Association of Epstein-Barr virus genome with mixed cellularity and cellular phase nodular sclerosis Hodgkin's disease subtypes. Association of Epstein-Barr virus (EBV) genome with Hodgkin's disease (HD) histological subtypes was investigated using the polymerase chain reaction (PCR). A highly significant association of EBV genome with the mixed cellularity (MC) subtype (10 positive out of 15 cases; 66%) was observed, suggesting a possible etiopathogenetic role of EBV in the induction of this subset. By contrast, a markedly lower frequency of association with EBV genome was found in nodular sclerosis (NS) (12 positive out of 46 cases; 26%) and in nodular lymphocytic predominance (NLP) (0 positive out of 5 cases) HD subtypes. In addition, in the NS series, the presence of EBV genome was mainly restricted to the 'cellular phase' NS subset. This finding strengthens the possibility, suggested by clinico-pathological features and survival rates, that 'cellular phase NS' is a disease more akin to MC than to typical NS HD.


Combined-modality therapies for non-small cell lung cancer. Regionally advanced stage III non-small cell lung cancer (NSCLC) accounts for nearly 40% of all presentations of NSCLC. In the past, such patients received radiotherapy alone, but the median and long-term survival durations were disappointingly poor. Past attempts at combining chemotherapy and radiation were also disappointing, and were troubled by low doses of radiation or orthovoltage equipment or both. Recently, cisplatin-containing regimens have shown some efficacy in stage IV disease. The response rate for these combinations in stage III disease is nearly double that in stage IV disease. The greater response in stage III has led to a series of trials of sequenced chemotherapy and radiotherapy for treatment of regionally advanced (unresectable stage IIIA and IIIB) NSCLC. Several randomized trials have now shown a statistically significant advantage for the combined modality over radiation alone regarding time to treatment failure, median survival duration, and percent of long-term survivors. Other trials have focused on the concurrent use of chemotherapy and radiotherapy. Several pilot studies have suggested that concurrent cisplatin plus chest irradiation can produce apparently beneficial results with respect to local control and are the subject of ongoing clinical trials. At the University of Maryland Cancer Center, we have combined weekly carboplatin 100 mg/m2 with concurrent chest irradiation. The preliminary results are very encouraging. The toxicity of this treatment program is very manageable, and preliminary data suggest excellent local control and survival. Other pilot studies have suggested that combination chemotherapy with concurrent radiotherapy is also technically feasible.(ABSTRACT TRUNCATED AT 250 WORDS)


Preclinical and clinical experience with cisplatin and carboplatin and simultaneous radiation in non-small cell lung cancer. Preclinical experiments with cisplatin and carboplatin showed radiosensitizing capability for these drugs against non-small cell lung cancer (NSCLC) cell lines and phase II clinical investigations were undertaken in patients with stage IIIA/B NSCLC. In the first trial, cisplatin 20 mg/m2 was infused on the first day of every treatment week, followed in about 1 hour by radiotherapy. Radiotherapy was given in single daily 2-Gy doses 5 days a week for 3 weeks, and, after a 2-week interval, for an additional 2 weeks. There were 5 complete responses (CRs) and 15 partial responses (PRs), while 6 patients had no change and 4 progressive disease, for a 67% overall response. Median survival was 14 months; the 2-year survival rate was 20%. To investigate the maximum tolerated dose of carboplatin, escalating doses were given on day 1 of each treatment week; simultaneous radiotherapy was administered as in the cisplatin study. Drug doses began at 100 mg/m2 and were increased by increments of 10 or 20 mg/m2 to a maximum of 200 mg/m2, with 5 patients treated at each dose level. Dose escalation was to stop when 3 of 5 patients developed intolerable World Health Organization (WHO) grade 3 myelosuppression. No grade 3 or 4 leukopenia occurred at carboplatin doses less than or equal to 140 mg/m2. Grade 3 leukopenia was seen in only 1 patient from the groups receiving 150, 160, or 180 mg/m2. Of the 35 patients evaluable thus far, 1 achieved CR and 18 PR, while 10 had no change and 6 progressive disease despite treatment, for a 54% overall response rate. Response rate in patients with stage IIIB disease was 48%.(ABSTRACT TRUNCATED AT 250 WORDS)


Prophylactic cranial irradiation: advantages and disadvantages. Anaplastic small cell carcinoma of the lung is a disease that responds to multiagent systemic therapy. Unfortunately, a high incidence of central nervous system (CNS) relapse has been observed by many investigators. Cranial irradiation has been successfully used in acute lymphoblastic leukemia to reduce CNS relapse and improve survival. A similar application of prophylactic cranial irradiation in patients undergoing therapy for small cell anaplastic carcinoma of the lung has been noted to decrease CNS relapse from approximately 25% to approximately 5%. However, CNS toxicity has been noted to occur with CNS prophylaxis. We can reduce the incidence of CNS toxicity by using modern treatment schedules and avoiding the use of doxorubicin with irradiation. Prophylactic cranial irradiation delivered to those patients who obtain a complete remission in response to induction chemotherapy can reduce the incidence of CNS relapse and can usually be delivered with minimal CNS toxicity. Unfortunately, long-term disease control with systemic agents is poor. Until more effective systemic agents are developed, the role of prophylactic cranial irradiation is one of palliation rather than improving survival.


Oral etoposide in oncology: an evolving role. Etoposide has been in clinical trials for over 2 decades. From the inception of these investigations, both the oral and intravenous routes have been employed. The oral route has been shown to be convenient, safe, and effective in the trials of diseases such as small cell lung cancer and lymphomas. A recent resurgence of interest has surfaced with its oral application in the elderly and in protracted dosing schedules. Investigations are now under way assessing the relative value of the protracted oral dosing schema in a variety of diseases.


Primary solitary tuberculosis of the liver. Primary solitary tuberculous involvement of the liver is a rare condition. We present the case of a patient who was operated on with a preoperative diagnosis of hepatocellular carcinoma. Liver resection was performed and antituberculous therapy was started. It is difficult to make the correct diagnosis preoperatively except when a successful needle biopsy can be performed. Despite the rarity of the condition primary solitary tuberculosis should be considered among the space occupying lesions of the liver.


[Uterine cervical carcinoma and human papillomaviruses]. For many years it has been thought that a significant proportion of cervical cancer could be attributed to sexually transmitted agents, such as sperm, smegma, Treponema pallidum, Gonococcus and herpes simplexvirus type 2. Recent advances of molecular biology, however, have revealed that human papillomavirus (HPV) might be the most causative virus of the disease. Since HPV type 16 DNA was found in a patient with cervical cancer in 1983, many HPV types have been cloned from cervical cancers, also from premalignant lesions (intraepithelial neoplasias). In Japan, we have found 6 new types of HPV (HPV 58, 59, 61, 62, 64, 67) in the female genital tract so far. Especially, HPV 58, which was cloned from a patient with cervical squamous cell carcinoma and was already fully sequenced, is thought to be an important agent for the development of cervical cancer as well as HPV 16. Now we are investigating extensively to clarify the real relationship between genital HPV infection and cervical cancer.


Complement proteins and soluble immune complex levels in Nigerians having primary liver cell carcinoma. Serum Complement (C3), proteins and circulating immune complex levels were estimated in Nigerians having primary liver cell carcinoma and control subjects by immunodiffusion in agar, biuret and polyethylene glycol precipitation methods respectively. The patients were observed to have diminished mean C3 and albumin concentrations whereas the mean total proteins, globulins and soluble immune complex levels were elevated. About 80 percent of the patients who had depressed serum C3 concentrations also had elevated levels of circulating immune complexes. There is however no correlation between the complement (C3) concentrations and the serum levels of immune complexes.


[Gene expression of hepatitis viruses in the liver and hepatocarcinogenesis]. Human hepatitis B virus (HBV) carriers run an increased risk of hepatocellular carcinoma (HCC), where the expression of HBV genes play the most important role in the initial stage of hepatocarcinogenesis. As the integration of HBV DNA into the cellular DNA of HCC as well as chronic hepatitis was demonstrated very frequently, the virus-cell fusion gene was considered to be most essential for hepatocarcinogenesis. Among the virus-cell fusion genes, the X gene is known to function as a transactivator for viral and cellular genes at the time of chronic infection. One mechanism for hepatocarcinogenesis that appears particularly reasonable is transactivation of cellular oncogenes by the X-cell fusion protein. In 1990, we found a part of the amino acid sequences in the X protein to be highly homologous to functionally essential sequences in the Kunitz domain, characteristic of Kunitz-type serine protease inhibitors. It has been recently demonstrated that X protein expressed in E. coli or from the in vitro translation system binds to a specific serine protease from the liver cells. These results indicate that transactivation function of X protein may be exerted by acting as a protease inhibitor analogue to control the proteolytic pathway of cellular transcription factor(s). On the other hand, viral hepatitis resulting from viruses other than hepatitis A virus and HBV has been referred to as non-A, non-B hepatitis. In 1989, the viral genome was molecularly cloned as a positive-strand RNA having about 10 kb in size and named as hepatitis C virus (HCV). Details of genetic structure and mechanism of expression are currently under investigation at molecular level.


Transmission of cytomegalovirus in the Gambia. One hundred and seventy-eight Gambian women in their late 3rd trimester plus babies born to them, and later on some 11 randomly selected CMV positive and 11 CMV negative infants with their mothers and siblings, were examined serologically, and virologically for the evidence of cytomegalovirus infection with a view to determining who is infecting who and how, in this community. There was significant correlation between CMV positive infants and CMV infection in their siblings, whereas there was no such correlation between these infants and their CMV positive mothers. Children under five years excreted more CMV and for a longer time in all their samples than the corresponding samples and in all specimens from their mothers. Urine had the highest amount of CMV both in the children and adults. It is concluded that children were more likely to transmit CMV to the infants and that urine through bed-wetting, was the most likely route of such transmission in Gambia and probably also in West Africa.


[Skin cancer and virus].

