 0.7-1.3 mM. Both the free and complexed forms ofphosphoenolpyruvate carboxylase have a K-m for phosphoenolpyruvate of 0.9-1.7 mM. The latter enzyme in both the complex and free forms is stimulated by NADH, acetyl-CoA, and ATP. In the free phosphoenolpyruvate carboxylase, the stimulation passes through a maximum depending on effector concentration. The effect of NADH is to increase V-max while K-m values remain unmodified.


Effect of pH on the liver alcohol dehydrogenase reaction.

New transient kinetic methods, which allow kinetics to be carried out under conditions of excess substrate, have been employed to investigate the kinetics of hydride transfer from NADH to aromatic aldehydes and from aromatic alcohols to NAD+ as a function of pH. The hydride transfer rate from 4-deuterio-NADH to beta-naphthaldehyde is nearly pH independent from pH 6.0 to pH 9.9; the isotope effect is also pH independent with kappa-H/kappaD congruent to 2.3. Likewise, the rate of oxidation of benzyl alcohol by NAD+ changes little with pH between pH 8.75 and pH 5.9; the isotope effect for this process is between 3.0 and 4.4. Earlier substituent effect studies on the reduction of aromatic aldehydes were consistent with electrophilic catalysis by either zinc or a protonic acid. The pH independence of hydride transfer is consistent with electrophilic catalysis by zinc since such catalysis by protonic acid (with a pK between 6.0 and 10.0) would show strong pH dependence. However, protonic acid catalysis cannot be excluded if the pKa of the acid catalyst in the ternary NADH-E-RCOH complex were smaller than 6.0 or smaller than 10.0. The two kinetic parameters changing significantly with pH are the kinetic binding constant for ternary complex formation with aromatic alcohol and the rate of dissociation of aromatic alcohols from enzyme. This is consistent with base-catalyzed removal of a proton from alcohol substrated and consequent acid catalysis of protonation of a zinc-alcoholate complex. The equilibrium constant for hydride transfer from benzaldehyde to benzyl alcohol at pH 8.75 is K-eq equals kappa-H/kappa-H equals 42; this constant has important consequences concerning subunit interactions during liver alcohol dehydrogenase catalysis.


19F nuclear magnetic resonance studies of structure and function relationships in trifluoroacetonylated rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.

Specific reaction of Cys-149 with 3,3,3-trifluorobromacetone allows one to probe symmetry relation between the active center regions of tetrameric glyceraldehyde-3-phosphate dehydrogenase by 19F nuclear magnetic resonance (nmr) techniques. Nmr titration studies in the pH range of greatest enzymic activity reveal the existence of species with an (alphaalpha')2 structure; this symmetry is not induced by the coenzyme. Addition of NADH to the ketone-labeled protein causes the enzymic reduction of the ligand in a stereospecific manner and is used to demonstrate the functionality of residues other than Cys-149 that are essential for catalysis. The interpretation of chemical shift characteristics found for the trifluoroacetonyl group together with the kinetics of its reduction allows the derivation of a dynamic model for the enzymic structure which may contribute to understanding of the half-of-the-sites phenomenon.


Enzymic and physicochemical properties of Streptomyces griseus trypsin.

Streptomyces griseus trypsin has been isolated from Pronase by ion-exchange chromatography on CM-Sephadex and SE-Sephadex. The isolated enzyme was homogeneous by the criteria tested except for a low degree of contamination by an enzyme with nontryptic activity. The latter could be partially resolved by chromatography on Bio-Rex 70. The molar absorbancy at 280 nm was found to be 3.96 times 10-4 M-1/cm and the E1cm1% was found to be 17.3. The molecular weight was 22,800 plus or minus 800. The enzyme was found to be stable at 0 degrees from pH 2 to 10. At 30 degrees the enzyme was maximally stable at pH 3-4 and significantly stabilized in the neutral and alkaline range by 15 mM Ca2+. Some evidence was obtained for a reversible denaturation of the enzyme at pH 12.0 and 2.0. The K-m for N-alpha-benzoyl-L-arginine ethyl ester at pH 8.0 in 20 mM CaCl2-0.1 M KCl-10 mM Tris-HCl buffer at 30 degrees was found to be 7.7 plus or minus 1.9 times 10-6 M and the esterase activity was observed to be dependent on an ionizing group with pK-a equals 5.85. In 2H2O this pKa was increased to 6.35 and the rate of hydrolysis dicreased threefold. The rate of hydrolysis was independent of pH between 8 and 10. The inhibition of the enzyme with L-1-chloro-3-tosylamido-4-phenyl-2-butanone was shown to be associated with the alkylation of its single histidine residue. This residue is present in a homologous amino acid sequence as the active-site histidine in trypsin and chymotrypsin. Optical rotatory dispersion and circular dichroism measurements over the pH range 5.3-10.5 indicated no significant conformational change until the pH was increased above 10.1. The observation that, under the conditions tested, acetylation and carbamylation of the NH2-terminal valine were incomplete is consistent with the view that this group is buried as an ion pair and only becomes available for deprotonation and reaction upon denaturation of the enzyme at pH values greater than 10.0.