[Skin cancer and virus]. Human papillomaviruses (HPV) generally associated with benign skin and anogenital warts. Because several of skin cancers were found to contain HPV-DNA, it has been speculated that certain types of HPV could be specifically associated with cancers. Although HPV-DNAs are not isolated from most of skin cancers, they are often isolated from penile cancers, vulval cancers and anogenital Bowen's diseases. Patients with epidermodysplasia verruciformis start to suffer from disseminated incurable warts during their childhood, and some of these benign lesions often convert to skin cancer in adulthood. Although the disease is very rare, HPV may also play a role in malignant transformation in epidermodysplasia verruciformis. More than 60 types of HPVs distinguished by molecular hybridization techniques. The type of HPV determines the clinical picture of wart and natural fate of HPV-associated lesion. There are two groups of HPVs, which are benign types and malignant types. Viral DNA of malignant type of HPV transforms human keratinocytes in vitro, and the transforming activity has been mapped to the E6 and E7 genes.


Glutamate antagonists that block hyperpolarizing bipolar cells increase the release of dopamine from turtle retina. Some neurochemical features of the neuronal circuitry regulating dopamine release were examined in the retina of the turtle, Pseudemys scripta elegans. Glutamate antagonists that block hyperpolarizing bipolar cells, such as 2,3 piperidine dicarboxylic acid (PDA), produced dose-dependent dopamine release. In contrast, the glutamate agonist 2-amino-4-phosphonobutyric acid (APB), which blocks depolarizing bipolar cell responses with high specificity, had no effect on the release of dopamine. The gamma-aminobutyric acid (GABA) antagonist, bicuculline, also produced potent dose-dependent release of dopamine. The release of dopamine produced by PDA was blocked by exogenous GABA and muscimol, suggesting that the PDA-mediated release process was polysynaptic and involved a GABAergic synapse interposed between the bipolar and dopaminergic amacrine cells. The only other agents that produced dopamine release were chloride-free media and high extracellular K+; in particular, kainic acid and glutamate itself were ineffective. These results suggest that the primary neuronal chain mediating dopamine release in the turtle retina is: cone----hyperpolarizing bipolar cell----GABAergic amacrine cell----dopaminergic amacrine cell.


Clinical and laboratory presentation of lichen planus patients with chronic liver disease. A recent case-control study on 577 lichen planus (LP) patients and 1008 controls confirmed that LP patients may significantly associate with a chronic liver disease (CLD) which is independent from drug or alcohol intake and has some connection with hepatitis B virus (HBV) infection. The study, however, failed to define the nature of CLD. This has been investigated through the clinical and laboratory features of 50 patients with LP and impaired liver function tests. Overall, the laboratory signs of cell necrosis prevailed over those of cholestasis and a good relationship with the HBV and HCV infections was found. Ninety percent of patients with LP and CLD had antibodies to one or another of the major viruses involved in infectious hepatitis. No patient had anti-liver kidney microsomal antibodies type 1. Liver biopsies were done in 12 cases and mostly revealed a chronic active hepatitis evolving into cirrhosis. No evident cases of primary biliary cirrhosis were found. It appears that LP associated CLD is post-viral in nature.


Actions of GABAergic ligands on brisk ganglion cells in the cat retina. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian retina. We tested the actions of iontophoretically applied GABAergic ligands on the spontaneous and stimulus-evoked activity of retinal ganglion cells recorded extracellularly in the in vivo cat eye. GABA as well as GABAA receptor agonists inhibited all brisk ganglion cell types. This action was antagonized by bicuculline. Bicuculline on its own increased the activity of ON-ganglion cells but suppressed OFF-ganglion cells. This suppression effect was abolished during the blockade of glycinergic transmission by strychnine. The GABAB receptor agonist baclofen inhibited OFF-ganglion cells whereas the activity of ON-ganglion cells was either increased or decreased depending on the stimulus contrast. The antagonists, phaclofen and 2-hydroxy saclofen, produced opposite effects to baclofen and antagonized its action. The present study demonstrates that both GABAA and GABAB receptors modulate the activity of ON- and OFF-ganglion cells in the cat retina.


25 mg oestradiol implants--the dosage of first choice for subcutaneous oestrogen replacement therapy? To assess the oestrogen concentrations and symptom relief obtained with 25 mg oestradiol implants. Open, observational study. Twelve symptomatic, post-menopausal women seen in a designated menopause clinic. A 25 mg oestradiol pellet was inserted subcutaneously, blood samples were obtained before implantation and at regular intervals (2-4 weeks) until symptoms refused as hypoestrogenaemia developed. Change in symptom score following implant treatment. Concentrations of oestrogens and their metabolites before and during low dose subcutaneous oestradiol therapy. Ten of the 12 women had excellent symptom relief, associated with oestradiol concentrations in the follicular range for between 28 and 35 weeks. The ratio of circulating oestrogen metabolites remained physiological, despite the oestradiol concentrations being substantially higher on treatment. We suggest that 25 mg pellets should be used as the initial dose for subcutaneous oestrogen treatment, and a combination of return of symptoms and weeks since insertion used to judge the timing of reimplantation.


Localisation of prostaglandin F2 alpha and E2 binding sites in the human eye. Prostaglandin F2 alpha reduces intraocular pressure possibly by increasing uveoscleral outflow. To further understand the mechanism of its action binding sites for prostaglandin F2 alpha and, for comparison, prostaglandin E2 were localised in sections of human cadaveric eyes using an in vitro ligand-binding technique and autoradiography. Specific binding sites for both prostaglandin F2 alpha and E2 were co-localised at a high level in the areas of the ciliary muscles and iris sphincter muscles, and at a lower level in the iris epithelium and the retina. The results suggest that prostaglandin F2 alpha and also prostaglandin E2, could modulate uveoscleral outflow by binding to their receptors located on the ciliary muscles and inducing their relaxation.


A monogalactosylated cholesterol derivative that specifically induces uptake of LDL by the liver. We described earlier the effect of tris-gal-chol (a triantennary galactose structure coupled to cholesterol) on the fate of low density lipoprotein (LDL) and high density lipoprotein (HDL). Tris-gal-chol-loaded LDL and HDL are both efficiently cleared from blood by hepatic galactose-specific receptors. Thus, tris-gal-chol combines a beneficial LDL-reducing effect with an equally effective but undesirable HDL-lowering effect. We recently synthesized a cholesterol derivative with a single terminal galactose residue, denoted mono-gal-chol. In the present study we show that this compound, which incorporates readily into both LDL and HDL, induces rapid association of LDL and HDL to the liver. The mono-gal-chol-stimulated hepatic association of HDL, however, was about fivefold lower than that of LDL. In the liver, Kupffer cells were mainly (90%) responsible for the liver uptake of mono-gal-chol-loaded LDL, whereas the complex of mono-gal-chol with HDL was predominantly (95%) taken up by parenchymal cells. Uptake by both cell types proceeded via galactose-specific receptors and was followed by degradation of the apolipoproteins in the lysosomes. Thus, compared with tris-gal-chol, mono-gal-chol is equally effective in the induction of galactose-specific uptake of LDL by Kupffer cells. However, the galactose-specific receptor on parenchymal cells recognizes mono-gal-chol-loaded HDL less efficiently than tris-gal-chol-containing HDL. These results indicate that mono-gal-chol might be used to specifically lower LDL levels in patients with a high LDL cholesterol level.


Regulation of the urokinase-type plasminogen activator receptor on vascular smooth muscle cells is under the control of thrombin and other mitogens. The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombin's stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.


Contribution of the endothelium to intimal thickening in normocholesterolemic and hypercholesterolemic rabbits. Endothelial cell injury is considered to be a primary event in the pathogenesis of atherosclerosis. In this study, we investigated the aortic intimal lesion after balloon catheterization in hypercholesterolemic and normocholesterolemic rabbits with or without probucol, an antioxidant. After deendothelialization, the rabbits were divided into four groups: 1) a control group fed a standard diet; 2) a probucol-treated group; 3) a cholesterol-fed group; and 4) a group fed a mixed cholesterol and probucol diet. Four animals from each group were killed at 2, 4, and 8 weeks after deendothelialization. The aortic segments of nonendothelialized areas, borderline areas, and uninjured areas were histologically and immunohistochemically examined. Deendothelialized areas showed various degrees of intimal thickening, which was mainly composed of smooth muscle cells in rabbits from groups 1 and 2. The intimal thickness of group 3 was significantly larger than that of other groups in any area examined. The intimal thickness of group 4 was less than that of group 3 despite the hypercholesterolemic state in the former group. The intima of borderline areas was generally thicker than that of nonendothelialized areas. Although the borderline lesions of groups 3 and 4 contained numerous macrophages, the number of macrophages was lower in the nonendothelialized compared with the reendothelized lesion. These data indicate that endothelial cell injuries can cause intimal thickening. The regenerated endothelial covering is favorable for monocyte migration and attachment. This process, together with the proliferation of smooth muscle cells, greatly contributes to the progression of atherosclerosis, which appears to involve lipid oxidation. Probucol prevented intimal thickening to a certain degree in this experiment in the normocholesterolemic as well as the hypercholesterolemic state.


Relationship between structure and function of dietary fibre: a comparative study of the effects of three galactomannans on cholesterol metabolism in the rat. Male adult rats were fed on diets containing 80 g/kg galactomannans with different galactose (G): mannose (M) ratios/kg. The galactomannans were compared with purified cellulose (Solkaflok) and the animal were also fed on a basal diet free from fibre. All diets contained cholesterol (10 g/kg) and sodium cholate (2 g/kg). The three galactomannans were fenugreek gum (1G:1M), guar gum (1G:2M) and locust-bean gum (1G:4M). In comparison with the fibre-free and Solkaflok diets, all three galactomannans lowered the concentrations of cholesterol in both liver and blood plasma. The galactomannans also decreased the rate of hepatic synthesis of cholesterol. Dietary galactomannans increased caecal volatile fatty acids, particularly propionic, increased the weight of the caecum and its contents and increased the amount of water in the faeces. The increase in propionic acid production was significantly related to a decrease in caecal pH, but not to changes in plasma cholesterol or hepatic cholesterol synthesis. These effects were significantly influenced by chemical composition and structure of the galactomannan; they were most evident when the proportion of galactose in the galactomannan was highest (i.e. fenugreek gum). The three galactomannans also differed markedly in their effects on the viscosity of the digesta, but the galactomannan which gave the highest viscosity was least effective in lowering plasma cholesterol. A separate experiment with perfused loops of small intestine in vivo showed that the most effective galactomannan, fenugreek gum, had no direct effect on cholesterol absorption.