Nuclear magnetic resonance studies of histone IV solution conformation.

The 220-MHz high-resolution proton magnetic resonance (PMR) spectrum of histone IV has been examined as a function of histone concentration, salt concentration, and pD. The hydrophobic C-terminal portion of the histone IV monomer appears to be largely PMR "invisible" indicating that this region of the polypeptide contains rigid secondary structure. Further loss of PMR resonance areas with increased histone IV concentration in neat D2O has been attributed to self-aggregation involving a monomer-dimer equilibrium. An equilibrium between the monomer and large aggregates, on the other hand, appears to dominate at NaCl concentrations above 0.01 M. pD studies reveal an abrupt increase in histone IV aggregation at pD smaller than 0.8 and precipitation of histone IV at pD values in the neighborhood of its isoelectric point, pD similar to 11.


Surface properties of monomolecular films of oxidized and reduced cytochrome c and f.

The surface properties of monomolecular films of oxidized and reduced cytochromes f and c were measured at an air-water interface. Area/molecular (A) and surface potential (deltaV) for oxidized and reduced forms of the cytochromes were measured as a function of pH. Oxidized cyt f has a maximum for both A and deltaV at pH 7.5. At a surface pressure of 6 dyn/cm the maximum A equals 2600 plus or minus 50 A2 and the maximum deltaV equals 200 plus or minus 10 mV. Reduced cyt f as a function of pH has a minimum value for both A (2200 A2) and deltaV (95 mV). Oxidized cyt c as a function of pH has minima for A (140 A2) and deltaV (188 mV) at pH 7.0 and 7.3, respectively. On the other hand, reduced cyt has maximum values for A (220 A2) and deltaV (260 mV) at pH 7.0 and 7.3, respectively.


The buoyant titration of native and carbamylated bovine serum mercaptalbumin.

The buoyant density titration curves of native and carbamylated bovine serum mercaptalbumin were measured throughout the pH range 5.3-12.7. Large increments in the buoyant density were observed above pH 10, with inflection pH values of 11.2 and 11.4 for native and carbamylated bovine serum mercaptalbumin, respectively. For the modified protein in which 25 out of 58 lysine residues were carbamylated, the buoyant densities were 0.048 g/ml higher at neutral pH and 0.024 g/ml higher at the extrapolated pH 13. The carbamyl groups apparently produce a larger residual density at pH 13 than they did in the case of ovalbumin. Homopolymer buoyant density titration data were demonstrated to be of value in calculating the contributions of titratable residues to the buoyant density of both proteins. The buoyant density increment at high pH was due largely to the deprotonation of the lysines as indicated by the diminished change in buoyant density between pH 10 and 12.7 for the modified protein. These density changes were attributable primarily to a gain of cesium ions. The limited modification of the lysine residues under mild reaction conditions and the rather high intrinsic dissociation constant of tyrosine residues in mercaptalbumin may indicate a preferential modification of easily accessible lysine residues. Phenolic deprotonation is facilitated by the neutralization of normally charged lysine residues and demonstrates ionic interactions between internal lysines and certain carboxyl and tyrosine residues thereby stabilizing the native state of the protein.


Kinetics and specificity of T4 polynucleotide kinase.