Hypocholesterolaemic effect of banana (Musa sapientum L. var. Cavendishii) pulp in the rat fed on a cholesterol-containing diet. The pulp of banana fruit (Musa sapientum L. var. Cavendishii) was examined for its cholesterol-lowering effect with male rats fed on a diet containing lard (50 g/kg) and cholesterol (5 g/kg). Freeze-dried banana pulp showed a marked cholesterol-lowering effect when incorporated into a diet at the level of 300 or 500 g/kg, while the banana pulp dried in a hot-air current (65 degrees) did not. Starch and tannin prepared from banana pulp were not responsible for the cholesterol-lowering effect. The results also suggest that banana lipids did not affect the concentration of serum cholesterol. Feeding of dopamine, n-epinephrine and serotonin tended to raise the concentration of serum cholesterol. Thus, all the substances tested which were thought to be susceptible to influence by hot-air drying were unlikely to be responsible for the hypocholesterolaemic effect. However, both soluble and insoluble fibres fractionated from banana pulp had a cholesterol-lowering effect, with the exception of cellulose. It was assumed that a browning reaction undergone during hot-air drying might be related to the disappearance of the hypocholesterolaemic effect of banana pulp dried in a hot-air current. The results obtained support the conclusion that soluble and insoluble components of dietary fibre participate in the hypocholesterolaemic effect of banana pulp.


Alterations in endothelium-associated proteins and serum thyroid hormone concentrations in anorexia nervosa. Plasma concentrations of endothelium-associated proteins (EAP) (plasma fibronectin (PFN), angiotensin-converting enzyme, factor VIII-related antigen (F VIII-R:Ag)) and tissue plasminogen activator and serum thyroid hormone concentrations were studied in nine patients with anorexia nervosa (AN), before and after weight gain. Before weight gain (-35.9 (SE 2.3)% of standard body-weight) PFN was significantly reduced and F VIII-R:Ag was significantly increased in AN patients compared with the concentrations in control subjects (211.5 (SE 14.9) v. 274.7 (SE 16.6) micrograms/ml, P < 0.05; 129.2 (SE 14.1) v. 88.2 (SE 9.7) %, P < 0.05 respectively). Serum triiodothyronine (T3) and free T3 levels were also significantly lower before weight gain in AN patients (0.85 (SE 0.07) v. 1.53 (SE 0.08) nmol/l, P < 0.001; 2.57 (SE 0.23) v. 5.31 (SE 0.34) pmol/l, P < 0.001 respectively), although serum thyroxine (T4), free T4, and thyrotropin concentrations were within the normal range throughout the study periods. Following weight gain, PFN and F VIII-R:Ag concentrations normalized as did the thyroid hormone levels. The incremental changes in PFN levels correlated significantly with those in serum thyroid hormone concentrations (T3, r 0.79, P < 0.01; free T3, r 0.84, P < 0.01). These findings suggest that PFN levels may be directly related to serum T3 concentrations in AN patients.


Kinetics and energetics of base-pair opening in 5'-d(CGCGAATTCGCG)-3' and a substituted dodecamer containing G.T mismatches. Proton nuclear magnetic resonance (NMR) spectroscopy is used to characterize the kinetics and energetics of base-pair opening in the dodecamers 5'-d(CGCGAATTCGCG)-3' and 5'-d(CGCGAATTTGCG)-3'. The latter dodecamer contains two symmetrical G.T mismatched base pairs. The exchange kinetics of imino protons is measured from resonance line widths and selective longitudinal relaxation times. For the G.T pair, the two imino protons (G-N1H and T-N3H) provide probes for the opening of each base in the mismatched pair. The lifetimes of individual base pairs in the closed state and the equilibrium constants for formation of the open state are obtained from the dependence of the exchange rates on the concentration of ammonia catalyst. The activation energies and standard enthalpy changes for base-pair opening are obtained from the temperature dependence of the lifetimes and equilibrium constants, respectively. The results indicate that the G.T mismatched pairs are kinetically and energetically destabilized relative to normal, Watson-Crick base pairs. The lifetimes of the G.T pairs are of the order of 1 ms or less, over the temperature range from 0 to 20 degrees C. The equilibrium constants for base-pair opening, at 20 degrees C, are increased up to 4000-fold, relative to those of normal base pairs. The energetic destabilization of the G.T base pairs is, at least in part, enthalpic in origin. The presence of the G.T mismatched base pairs destabilizes also neighboring base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparative properties of hydroquinone and hydroxylamine reduction of the Ca(2+)-stabilized O2-evolving complex of photosystem II: reductant-dependent Mn2+ formation and activity inhibition. Calcium binding to photosystem II slows NH2OH inhibition of O2 evolution; Mn2+ is retained by the O2-evolving complex [Mei, R., & Yocum, C. F. (1991) Biochemistry 30, 7836-7842]. This Ca(2+)-induced stability has been further characterized using the large reductant hydroquinone. Salt-washed photosystem II membranes reduced by hydroquinone in the presence of Ca2+ retain 80% of steady-state O2 evolution activity and contain about 2 Mn2+/reaction center that can be detected at room temperature by electron paramagnetic resonance. This Mn2+ produces a weak enhancement of H2O proton spin-lattice relaxation rates, cannot be easily extracted by a chelator, and is reincorporated into the O2-evolving complex upon illumination. A comparison of the properties of Ca(2+)-supplemented photosystem II samples reduced by hydroquinone or NH2OH alone or in sequence reveals the presence of a subpopulation of manganese atoms at the active site of H2O oxidation that is not accessible to facile hydroquinone reduction. At least one of these manganese atoms can be readily reduced by NH2OH following a noninhibitory hydroquinone reduction step. Under these conditions, about 3 Mn2+/reaction center are lost and O2 evolution activity is irreversibly inhibited. We interpret the existence of distinct sites of reductant action on manganese as further evidence that the Ca(2+)-binding site in photosystem II participates in regulation of the organization of manganese-binding ligands and the overall structure of the O2-evolving complex.


Pathways of proton release in the bacteriorhodopsin photocycle. The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.


Investigation of the solution conformation of cytochrome c-551 from Pseudomonas stutzeri. 1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri (ATCC 17588). Resonance assignments are proposed for all main-chain and most side-chain protons. Distance constraints were determined on the basis of nuclear Overhauser enhancements between pairs of protons. Dihedral angle constraints were determined from estimates of scaler coupling constants. Twenty-four structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1033 interproton distance and 57 torsion angle constraints. Both the main-chain and side-chain atoms are well defined except for a loop region around residues 34-40, the first two residues at the N-terminus and the last two at the C-terminus, and some side chains located on the molecular surface. The average root mean squared deviation in position for equivalent atoms between the 24 individual structures and the mean structure obtained by averaging their coordinates is 0.54 +/- 0.08 A for the main-chain atoms and 0.97 +/- 0.09 A for all non-hydrogen atoms of residues 3-80 plus the heme group. These structures were compared to the X-ray crystallographic structure of an analogous protein, cytochrome c-551 from Pseudomonas aeruginosa [Matsuura, Takano, & Dickerson (1982) J. Mol. Biol. 156, 389-409). The main-chain folding patterns are very consistent, but there are some differences. The largest difference is in a surface loop segment from residues 34 to 40.


Membrane interactions of the sodium channel S4 segment and its fluorescently-labeled analogues. A 24-amino acid peptide corresponding to the S4 segment of the sodium channel was synthesized. In order to perform fluorescence energy transfer measurements and to monitor the interaction of the peptide with lipid vesicles, the peptide was selectively labeled with fluorescence probes at either its N- or C-terminal amino acids. The fluorescent emission spectra of 7-nitrobenz-2-oxa-1,3-diazol-4- yl-(NBD-)labeled analogues displayed blue shifts upon binding to small unilamellar vesicles (SUV), reflecting the relocation of the fluorescent probe to an environment of increased apolarity. The results revealed that both the N- and C-terminus of the S4 segment are located within the lipid bilayer. Titration of solutions containing NBD-labeled peptides with SUV was used to generate binding isotherms, from which surface partition constants, in the range of 10(4) M-1, were derived. The shape of the binding isotherms as well as fluorescence energy transfer measurements suggest that aggregation of peptide monomers within the membrane readily occurs in acidic but not in zwitterionic vesicles. Furthermore, the results provide good correlation between the incidence of aggregation in PC/PS vesicles and the ability of the peptides to permeate the vesicle's membrane. However, a transmembrane diffusion potential had no detectable effect on the location of the peptide within the lipid bilayer or on its aggregation state.(ABSTRACT TRUNCATED AT 250 WORDS)


Substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus examined using the resolved enantiomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate). The substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus is examined using the resolved optical isomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate), a chromogenic substrate for the phospholipase. The synthetic route employs mild acid-labile protecting groups and separation of the substituted myo-inositol enantiomers as the (-)-camphanyl ester diastereomers. Measurements of the initial rates of cleavage of the D and L enantiomers of the nitrophenyl substrate by phosphatidylinositol-specific phospholipase C from B. cereus show that this enzyme is essentially stereospecific for the D enantiomer. Under identical conditions, the rate of cleavage of the L isomer is less than 0.2% of that observed for the D isomer. The same is observed for the highly homologous enzyme from Bacillus thuringiensis. There is no measurable inhibition by the L enantiomer of the B. cereus enzyme acting on the D enantiomer, even when the molar ratio of L:D is 5, indicating that binding of the L enantiomer to the phospholipase is negligible. Thus, the enzyme active site is exquisitely sensitive to the stereochemistry of the myo-inositol group of the substrate.


DNA helicase E and DNA polymerase epsilon functionally interact for displacement synthesis. A functional interaction between DNA helicase E and DNA polymerase epsilon from calf thymus has been detected which results in the extension of an upstream 3' OH through a downstream primer to the end of a synthetic template. DNA synthesis resulting in full-length extension products was dependent on the addition of DNA helicase E and hydrolysis of ATP, suggesting that displacement of the downstream primer was required. Identical reactions using DNA polymerases alpha and delta in place of DNA polymerase epsilon showed no full-length products dependent on helicase E, indicating that polymerases alpha and delta were incapable of functionally interacting with the helicase. The reaction leading to full-length extension products was time dependent and dependent on the concentration of added polymerase epsilon and helicase E. Exonucleolytic degradation of the downstream primer, or ligation of the downstream primer to the upstream 3' OH, were not responsible for the full-length products observed. Displacement of the downstream primer by DNA helicase E was not affected by the addition of polymerase epsilon to the reactions. Template dilution experiments demonstrated that DNA polymerase epsilon and helicase E were acting in concert to perform displacement synthesis. Additional evidence for functional coordination was obtained by demonstration that DNA helicase E stimulated DNA polymerase epsilon in a standard DNA synthetic assay using dA3000.dT16 as the template-primer. The results presented are consistent with the hypothesis that DNA helicase E and DNA polymerase epsilon are capable of coordinated activities that result in displacement synthesis. A functional interaction of this sort may be involved at the eukaryotic replication fork or in DNA repair.


Minimal kinetic mechanism for misincorporation by DNA polymerase I (Klenow fragment). The minimal kinetic mechanism for misincorporation of a single nucleotide (dATP) into a short DNA primer/template (9/20-mer) by the Klenow fragment of DNA polymerase I [KF(exo+)] has been previously published [Kuchta, R. D., Benkovic, P., & Benkovic, S.J. (1988) Biochemistry 27, 6716-6725]. In this paper are presented refinements to this mechanism. Pre-steady-state measurements of correct nucleotide incorporation (dTTP) in the presence of a single incorrect nucleotide (dATP) with excess KF-(exo+) demonstrated that dATP binds to the KF(exo+)-9/20-mer complex in two steps preceding chemistry. Substitution of (alpha S)dATP for dATP yielded identical two-step binding kinetics, removing nucleotide binding as a cause of the elemental effect on the rate of misincorporation. Pyrophosphate release from the ternary species [KF'(exo+)-9A/20-mer-PPi] was found to occur following a rate-limiting conformational change, with this species partitioning equally to either nucleotide via internal pyrophosphorolysis or to misincorporated product. The rate of 9A/20-mer dissociation from the central ternary complex (KF'-9A/20-mer-PPi) was shown to be negligible relative to exonucleolytic editing. Pyrophosphorolysis of the misincorporated DNA product (9A/20-mer), in conjunction with measurement of the rate of dATP misincorporation, permitted determination of the overall equilibrium constant for dATP misincorporation and provided a value similar to that measured for correct incorporation. A step by step comparison of the polymerization catalyzed by the Klenow fragment for correct and incorrect nucleotide incorporation emphasizes that the major source of the enzyme's replicative fidelity arises from discrimination in the actual chemical step and from increased exonuclease activity on the ternary misincorporated product complex owing to its slower passage through the turnover sequence.


Molecular interactions between human lactotransferrin and the phytohemagglutinin-activated human lymphocyte lactotransferrin receptor lie in two loop-containing regions of the N-terminal domain I of human lactotransferrin. Fluorescein isothiocyanate derivatization of the human lactotransferrin on Lys-264 inhibits the binding of the protein of human PHA-activated lymphocytes [Legrand, D., Mazurier, J., Maes, P., Rochard, E., Montreuil, J., & Spik, G. (1991) Biochem. J. 276, 733-738], indicating that part of the receptor-binding site is located in the N-terminal domain I of lactotransferrin. In the present study, a 6-kDa peptide (residues 4-52) was isolated from the N-terminal lobe of human lactotransferrin which inhibited the binding of the protein to its cell receptor. In addition, lactotransferrin was derivatized using sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD) and sulfosuccinimidyl 6-((4'-azido-2'-nitrophenyl)amino)hexanoate (sulfo-SANPAH), two heterobifunctional reagents generally used for receptor-ligand cross-linking. The azide group of these two reagents was inactivated by photolysis, and only the succinimidyl ester group was allowed to react with lysine residues of the protein. The binding of the derivatized lactotransferrins to the human lymphocyte receptor was assayed. SASD, which binds to Lys-74, was able to inhibit the binding of lactotransferrin to the cell receptor, in contrast to Lys-281-binding sulfo-SANPAH. Molecular modeling showed the position of SASD, sulfo-SANPAH, and fluorescein molecules at the surface of the protein and suggested that SASD and fluorescein could mask residues 4-6 and two loop-containing regions of human lactotransferrin (residues 28-34 and 38-45). The comparison of the primary and tertiary structures of human lactotransferrin and serotransferrin, which bind to specific cell receptors, shows that the above-mentioned regions, which are likely involved in protein-receptor interactions, possess specific structural features.


Preparation and spectroscopic characterization of a coupled binuclear center in cobalt(II)-substituted hemocyanin. A binuclear cobalt derivative of arthropod hemocyanin (Hc) has been prepared by the reaction of apo-Hc with Co(II) in the presence of thiocyanate. The crude product of the reaction contains specifically and adventitiously bound metal, the latter being removable by EDTA treatment. The specifically bound Co(II) constitutes a binuclear metal center that exhibits optical and CD spectra typical in their absorption maxima and extinction coefficients of Co(II) complexes with near-tetrahedral geometry. The EPR spectrum of the binuclear Co(II) derivative contains a resonance at g approximately 13, which is characteristic of integer spin systems and indicates coupled metal ions; the excess Co(II) bound to crude products exhibits an EPR signal at g approximately 4. The time course of derivative formation was followed by EPR, optical and atomic absorption techniques, and by fluorimetry. The intensity of the optical absorption in the visible region due to Co(II) increases with increasing stoichiometry of specifically bound metal [up to 2 Co(II) per protein monomer], but the intensity of the Co(II) EPR signal increases only during the formation of a mononuclear derivative. As the reaction proceeds over approximately 100 h to the formation of the binuclear derivative, the EPR signal intensity decreases to 10% of the value expected for 2 mol of EPR-active Co(II)/mol of protein. The binuclear cobalt derivative cannot be reconstituted to native Hc with Cu(I), indicating the stable loading of Co(II) in the active site. EPR and optical spectroscopic evidence is presented showing that the binuclear derivative does not bind oxygen.


A potential gene target in HIV-1: rationale, selection of a conserved sequence, and determination of NMR distance and torsion angle constraints. Recently, the capability for determining the high-resolution, sequence-dependent structure of oligonucleotides in solution via careful analysis of multidimensional NMR spectra and structure refinement procedures has been developed. Consequently, the rationale for selection of a genome sequence as a target for drug design based on the detailed three-dimensional structure of the target is presented. The concept is illustrated by the successful search for a highly conserved region of the HIV-1 genome's long terminal repeat which could serve as a molecular target. A compound which could selectively bind the target sequence could inhibit both RNA transcription from the integrated provirus and the reverse transcription process. Of 148 unique HIV-1 sequences examined, 147 exhibit a 21-base conserved sequence (nucleotides 70-90 in HIVHXB2R) in the R region of the long terminal repeat. The only exception, a minor constituent for one individual, has a change in the penultimate base. A 13 base pair duplex sequence, [d(AGCTTGCCTTGAG).d(CTCAAGGCAAGCT)], from this conserved region was selected and synthesized for NMR structure studies. Phase-sensitive proton two-dimensional nuclear Overhauser enhancement (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra were obtained at 500 MHz for the DNA duplex. Exchangeable and nonexchangeable proton resonances were assigned. Quantitative assessments of the 2D NOE cross-peak intensities for different mixing times were carried out using conventional Fourier transform NMR and the maximum likelihood method (MLM). Distance constraints, along with upper and lower bounds, were obtained from the 2D NOE intensities using the iterative complete relaxation matrix algorithm MARDIGRAS. Distances entailing both exchangeable and nonexchangeable protons were determined: 7-11 experimental distance constraints per residue including interresidue and interstrand distances. Simulations of the scalar coupling effects manifest in 2QF-COSY cross-peaks by means of the program SPHINX/LINSHA were compared with experimental data to yield torsion angle constraints for the sugar rings. A single conformer was inadequate to describe any of the sugar puckers, but a rapid two-state equilibrium with one conformer strongly dominant (75-95%) provided a good fit of the 2QF-COSY cross-peaks. The sugar pucker of the major conformer exhibited significant variability for the various nucleotides but was roughly 2'-endo. Though derived independently and subject to different time-averaging effects, the 2QF-COSY and 2D NOE results are in accord.


Magnetic circular dichroism study of cytochrome ba3 from Thermus thermophilus: spectral contributions from cytochromes b and a3 and nanosecond spectroscopy of CO photodissociation intermediates. Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdóttir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.


An application of 3D-QSAR to the analysis of the sequence specificity of DNA alkylation by uracil mustard. The sequence specificity of DNA alkylation by uracil mustard was examined using a novel three-dimensional QSAR method known as HASL, or the hypothetical active site lattice. The structures of a variety of 4-mer sequences obtained from pBR322 and SV40 were related to their degree of guanine-N7 alkylation by uracil mustard. The resulting correlations were found to point to a significant contribution from bases on the 3' side of the target guanine nucleotide. The HASL models derived from the analysis of 52 guanine-containing 4-mer sequences were used to highlight those atomic features in the favored TGCC sequence that were found most important in determining specificity. It was found that the NH2-O systems present in the two GC base pairs on the 3' side of the target guanine were significantly correlated to the degree of alkylation by uracil mustard. This finding is consistent with a prealkylation binding event occurring between these sites along the major groove and the uracil mustard O2/O4 system.