The kinetics of T4 polynucleotide kinase has been investigated at pH 8.0 and 37 degrees. Double reciprocal plots of initial rates vs. substrate concentrations as well as product inhibition studies have indicated that the enzyme reacts according to the ordered sequential mechanism shown in eq 2 in the text for phosphorylation of a DNA molecule. Based on this mechanism the rate equation for the overall reaction was deduced and the various kinetic constants estimated. Hill plots indicated little or no interaction between active sites in the enzyme. The apparent Michaelis constants and V-max were determined at a fixed ATP concentration, 66 muM, for a number of different substrates varying in chain length, base composition, and nature of the sugar, and a wide variation was found. For the nucleoside 3'-monophosphates tested both the apparent Michaelis constant and V-max values were from approximately 2 to 5 times larger than for the corresponding oligonucleotide. The following orders were obtained with regard to apparent Michaelis constants and V-max for the nucleoside 3'-monophosphates investigated: Michaelis constant, rGP greater than rUp greater than rCp greater than rAp greater than dTp; V-max, rGp greater than rCp greater than rAp greater than dTp greater than rUp. Somewhat similar results were also obtained with the deoxyoligonucleotides tested.


Formaldehyde as a probe of DNA structure. I. Reaction with exocyclic amino groups of DNA bases.

A comprehensive description is given of both the equilibrium and the kinetic aspects of the reaction of formaldehyde with the exocyclic amino groups of derivatives of adenine, cytosine, and guanine; the results extend previous data in the literature to the point where formaldehyde can now be used as a quantitative probe of DNA structure and dynamic behavior. The main results are: (i) the reaction product is proven (by isolation followed by nuclear magnetic resonance (NMR) spectroscopy) to be a hydroxymethyl group; (ii) a dihydroxymethyl adduct is shown to exist at high formaldehyde concentrations; (iii) equilibrium constants at 25 degrees for forming the monoadduct with adenine and cytosine compounds are about 12 (M-1), while those for forming the dihydroxymethyl adduct are about 0.4 (M-1); (iv) the standard enthalpies for forming the monoadducts with adenine and cytosine compounds are about minus 4 to minus 6 kcal/mol; (v) indirect evidence is presented suggesting that a monohydroxymethyl group on adenine or cytosine derivatives exists preferentially as that rotational isomer which blocks Watson-Crick hydrogen bonding; (vi) in derivatives of guanine, it is shown that the N-1 endocyclic imino group can react with formaldehyde, as well as the amino group, the overall equilibrium constant being about 6 (M-1); (vii) all rate constants are reported, as well as their response to temperature, pH, and various solvent additives known to perturb DNA structure; (viii) using a series of substituted anilines, a linear free energy relation is obtained between the logarithm of both the forward and the reverse rate constant for the formaldehyde reaction and the amine pK, over a range of 10-8 change in amie basicity; (ix) using this relation, the pK's for protonating the nucleoside amino groups are estimated to lie in the range of minus 2 to minus 4; (x) a reaction mechanism is proposed; and (xi) some implications of these results forpolynucleotide studies are discussed.


Formaldehyde as a probe of DNA structure. II. Reaction with endocyclic imino groups of DNA bases.

We describe the equilibrium and kinetic aspects of the formaldehyde reaction with the endocyclic imino groups of derivatives of thymine, uracil, and a series of halogenated uracils, as well as poly(uridylic acid) and poly(inosinic acid). The main results are: (i) the equilibrium constants for forming a hydroxymethyl adduct remain quite constant at about 2-2.5 (M-1) for all the compounds studied, independent of their pK; (ii) both forward and reverse rate constants with 5'-TMP are specific base catalyzed in the pH range of about 4-9; (iii) the response of the rate constants to temperature and to several solvent additives are measured; (iv) at neutral pH, for the series of pyrimidine compounds, a linear free energy relation is observed between the logarithm of both the forward and the reverse rate constant and the pK for deprotonation; (v) the unstructured polynucleotides, poly(U) and poly(I) react very similarly to their constituent monomers; (vi) a reaction mechanism is proposed; and (vii) some implications for polynucleotide studies are discussed. In an appendix, a method of spectral analysis is derived to obtain accurate estimates of the quite small equilibrium constants; this should be applicable to all similar two-component systems in which the final product is unobtainable, either by isolation or by saturation. Together with the results of the previous paper on the formaldehyde reaction with exocyclic amino groups (J. D. McGhee and P. H. von Hippel, preceding paper), these results form a reasonably comprehensive account of the basic chemical controls required to use formaldehyde as a quantitative probe of DNA structure.