Inhibition of chymotrypsin by fluorinated alpha-keto acid derivatives. A series of fluorinated alpha-keto acid derivatives [PhCHFCOCO2R,PhCH2CHFCOCO2R,PhCF2-COCO2R, and PhCH2CF2COCO2R (R = H, Me, and Et)] was synthesized. They were inhibitors of chymotrypsin, with Ki values ranging from 4700 to 15 microM. Benzylpyruvic derivatives were generally more potent than the corresponding phenylpyruvic analogs. Esters of the first series were also more potent than their corresponding acids, and potency increased with the number of fluorine atoms. By replacing the ethoxy group of PhCH2CF2COCO2Et (15b) with an amino acid chain (i.e., alanyl-leucyl-arginine methyl ester hydrochloride and alanyl-leucyl-valine ethyl ester), the resultant peptides PhCH2CF2COCO-Ala-Leu-Arg-OMe.HCl.H2O (20) and PhCH2CF2COCO-Ala-Leu-Val-OEt.H2O (23) were found to be slow-binding inhibitors of chymotrypsin with considerably lower Ki values (0.19 and 3.6 microM, respectively). 19F NMR studies indicate, in the case of 20, the presence of an enzyme-inhibitor complex with a hemiketal structure similar to those observed between trifluoromethyl ketones and chymotrypsin. The results illustrate that effective protease inhibitors can be designed by enhancing the electrophilic character of the reactive carbonyl group (with an electron-withdrawing group placed on each side of the carbonyl group). Their potency and/or selectivity can also be improved by taking advantage of binding interactions at S' subsites of the protease.


Membrane stimulation of cGMP phosphodiesterase activation by transducin: comparison of phospholipid bilayers to rod outer segment membranes. To clarify the role of phospholipids in G protein-effector interactions of vertebrate phototransduction, transducin activation of cGMP phosphodiesterase (PDE) has been reconstituted on the surface of well-defined phosphatidylcholine (PC) vesicles, using purified proteins from bovine rod outer segments (ROS). PC vesicles enhanced PDE stimulation by the GTP-gamma S-bound transducin alpha subunit (T alpha-GTP gamma S) as much as 17-fold over activation in the absence of membranes. In the presence of 3.5 microM accessible PC in the form of large (100 nm) unilamellar vesicles, 500 nM T alpha-GTP gamma S stimulated PDE activity to more than 70% of the maximum activity induced by trypsin. Activation required PC, PDE, and T alpha-GTP gamma S, but did not require prior incubation of any of the components, and occurred within 4 s of mixing. The PC vesicles were somewhat more efficient than urea-washed ROS membranes in enhancing PDE activation. Half-maximal activation occurred at accessible phospholipid concentrations of 3.8 microM for PC vesicles, and 13 microM for ROS membranes. Titrations of PDE with T alpha-GTP gamma S in the presence of membranes indicated a high-affinity (Kact less than 250 pM) activation of PDE by a small fraction (0.5-5%) of active T alpha-GTP gamma S, as did titrations of ROS with GTP gamma S. When activation by PC vesicles was compared to PDE binding to membranes, the results were consistent with activation enhancement resulting from formation of a T alpha-GTP gamma S-dependent PDE-membrane complex with half-maximal binding at phospholipid concentrations in the micromolar range. The value of the apparent dissociation constant, KPL, associated with the activation enhancement was estimated to be in the range of 2.5 nM (assuming an upper limit value of 1600 phospholipids/site) to 80 nM (for a lower limit value of 50 phospholipids/site). Another component of membrane binding was more than 100-fold weaker and was not correlated with activation by T alpha-GTP gamma S. Low ionic strength disrupted the ability of ROS membranes, but not PC vesicles, to bind and activate PDE. Removal of PDE's membrane-binding domain by limited trypsin digestion eliminated both the binding of PDE to vesicles and the ability of PDE to be activated by T alpha-GTP gamma S and membranes. These results suggest that ROS membrane stimulation of PDE activation by T alpha-GTP gamma S is due almost exclusively to the phospholipids in the disk membrane.


Functional characterization of human hepatocyte growth factor mutants obtained by deletion of structural domains. Human hepatocyte growth factor (hHGF) consists of characteristic structural domains. In this study, we prepared mutant proteins lacking each of these domains and examined their biological activities for stimulation of hepatocyte DNA synthesis, inhibition of Meth A cell growth, and induction of MDCK cell dissociation. We also examined their interactions with the c-met/HGF receptor by competition analysis and by analysis of levels of tyrosine phosphorylation. The mutant proteins lacking the N-terminal, the first kringle, or the second kringle domain were not biologically effective and could not compete with hHGF bound to the c-met/HGF receptor. The results indicate that these domains are necessary for the biological activities of hHGF mediated by binding to the c-met/HGF receptor. The mutant proteins lacking the third or fourth kringle domain moderately retained biological activities and the receptor binding. The relative levels of the tyrosine phosphorylation of the c-met/HGF receptor by these mutant proteins correlated well with the relative potencies of the biological activities when compared with that of the wild-type hHGF. The mutant protein lacking the light chain was not effective in the biological activities and tyrosine phosphorylation of the c-met/HGF receptor, but competed with hHGF bound to the c-met/HGF receptor. These results suggest that the heavy chain plays an important role in the interaction of hHGF with the c-met/HGF receptor and that the light chain is further required for the tyrosine phosphorylation of the c-met/HGF receptor.


Sequential 1H NMR assignments and secondary structure of the kringle domain from urokinase. The sequence-specific 1H NMR assignments of the 89-residue recombinant kringle domain from human urokinase are presented. These were achieved primarily by utilizing TOCSY and NOESY spectra in conjunction with COSY spectra recorded at 500 MHz and 600 MHz. Regular secondary structure elements have been derived from a qualitative interpretation of nuclear Overhauser enhancement, JNH alpha coupling constant, and amide proton exchange data. Two helices have been identified. One helix, involving Ser40-Gly46, corresponds to that reported for t-PA kringle 2 (Byeon et al., 1991), but does not exist in other kringles with known structures. The second helix, in the region Asn26-Gln33, is thus far unique to the urokinase kringle. Three antiparallel beta-sheets and three tight turns have also been identified, which correspond exactly to those identified in t-PA kringle 2 both in solution and in the crystalline state (de Vos et al., 1992). Despite the very different ligand binding properties of the urokinase kringle, NOE data indicate that the tertiary fold of the molecule conforms closely to that found for other kringles.


Solution conformation of tuftsin.

Solution conformation of tuftsin. Tuftsin, a natural linear tetrapeptide (Thr-Lys-Pro-Arg) of potential antitumor activity, has been studied in DMSO-d6 solution by 2D NMR spectroscopy. 1H and 13C spectra show the presence of two families of conformations characterized by a trans or cis Lys-Pro bond, respectively. The family of conformers containing the cis peptide bond is a mixture of extended structures as expected for a short linear peptide. On the contrary, the trans isomer appears to be a rigid, folded conformer, as indicated by crucial NOEs and by the exceptionally low temperature coefficient of Arg NH. Analysis of the solution data by means of energy calculations leads to a unique structure, characterized by a Lys-Pro inverse gamma-turn.


NMR structural studies of a 15-mer DNA sequence from a ras protooncogene, modified at the first base of codon 61 with the carcinogen 4-aminobiphenyl. Proton NMR studies were conducted on the complementary 15-mer duplex d(5'-TACTCTTCTTGACCT).(5'-AGGTCAAGAAGAGTA) (designated as unmodified 15-mer duplex) spanning a portion of the mouse c-Ha-ras protooncogene centered around codon 61. Identical studies were carried out on the same sequence, after specific modification with a reactive derivative of the carcinogen 4-aminobiphenyl (ABP), which resulted in incorporation of a single N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) adduct in the noncoding strand (designated as ABP-modified 15-mer duplex). The adduct was located at the position corresponding to the first base of codon 61. The NMR data for the unmodified 15-mer duplex were fully consistent with a standard right-handed B-type DNA duplex conformation, with the possible exception of the frayed terminal base pairs. The ABP-modified 15-mer duplex was found to adopt one major conformation, although at least one additional conformation could be detected especially near room temperature. The major form, which exhibited strikingly similar NOE patterns as to those of the parent oligomer, both in H2O and D2O spectra, assumed a standard Watson-Crick base pairing throughout the entire length of the duplex, including the modification site and its flanking base pairs. Although some local perturbation of the helix could be detected in the vicinity of the modified guanosine, the NOE distance constraints established that the helix was globally right-handed and that the glycosidic torsion angles had the normal anti orientation, both at the modified base and its partner cytidine. Furthermore, the absence of strong NOE interactions between protons in the ABP moiety, which was rapidly rotating, and the nucleic acid protons was consistent with positioning of the arylamine moiety in the major groove of a weakly distorted double-helical structure. Although insufficient data prevented a detailed characterization of the minor conformer(s), the observation of significant shieldings for all the arylamine protons indicated a different orientation at the modified site in the minor contributor(s), possibly with extensive stacking between the ABP fragment and the neighboring bases.


Electron paramagnetic resonance studies on the high-salt form of bovine spleen purple acid phosphatase. The EPR spectra of the high-salt form of reduced bovine spleen purple acid phosphatase (BSPAPr) and its complexes with inhibitory tetrahedral oxyanions, AMP, and fluorine have been examined in the 4-30 K temperature range. The EPR spectrum of the high-salt form of BAPAPr is identical to that previously reported for the low-salt form (Averill et al. (1987) J. Am. Chem. Soc. 109, 3760-3767), indicating that the substantial differences in conformation of the two forms result in undetectable alterations in the electronic structure of the binuclear iron center. Phosphate, AMP, and arsenate all result in broadened, highly anisotropic EPR spectra with decreased values of the antiferromagnetic coupling constant, -2J, while molybdate and tungstate produce a sharp axial or slightly rhombic spectrum, respectively, and fluoride produces an anomalous spectrum with an inverted g-tensor. These results are consistent with binding of the two classes of oxyanions (and AMP) to distinct sites at or near the binuclear iron center, while fluoride binds in yet a third mode. EPR spectra of the BSPAPr complex with molybdate show altered relaxation behavior in the presence of phosphate, consistent with a 50% decrease in the magnitude of -2J, suggesting that phosphate binds to the molybdate complex to produce a ternary complex analogous to that proposed for molybdate inhibition on the basis of kinetics studies.