The binding of hemoglobin to membranes of normal and sickle erythrocytes.

The binding of hemoglobins A, S, and A2 to red cell membranes prepared by hypotonic lysis from normal blood and blood from persons with sickle cell anemia was quantified under a variety of conditions using hemoglobin labelled by alkylation with 14C-labelled Nitrogen Mustard. Membrane morphology was examined by electron microscopy. Normal membranes were found capable of binding native hemoglobin A and hemoglobin S in similar amounts when incubated at low hemoglobin: membrane ratios, but at high ratios hemoglobin saturation levels of the membranes increased progressively for hemoglobin A, hemoglobin S and hemoglobin A2, respectively, in order of increasing electropositivity. Binding was unaffected by variations in temperature (4-22 degrees C) and altered little by the presence of sulfhydryl reagents, but was inhibited at pH levels above 7.35; disrupted at high ionic strength; and dependent on the ionic composition of the media. These findings suggest that electrostatic, but not hydrophobic or sulfhydryl bonds are important in membrane binding of the hemoglobin under the conditions studied. An increased retention of hemoglobin in preparations of membranes from red cells of patients with sickle cell anemia (homozygote S) was attributable to the dense fraction of homozygote S red cells rich in irreversibly sickled cells, and the latter membranes had a smaller residual binding capacity for new hemoglobin. This suggests that in homozygote S cells which have become irreversibly sickled cells in vivo, there are membrane changes which involve alteration and/or blockade of hemoglobin binding sites. These findings support the notion that hemoglobin participates in the dynamic structure of the red cell membrane in a manner which differs in normal and pathological states.


Comparison of the cerebroside sulphatase and the arylsulphatase activity of human sulphatase A in the absence of activators.

A cerebroside sulphatase (cerebroside-3-sulphate 3 sulphohydrolase, EC 3.1.6.8) assay based on radio thin-layer chromatography is described. The substrate was labelled by the catalytic addition of tritium to cerebroside sulphate. Using this assay the cerebroside sulphatase activity of sulphatase A (Aryl-sulphate sulphohydrolase, EC 3.1.6.1) from human liver and kidney in the absence of activators was investigated. The pH optimum of this reaction depends on the buffer concentration, being pH 4.5 at 50 mM and 5.3 at 10 mM sodium formate. With the latter concentration the apparent Km for cerebroside sulphate is 0.06 mM; SO2-4 and nitrocatechol sulphate inhibit noncompetitively with a Ki of 4.51 mM for Na2SO4 and 0.43 mM for nitrocatechol sulphate. The cerebroside sulphatase activity of sulphatase A is highly dependent on the ionic strength. The optimum sodium formate concentration is 10 mM, and the cerebroside suophatase activity decreases rapidly with increasing buffer concentration. The same concentration dependence is observed in the inhibitory effect of cerebroside sulphate on the arylsulphatase reaction. The inhibition decreases at increasing buffer concentrations, becoming an activation at 70 mM sodium formate. The progress curve of the cerebroside sulphatase reaction shows a deviation from linearity similar to that of the arylsulphatase reaction. Investigation of the effect of preincubation with cerebroside sulphate on the arylsulphatase activity of the enzyme shows that cerebroside sluphatase activity and inactivation of the enzyme by cerebroside sulphate occur simultaneously. These observations are interpreted as supporting the assumption that cerebroside suophate and arylsulphates are degraded at an identical active site on the same enzyme. Differences in the properties of the cerebroside sulphatase and the arylsulphatase reaction of the enzyme may be attributed to the differences in the physiocochemical state of the two substrates.


Effect of alpha2 macroglobulin on some kinetic parameters of trypsin.