Binding of influenza virus hemagglutinin to analogs of its cell-surface receptor, sialic acid: analysis by proton nuclear magnetic resonance spectroscopy and X-ray crystallography. The interaction between influenza virus hemagglutinin and its cell-surface receptor, 5-N-acetylneuraminic acid (sialic acid), was probed by the synthesis of 12 sialic acid analogs, including derivatives at the 2-carboxylate, 5-acetamido, 4-, 7-, and 9-hydroxyl, and glycosidic positions. The equilibrium dissociation constants of these analogs were determined by nuclear magnetic resonance spectroscopy. Ligand modifications that reduced or abolished binding included the replacement of the 2-carboxylate with a carboxamide, the substitution of azido or N-benzyloxycarbonyl groups for the 5-acetamido group, and the replacement of the 9-hydroxyl with amino or O-acetyl moieties. Modifications having little effect on binding included the introduction of longer chains at the 4-hydroxyl position, the replacement of the acetamido methyl group with an ethyl group, and the removal of the 7-hydroxyl group. X-ray diffraction studies yielded 3 A resolution crystal structures of hemagglutinin in complex with four of the synthetic analogs [alpha-2-O-methyl-, 4-O-acetyl-alpha-2-O-methyl-, 9-amino-9-deoxy-alpha-2-O-methyl-, and alpha-2-O-(4'-benzylamidocarboxybutyl)-N-acetylneuraminic acid] and with the naturally occurring cell-surface saccharide (alpha 2-3)sialyllactose. The X-ray studies unambiguously establish the position and orientation of bound sialic acid, indicate the position of the lactose group of (alpha 2-3)sialyllactose, and suggest the location of an alpha-glycosidic chain (4'-benzylamidocarboxybutyl) that increases the binding affinity of sialic acid by a factor of about 3. Although the protein complexed with alpha-2-O-methylsialic acid contains the mutation Gly-135-->Arg near the ligand binding site, the mutation apparently does not affect the ligand's position. The X-ray studies allow us to interpret the binding affinities in terms of the crystallographic structure. The results suggest further experiments which could lead to the design of tight binding inhibitors of possible therapeutic value.


Energy coupling in DNA gyrase: a thermodynamic limit to the extent of DNA supercoiling. ATP alpha S (Rp) has been shown to support the supercoiling of plasmid pBR322 catalyzed by Escherichia coli DNA gyrase at comparable rates to the natural substrate ATP and is able to promote the introduction of one more superhelical turn than ATP. The difference in free energy change between consecutive rounds of supercoiling in gyrase-mediated reactions is calculated to be 2.6 kJ mol-1. The difference in free energy of hydrolysis of ATP and ATP alpha S (Rp) has been determined from the difference in the equilibrium constants for the phosphorylation of arginine established by arginine kinase. This equilibrium constant has been found to be displaced by a factor of about 1.5, corresponding to a greater free energy of hydrolysis of ATP alpha S (Rp) compared to ATP of approximately 1 kJ mol-1. This difference in free energy can be tentatively ascribed to a relative destabilization of the MgATP alpha S (Rp) complex with respect to MgATP. Assuming that the stoichiometry of the coupled reactions requires two ATPs hydrolyzed per round of supercoiling, ATP alpha S (Rp) should be capable of providing an additional ca. 2 kJ mol-1 of free energy for DNA supercoiling, which is in good agreement with estimates for the additional free energy required to achieve a further round of supercoiling. These results provide direct evidence to support the proposal that the extent of DNA supercoiling by DNA gyrase is limited by the free energy of hydrolysis of the nucleotide.


Transcription activation by cAMP receptor protein (CRP) at the Escherichia coli gal P1 promoter. Crucial role for the spacing between the CRP binding site and the -10 region. The cAMP-CRP complex activates the initiation of transcription at the Escherichia coli gal P1 promoter, and the activation efficiency is highly sensitive to the location of the complex on this promoter region. Moving the CRP binding site by one base pair toward the start of transcription significantly decreases the extent of activation in vivo and actually turns the cAMP-CRP complex into an inhibitor in in vitro experiments. A structural analysis of open complexes formed on the two promoter fragments at 37 degrees C has revealed three elements crucial for an optimal activation process: a strong upstream anchorage of RNA polymerase, a cooperative binding of CRP and RNA polymerase, and an accurate orientation of the two promoter regions located upstream and downstream of the CRP binding site. Furthermore, structural analysis of polymerase promoter complexes at lower temperatures suggests that RNA polymerase initially recognizes the upstream region of the gal P1 promoter and subsequently interacts with sequences from the -10 to +20 region to yield the final open complex structure. The involvement of CRP in these sequential events has been examined.


Redox reactions of apo mammalian ferritin. Apo horse spleen ferritin undergoes a 6.3 +/- 0.5 electron redox reaction at -310 mV at pH 6.0-8.5 and 25 degrees C to form reduced apoferritin (apoMFred). Reconstituted ferritin containing up to 50 ferric ions undergoes reduction at the same potential, taking up one electron per ferric ion and six additional electrons by the protein. We propose that apo mammalian ferritin (apoMF) contains six redox centers that can be fully oxidized forming oxidized apoferritin (apoMFox) or fully reduced forming apoMFred. ApoMFred can be prepared conveniently by dithionite or methyl viologen reduction. ApoMFred is slowly oxidized by molecular oxygen but more rapidly by Fe(CN)6(3-) to apoMFox. Fe(III)-cytochrome c readily oxidizes apoMFred to apoMFox with a stoichiometry of 6 Fe(III)-cytochrome c per apoMFred, demonstrating a rapid interprotein electron-transfer reaction. Both redox states of apoMF react with added Fe3+ and Fe2+. Addition of eight Fe2+ to apoMFox under anaerobic conditions produced apoMFred and Fe3+, as evidenced by the presence of a strong g = 4.3 EPR signal. Subsequent addition of bipyridyl produced at least six Fe(bipyd)3(2+) per MF, establishing the reversibility of this internal electron-transfer process between the redox centers of apoMF and bound iron. Incubation of apoMFred with the Fe(3+)-ATP complex under anaerobic conditions resulted in the formation and binding of two Fe2+ and four Fe3+ by the protein. The various redox states formed by the binding of Fe2+ and Fe3+ to apoMFox and apoMFred are proposed and discussed. The yellow color of apoMF appears to be an integral characteristic of the apoMF and is possibly associated with its redox activity.


A photoactivatable naltrexone derivative labels glycoproteins of different molecular weight corresponding to the mu- and kappa-opioid receptors. The synthesis and characterization of a novel opioid receptor photoaffinity probe [3H]naltrexyl urea phenylazido derivative ([3H]NUPA) is described. In the absence of light, [3H]NUPA binds with high affinity in a reversible and saturable manner to rat brain and guinea pig cerebellum membranes. Dissociation constants and binding capacities (Scatchard plots) are 0.11 nM and 250 fmol/mg of protein for rat brain and 0.24 nM and 135 fmol/mg of protein for guinea pig cerebellum. Competition experiments indicate that this ligand interacts with high affinity at both mu- and kappa-opioid binding sites while exhibiting low affinity at delta sites (Ki = 21 nM). On irradiation, [3H]NUPA incorporates irreversibly into rat brain and guinea pig cerebellum membranes. SDS gel electrophoresis of rat brain membranes reveals specific photolabeling of a 67-kDa molecular mass band. Conversely, a major component of 58 kDa and a minor component of 36 kDa are obtained from [3H]NUPA-labeled guinea pig cerebellum membranes. Different photolabeling patterns are obtained in rat brain (mu/delta/kappa, 4/5/1) and guinea pig cerebellum (mu+delta/kappa, 1,5/8,5) membranes in the presence of selective opioid ligands indicating labeling of mu and kappa sites, respectively. Thus, [3H]NUPA behaves as an efficient photoaffinity probe of mu- and kappa-opioid receptors, which are probably represented by distinct glycoproteins of 67 and 58 kDa, respectively.


Kinetics of electron transfer between cytochrome c and laccase. Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting Rhus vernicifera laccase as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 125 M-1 s-1 at 25 degrees C in 0.2 M phosphate buffer at pH 6.0 with the activation parameters delta H++ = 16.2 kJ mol-1 and delta S++ = 28.9 J mol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that electron transfer from cytochrome c to laccase is favored by the local electrostatic interaction (ZAZB = -0.9 at pH 6 and -1.3 at pH 4.8) between the basic proteins with positive net charges. From the increase of the rate of electron transfer with decreasing pH, one of the driving forces of the reaction was suggested to be the difference in the redox potentials between the type 1 copper in laccase and the central iron in cytochrome c. Further, on addition of one hexametaphosphate anion per cytochrome c molecule, the rate of the electron transfer was increased, probably because the association of both proteins became more favorable.


Difference FT-IR study of a novel biochemical preparation of photosystem II. There are two redox-active tyrosines in photosystem II, the water-splitting complex, that form neutral tyrosine radicals. One of these tyrosine radicals, D., is stable and has an unknown function. The other redox-active tyrosine, Z, acts to transfer oxidizing equivalents from the primary chlorophyll donor of photosystem II to the manganese cluster, where water oxidation occurs. In an attempt to obtain more information about Z and its interaction with its environment, we have begun a study using Fourier-transform infrared (FT-IR) vibrational spectroscopy. To facilitate these studies, we have developed a procedure to isolate spinach photosystem II complexes with an antenna size of approximately 100-110 chlorophylls per reaction center. These complexes show an approximately 2-fold increase in the specific activity of oxygen evolution over the activity of the starting material, photosystem II membranes. Although fully solubilized in detergent, these complexes retain the 24- and 18-kDa extrinsic proteins and exhibit no calcium chloride requirement for optimal oxygen evolution. In manganese-depleted photosystem II samples, Z. can be accumulated in the light. In the dark, the tyrosine radical is reduced and reprotonated to form the neutral tyrosine. Since this process is reversible and light-dependent, we have used light-minus-dark difference FT-IR spectroscopy to observe the vibrational difference spectrum that is associated with the oxidation of this residue. As a control, EPR spectra were measured under identical conditions to assess the amount of Z. that accumulated in the light. We also hope to use difference FT-IR to identify the amino acid with which Z may form a hydrogen bond.(ABSTRACT TRUNCATED AT 250 WORDS)


1H NMR investigation of manganese peroxidase from Phanerochaete chrysosporium. A comparison with other peroxidases. 1H NMR spectra at 200- and 600-MHz of manganese peroxidase from Phanerochaete chrysosporium and of its cyanide derivative are reported. The spectrum of the native protein is very similar to that of other peroxidases. The assignment of the spectrum of the cyanide derivative has been performed through 1D NOE, 2D NOESY, and COSY experiments. This protein is very similar to lignin peroxidase, the only meaningful difference being the shift of H delta 2 of the proximal histidine. The spectra of the cyanide derivative of these two proteins are compared with those of horseradish peroxidase and cytochrome c peroxidase. The shift pattern of the protons of the proximal histidine is discussed relative to the structural properties which affect the Fe3+/Fe2+ redox potential.