1. Complex formation of trypsin with alpha2 macroglobulin results in marked changes of the Michaelis-Menten constant, pH optimum and sensitivity to ionic strength in a system using N-carbobenzoxy-glycylglycyl-L-arginine-2-naphthylamide as substrate. 2. In contrasts to the inhibition (50%) observed when alpha2 macroglobulin-bound trypsin is assayed under conditions optimal for the free enzyme, there is minimal reduction of activity when determinations are performed at a substrate concentration and pH optimal for the bound enzyme. 3. The changes in substrate concentration and ionic environment required for maximum activity of alpha2 macroglobulin-bound trypsin are similar to those observed with enzymes embedded in polyelectrolyte matrices and may reflect alterations in the microenvironment of the enzyme resulting from conformational changes of the macromolecule during interaction with trypsin. 4. Enzymatic activity of trypsin towards casein is greatly reduced by alpha2 macroglobulin, even under assay conditions optimal for the bound enzyme, confirming previous findings that access to the active center for high-molecular weight substrates is sterically hindered by alpha2 macroglobulin.


Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.


Myocardial guanylate cyclase: properties of the enzyme and effects of cholinergic agonists in vitro.

The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig greater than man greater than cat greater than dog greater than rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 times g. The Km for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the Ka for Mn2+ in particulate preparations (0.3-0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8-2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005-10.0 mM Ca2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 times g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4-4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10-4-10-8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012-1.2 mM GTP, 0.3-10.0 mM Mn2+ and 0.005-10.0 mM Ca2+ or 0.0012-1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzyme-lipid suspensions were assayed in the presence of cholinergic agents.


Purification and characterization of xanthine oxidase from livers of vitamin E deficient rabbits.

Xanthine oxidase which increases in activity during vitamin E deficiency was purified from livers of deficient rabbits. The procedure incorporates preparative sucrose gradient centrifugation and yields a homogeneous preparation on acrylamide gel electrophoresis. The purified enzyme exhibits a pH optimum of 8.1 and a Km value of 22 muM. Gel filtration chromatography gave the molecular weight of 280 000. Acrylamide gel electrophoresis in the presence of sodium dodecylsulphate reveals two types of subunits of molecular weights 52 000 and 99 000.


Lysine-ketoglutarate reductase in human tissues.

Lysine-ketoglutarate reductase (saccharopine dehydrogenase (NADP+, lysine-forming) EC 1.5.1.8) from human liver has been partially purified and characterized. A spectrophotometric assay is described. The Michaelis constants have been determined for lysine (1.5-10-3 M), alpha-ketoglutarate (1-10-3 M) and NADPH (8-10-5 M). The pH optimum is 7.8. The enzyme is product inhibited. The specificity of the enzyme, response to inhibitors, pH and thermal stability are reported. Lysine-ketoglutarate reductase is present in high concentration in liver and heart, to a lesser degree in kidney and skin and in trace amounts in several other tissues. Saccharopine dehydrogenase (saccharopine dehydrogenase (NAD+, L-glutamate-forming) EC 1.5.1.9) was demonstrable only in liver and kidney. Lysine-ketoglutarate reductase reacts effectively with delta-hydroxylysine.


Effect of enzymic assay conditions on sulfite reduction catalysed by desulfoviridin from Desulfovibrio gigas.

The type and the amount of end products resulting from sulfite reduction catalysed by a single partially purified desulfoviridin preparation from Desulfovibrio gigas were shown to depend upon the enzymic assay conditions employed. Both manometric and spectrophotometric assays were used, with reduced methyl viologen serving as the electron donor in each system. Trithionate, thiosulfate, tetrathionate and sulfide were identified as possible end products. In the manometric assays, sulfide production was favoured by high reduced methyl viologen concentrations, low sulfite concentrations and a pH value of 7.0 as opposed to 6.0. In the spectrophotometric assays, results approaching the stoichiometric conversion of sulfite to sulfide were obtained only at high initial reduced methyl viologen concentrations.


Coupled optical rate determinations of amino acid oxidase activity.

Methods are described in which liberation of ammonia from amino acid substrates by the D- and L-amino acid oxidases may be coupled with the NADH-dependent reductive amination of 2-oxoglutarate catalysed by exogenous glutamate dehydrogenase (L-glutamate: NAD oxidoreductase (deaminating), EC 1.4.1.2). The inhibition of D-amino acid oxidase (D-amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) by ADP needed to activate and stabilise glutamate dehydrogenase was relieved by FAD, and the substrate was D-alanine at approximately 6-fold Km concentration. Neither FAD or FMN were required in the L-amino acid oxidase (L-amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.2) assay; this utilised L-leucine as substrate in a concentration approximately 7-fold the Km value. The methods were reasonably sensitive and precise, and a linear relationship between activity and enzyme concentration prevailed up to an absorbance change of 0.050 per min. They have the advantage of being amenable to automation and to employment of fluorescence techniques should greater sensitivity be required.


Kinetic properties of an enzyme highly enriched in carbon-13.

Preparations of Candida utilis 6-phosphogluconate dehydrogenase with extreme variation in carbon isotope composition have been obtained. Molecular activities, pH optima and substrate affinities of these enzyme preparations have been measured. It was observed that any effect of isotope composition on these physiologically important properties is minimal.


Glutamate dehydrogenase from Escherichia coli: induction, purification and properties of the enzyme.

When Escherichia coli was grown in a minimum medium with glucose as sole carbon source and a proper level of ammonia, NADP+ specific glutamate dehydrogenase (L-glutamate: NADP+ oxidoreductase (deaminating), ED 1.4.1.4) was induced. The enzyme was solubilized by French press treatment and purified to homogeneity by (NH4)2SO4 fractionation, heat treatment followed by DEAE-cellulose, hydroxylapatite and Bio-Gel chromatography with an overall yield of 30%. The enzyme proved to be heat stable and relatively resistant to protein denaturants. The optimum of enzymic activity for the reductive amination is at pH 8 and at pH 9 for the oxidative deamination. The activity is affected by adenine nucleotides. The molecular weight (about 250 000 for the native form and 46 000 for the inactive subunit) and amino acid composition, suggest strict similarities with the NADP+ enzyme from fungal origin.


A purification procedure for the isolation of homogeneous preparations of bovine aorta amine oxidase and a study of its lysyl oxidase activity.

It has been reported that bovine aorta amine oxidase oxidizes lysine residues in tropoelastin to allysine (Rucker, R.B. and O'Dell, B.L. (1971) Biochim. Biophys. Acta 235, 32-43). Pure bovine aorta amine oxidase was isolate by DEAE-cellulose, hydroxylapatite, Bio-Gel A-1.5 m and concanavalin A-Sepharose 4B chromatography. Enzymatic, chromatographic and immunochemical tests disclosed that pure bovine aorta amine oxidase was not a lysyl oxidase capable of oxidizing the lysine residues of tropoelastin to allysine; The bovine aorta amine oxidase preparation used by Rucker and O'Dell appears to have been contaminated with lysyl oxidase which is the emzyme that oxidizes some of the lysine residues in tropoelastin and tropocollagen to allysine.


Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora.

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 muM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH2-nitrate reductase of Neurospora suggests some differences in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 mu M) markedly enhanced the action of NAD(P)H on the maize enzyme; Cyanide at a higher level (6 mu M) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no effect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.


Studies on the in vitro inactivation of the Neurospora crassa assimilatory nitrite reductase in the presence of reduced pyridine nucleotides plus flavin.

In vitro inactivation of Neurospora crassa nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4) can be obtained by preincubation of the enzyme with reduced pyridine nucleotide plus FAD. The presence of nitrite or hydroxylamine, electron acceptors for the N. crassa nitrite reductase, or cyanide, sulfite or arsenite, competitive inhibitors with respect to nitrite of this enzyme, protects the enzyme against this inactivation. Anaerobic experiments reveal that oxygen is required in order to obtain complete inactivation of nitrite reductase by preincubation with reduced pyridine nucleotide plus FAD. Also, inactivation is prevented if catalase is included in the preincubation mixture. The presence of hydrogen peroxide in the preincubation mixture increases the sensitivity of nitrite reductase to the in vitro FAD-dependent NAD(P)H inactivation. Neither electron acceptors, competitive inhibitors nor catalase, agents which protect the enzyme against the FAD-dependent NAD(P)H inactivation, can reverse this process once it has occurred.