Optical and resonance Raman spectroscopy of the heme groups of the quinol-oxidizing cytochrome aa3 of Bacillus subtilis. The cytochrome aa3-type terminal quinol oxidase of Bacillus subtilis catalyzes the four-electron reduction of dioxygen to water. It resembles the aa3-type cytochrome-c oxidase in using heme A as its active-site chromophores but lacks the CuA center and the cytochrome-c oxidizing activity of the mitochondrial enzyme. We have used optical and resonance Raman spectroscopies to study the B. subtilis oxidase in detail. The alpha-band absorption maximum of the reduced minus oxidized enzyme is shifted by 5-7 nm to the blue relative to most other aa3-type oxidases, and accordingly, we designate the Bacillus enzyme as cytochrome aa3-600. The shifted optical spectrum cannot be ascribed to an alteration in the strength of the hydrogen bond between the formyl group of the low-spin heme and its environment, as the Raman line assigned to this mode in aa3-600 has the same frequency and degree of resonance enhancement as the low-spin heme a formyl mode in most other aa3-type oxidases. Raman modes arise at 194 and 214 cm-1 in aa3-600, whereas a single band at about 214 cm-1 is assigned to the iron-histidine stretch for the other aa3-type oxidases. Possible explanations for the occurrence of these two modes are discussed. Comparison of formyl and vinyl modes and heme skeletal vibrational modes in different oxidation states of aa3-600 and of beef heart cytochrome-c oxidase shows a strong similarity, which suggests conservation of essential features of the heme environments in these oxidases.


Interaction of gelsolin with covalently cross-linked actin dimer. One of the two actin molecules in the ternary actin-gelsolin complex was selectively cross-linked to gelsolin when benzophenonemaleimide-actin (BPM-actin) was used [Doi, Y., Banba, M., & Vertut-Doi (1991a) Biochemistry 30, 5769-5777]. Here, we examine the interaction between gelsolin and BPM-actin dimer in which BPM-actin is covalently conjugated to unlabeled actin by p-phenylenedimaleimide (pPDM). BPM-actin dimer having an apparent molecular mass of 115 kDa is photo-cross-linked to gelsolin (90 kDa) more effectively than BPM-actin monomer in the presence of Ca2+, forming a cross-linked actin dimer-gelsolin (1:1) complex with a molecular mass of 210 kDa. The tight direct association of the dimer to gelsolin is shown by the titration of gelsolin with the fluorescently labeled dimer and by the higher concentration of phosphatidylinositol 4,5-bisphosphate required to inhibit the formation of BPM-dimer complex with gelsolin than that of BPM-monomer complex. However, an attempt to cross-link the two actin molecules in the ternary actin-gelsolin (2:1) complex by pPDM fails. The results argue that the topography of the two actin molecules in the actin-gelsolin (2:1) complex is similar, but not identical, to that of the barbed end of an actin filament.


Role of viral envelope sialic acid in membrane fusion mediated by the vesicular stomatitis virus envelope glycoprotein. Fusion of vesicular stomatitis virus (VSV) with cells and liposomes before and after treatment with neuraminidase was studied using the R18 dequenching assay. Desialylation of VSV significantly enhanced the extent of fusion with Vero cells but affected neither the pH dependence nor the binding of VSV to Vero cells. The enhanced fusion of asialo-VSV was observed both at the plasma membrane as well as via the endocytic pathway. Both VSV and asialo-VSV fused with liposomes made of neutral phospholipid, but only asialo-VSV fused with liposomes containing a 1:1 mixture of neutral and negatively charged phospholipid. To examine factors which contribute to the extent of fusion, we analyzed the various activation and inactivation reactions that take place as a result of low-pH triggering of VSV prebound to the target membrane. Lag times for the onset of fusion were similar for VSV and asialo-VSV, indicating that desialylation did not affect the activation reactions. However, exposure of VSV bound to target membranes at pH 6.5 for 400 s led to considerable inactivation, whereas little inactivation was seen after desialylation of VSV. These results are analyzed in terms of a model which allows us to determine which components of the overall fusion process are dominated by viral envelope sialic acid.


Lipoprotein lipase expression in undifferentiated hepatoma cells is regulated by progesterone and protein kinase A. Recently, it was shown that lipoprotein lipase (LPL) was produced in neonatal but not in adult rat liver. In an attempt to further define the mechanism involved in liver LPL expression, we identified a neonatal mouse hepatoma cell line, BWTG3, capable of producing LPL. The regulation of LPL expression by various extracellular stimuli was investigated in this cell line. Progesterone caused a rise in LPL production by BWTG3 cells. Other hormones tested, such as insulin, glucagon, adrenalin, testosterone, and thyroid hormone, had no effect on LPL production. The effects of progesterone on LPL production showed slow kinetics reaching a maximum 24 h after addition. Cotransfection of a progesterone receptor expression vector with a 5'-LPL-CAT reporter construct resulted in an induction of CAT activity, suggesting that the increase in LPL accumulation after progesterone was linked to transcriptional induction of the LPL gene. Stimuli causing an elevation of protein kinase A activity in the cells also increased LPL production. Three agents capable of elevating intracellular cAMP levels, i.e., forskolin, dBcAMP, and choleratoxin, caused an elevation of LPL production. The increase in LPL activity caused by forskolin and choleratoxin was paralleled by an elevation of LPL mRNA levels, while dBcAMP only induced a small elevation of LPL mRNA levels. The increase in LPL production was shown to be linked to the stimulation of the PKA signal transduction pathway and was apparently transmitted via the transcription factor CREB. No effect of the stimulation of protein kinase C or calcium/calmodulin-dependent kinase on LPL production was detected.(ABSTRACT TRUNCATED AT 250 WORDS)


Cytochrome c interactions with cardiolipin in bilayers: a multinuclear magic-angle spinning NMR study. The influence of cytochrome c binding to cardiolipin bilayers on the motional characteristics of each component has been analyzed by magic-angle spinning (MAS) NMR. Observations were made by NMR of natural abundance 31P, 13C, and 1H nuclei in the lipid as well as sites enriched with 13C in the protein. Analysis of methyl carbons enriched in ([epsilon-13CH3]methionine)cytochrome c at residues 65 and 80 reveal quite different behavior for these sites when the protein was bound at a 1:15 molar ratio with hydrated cardiolipin. Cross-polarization (CP) shows a single broad resonance downfield in the methyl region which corresponds to the spectral characteristics of methionine 65 in the solution protein when subjected to moderate thermal perturbations. These observations suggest that although methionine 65 remains motionally restricted when the protein binds to the lipid bilayers, this residue becomes less shielded and exposed to more chemically distinct environments than in the native state of the protein. In contrast to its behavior in native oxidized protein, the methionine 80 methyl could be detected following direct pi/2 pulse excitation, and this residue is assumed to be released from the axial ligand site on the heme iron to become more exposed and highly mobile in the protein-lipid complex. An analysis of the CP response for natural abundance 13C nuclei in the lipid reveals a general increase in motions with slower rates (tens of kilohertz) on binding with cytochrome c, except for sites within the region of fatty acyl chain unsaturation which appear to be selectively mobilized in the complex with protein. It is concluded that, aside from effects on the unsaturated segments, the bound protein induces new modes of slow motions in the lipid assemblies rather than restricting the overall reorientation freedom of the lipid. The strong paramagnetic effects observed previously on the relaxation of phosphorus in protein-bound lipid [Spooner, P.J.R., & Watts, A. (1991) Biochemistry 30, 3880-3885] were not extended to any carbon and proton sites observable by MAS NMR in the lipid, and this infers a specific interaction of lipid phosphate groups with the heme. However, when protein was bound to cardiolipin mixed at a 1:4 mole ratio with dioleoylphosphatidylcholine in bilayers, no direct interaction with the heme was apparent from the phosphorus NMR relaxation behavior in this component, resolved by MAS. Instead, the spectral anisotropy of cardiolipin phosphorus was determined to be reduced, indicating that, on binding with cytochrome c, the headgroup organization was perturbed in this component.(ABSTRACT TRUNCATED AT 400 WORDS)


Folding topology of the disulfide-bonded dimeric DNA-binding domain of the myogenic determination factor MyoD. The myogenic determination factor MyoD is a member of the basic-helix-loop-helix (bHLH) protein family. A 68-residue fragment of MyoD encompassing the entire bHLH region (MyoD-bHLH) is sufficient for protein dimerization, sequence-specific DNA binding in vitro, and conversion of fibroblasts into muscle cells. The circular dichroism spectrum of MyoD-bHLH indicates the presence of significant alpha-helical secondary structure; however, the NMR spectrum lacks features of a well-defined tertiary structure. There is a naturally occurring cysteine at residue 135 in mouse MyoD that when oxidized to a disulfide induces MyoD-bHLH to form a symmetric homodimer with a defined tertiary structure as judged by sedimentation equilibrium ultracentrifugation and NMR spectroscopy. Oxidized MyoD-bHLH retains sequence-specific DNA-binding activity, albeit with an apparent 100-1000-fold decrease in affinity. Here, we report the structural characterization of the oxidized MyoD-bHLH homodimer by NMR spectroscopy. Our findings indicate that the basic region is unstructured and flexible, while the HLH region consists of two alpha-helices of unequal length connected by an as yet undetermined loop structure. Qualitative examination of interhelical NOEs suggests several potential arrangements for the two helix 1/helix 2 pairs in the symmetric oxidized dimer. These arrangements were evaluated for whether they could incorporate the disulfide bond, satisfy loop length constraints, and juxtapose the two basic regions. Only a model that aligns helix 1 parallel to helix 1' and antiparallel to helix 2 was consistent with all constraints. Thus, an antiparallel four-helix bundle topology is proposed for the symmetric dimer. This topology is hypothesized to serve as a general model for other bHLH protein domains.


Kinetic mechanism of the adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit in the direction of magnesium adenosine 5'-diphosphate phosphorylation. In order to define the overall kinetic mechanism of adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit and also to elaborate the kinetic mechanism in the direction of peptide phosphorylation, we have determined its kinetic mechanism in the direction of MgADP phosphorylation. Studies of initial velocity as a function of uncomplexed Mg2+ (Mgf) in the absence and presence of dead-end inhibitors were used to define the kinetic mechanism. Data are consistent with the overall kinetic mechanism in the direction of MgADP phosphorylation being random with both the pathways allowed, i.e., the pathway in which MgADP binds to the enzyme prior to phosphorylated peptide and the pathway in which phosphorylated peptide binds to enzyme prior to MgADP. In addition, depending on the concentration of Mgf, one or the other pathway predominates. At low (0.5 mM) Mgf, the mechanism is steady-state ordered with the pathway in which phosphorylated peptide binds first being preferred; at high (10 mM) Mgf, the kinetic mechanism is equilibrium ordered, and the pathway in which MgADP binds first is preferred. This change in mechanism to equilibrium ordered at higher concentration of Mgf is due to an increase in affinity of the enzyme for MgADP and a decrease in affinity for the phosphorylated peptide. The Haldane relationship gives a Keq of 2 +/- 1 x 10(3) at pH 7.2, in agreement with the values obtained from 31P NMR (1.6 +/- 0.8 x 10(3)) and direct determination of reactant concentrations at equilibrium (3.5 +/- 0.6 x 10(3)).


Proton transfer in catalysis by fumarase. Using 3T[14C]malate it was possible to show intermolecular T-transfer to unlabeled fumarate. The rate of dissociation of ET derived from the malate was not rapid, only about as fast as required for KMcat. Because of the slow dissociation of ET derived from T-malate, the awkward complex ET-malate is readily formed. As shown by the effect of added malate on the partition of ET, otherwise captured by fumarate, ET.malate must be functional. Its rate of dissociation to E.M determines the V/Km value of malate. Hydrogen dissociation of the complex ET.F was linearly related to the concentration and basicity of the buffer provided, varying from < 10% to > 60% of the overall rate with alkyl phosphonates. Partition of EH.F to free malate or fumarate occurs in a ratio approximately 2:1 at both low and high buffer. This agrees well with the comparison of the equilibrium exchange rates: malate with [18O]water to malate with [14C]-fumarate [Hansen, J.N., Dinovo, E.C., & Boyer, P.D. (1969) J. Biol. Chem. 244, 6270-6279]. Therefore, the abstracted hydroxyl group is fully exchanged from the enzyme when the bound hydrogen and fumarate return to malate and must be much more accessible to the medium than the abstracted proton. The fact that buffer increases the rate of proton transfer to the medium in the central complex makes it appear that a proton relay connects the active site donor with a remote site that interfaces with the ultimate proton source, water.


In bacterial reaction centers protons can diffuse to the secondary quinone by alternative pathways. The mechanisms of proton conduction to the reduced secondary quinone in bacterial reaction centers were studied in wild-type and genetically modified reaction centers from Rhodobacter capsulatus. In the L212-213AA double mutant (L212Glu----Ala, L213Asp----Ala), reaction center function is severely altered. However, a photocompetent revertant of this strain which carries a third 'compensating' mutation, M231Arg----Leu, at about 15 A from the secondary quinone, displays the normal proton binding function of the reaction center. Furthermore, the apparent pK values of group(s) involved in the stabilization of the semiquinone anion are restored by that mutation. We conclude that L212Glu and L213Asp are not obligatory residues for proton donation to QB in Rb. capsulatus. We suggest that protons can be delivered to the QB site from the cytoplasm via a network of proton channels activated by compensatory mutations, possibly involving water molecules bound in the interior of the reaction center.


H+/glycyl-glycine cotransport in eel intestinal brush-border membrane vesicles: studies with the pH-sensitive dye Acridine orange. Monitoring the fluorescence quenching of the pH-sensitive dye Acridine orange, proton accumulation in the presence of an inside-negative transmembrane potential was measured in eel (Anguilla anguilla) intestinal brush-border membrane vesicles. It was demonstrated that the proton accumulation was specifically increased by the presence of the dipeptide glycyl-glycine in the extravesicular space, showing saturation kinetics at increasing dipeptide concentrations and was specifically inhibited by diethylpyrocarbonate. Data reported suggest the presence of an electrical-potential-dependent H+/glycyl-glycine cotransport system in the eel intestinal brush-border membrane vesicles.


Heterogeneity of pig kidney Na,K-ATPase as indicated by ADP- and ouabain-binding stoichiometry. A centrifugation method has been used for determination of [14C]ADP and [3H]ouabain binding to Na,K-ATPase from pig kidney with high specific activity. In the presence of K+, the fit of the [14C]ADP binding data to a two-site model gives a component with high affinity which accounts for 12 +/- 2% of the total sites. The figure is significantly different from 50%, i.e., two components of equal size cannot be assumed. This contrasts with a ratio between the sites of 1:1 obtained by the rate dialysis technique. The discrepancy may be due to the fact that the centrifugation method enables bound ADP to be determined at lower concentrations of free ligand. [3H]Ouabain binding in the absence of Na+ is compatible with a straight line in a Scatchard plot if the isotope is purified shortly before use. An unspecific binding of ouabain can be neglected if the concentration of free ouabain is not too high. In the presence of Na+, the isotherms become upward concave. An analysis of the binding data gives a 19:81% division, although equilibrium is not quite attained. This is a maximum value because the lack in equilibrium will be most pronounced at the small values of free ouabain. Thus the ADP-binding studies are supported. The finding here is in some agreement with the semiquantitative immunoassay showing that pig kidney enzyme contains the isoenzymes alpha 1, alpha 2 and alpha 3 in a proportion of 84:12:4, respectively. Determination of ADP- and ouabain-binding site stoichiometry favours a theory with one substrate site per (alpha beta)2.


Asymmetric fusion between synthetic di-n-dodecylphosphate vesicles and virus membranes. The interaction between vesicles, prepared from the synthetic amphiphile di-n-dodecylphosphate (DDP), with Sendai virus membranes was investigated. DDP vesicles fuse in the presence of Ca2+ ('symmetric' fusion). However, in the absence of Ca2+, DDP vesicles and Sendai virus, both displaying a high intrinsic fusion capacity with various target membranes, can also readily fuse with each other ('asymmetric' fusion). Under these conditions, fusion was found not to depend on specific viral proteins. Thus fusion occurs over a broad pH range (3.0-9.0) and is not affected by perturbation of viral protein structure. The overall interaction process was further analyzed with a mass action kinetic model. The analysis reveals that the destabilization and reorganization of the synthetic and viral bilayers are as fast as in pure phospholipid systems. Furthermore, the drastic effect of temperature on the overall reaction appears to be related to an effect of this parameter on fusion itself rather than on vesicle-virus aggregation. This could suggest that protein mobility constraints modulate the fusion reaction. The morphology of the fusion products, which consist of a single virus particle and several DDP vesicles, indicates a bilayer stabilization of the fusion product, rather than formation of tubular structures, as observed for symmetric DDP fusion products. The present results further emphasize the high susceptibility of vesicles composed of synthetic amphiphiles to engage in (protein-mediated) membrane fusion. This bears relevance to their potential application as carriers for biomolecules.


Characterization of CO2/carbonic acid mediated proton flux through phosphatidylcholine vesicles as model membranes. The apparent proton permeability coefficient for phospholipid vesicles measured in our laboratory (Norris, F. A. and Powell, G. L. (1990) Biochim. Biophys. Acta 1030, 165-171) for proton flux initiated by rapidly lowering of the external pH (acid jump) was a linear function of the reciprocal internal proton concentration. This behavior was ascribed to the presence of the weak acid carriers, carbonic acid/CO2/bicarbonate. In the present work, a theoretical description, appropriate for proton transport by any weak acid carrier, has been developed which lends itself to novel graphical treatment permitting the separate estimation of the permeability coefficients for protons, hydroxide ions and bicarbonate. The proton permeability coefficient determined by this method was 1.8 x 10(-5) (S.E. 1.3 x 10(-5)) cm/s; that for hydroxide ion was 3.8 x 10(-5) (S.E. 5.6 x 10(-6)) cm/s and a lower limit for the permeability of bicarbonate ion, 4.3 x 10(-6) (S.E. 3.6 x 10(-7) cm/s, can be set. The presence of negative surface charge on the lipid bilayer increased the observed proton permeability coefficient in accordance with Gouy-Chapman theory. The charge was introduced by preparing vesicles containing increasing amounts of negatively charged dioleoylphosphatidylglycerol. The observed proton permeability coefficient increased and the observed permeability coefficients for hydroxide ion and bicarbonate decreased. The addition of the lipophilic cations, valinomycin-K+ and tetrabutylammonium ion increased the slope of P vs. 1/[Hi+]. These changes are analogous to those reported for the permeant weak acid uncouplers FCCP and CCCP. These studies demonstrated that CO2/carbonic acid was an effective carrier of protons across phospholipid model membranes.


Identification of a regulated Na/K/Cl cotransport system in a distal nephron cell line. Lack of an adequate cell model has limited investigation of Na/K/Cl cotransporter regulation in the kidney. Using A6 cells, an amphibian distal renal cell line, we observed that 63% of rubidium uptake in confluent A6 monolayers was ouabain-insensitive. Ouabain-insensitive rubidium uptake was inhibited in a dose-dependent fashion by furosemide (IC50 6.6 microM) or bumetanide (IC50 1.7 microM). Kinetic studies confirmed that furosemide-sensitive rubidium uptake had features consistent with cotransporter activity in other cell lines. Furthermore, specific binding of [3H]bumetanide occurred with a capacity of 8.6 pmol/mg protein and a Kd of 1.6 microM bumetanide. Finally, furosemide-sensitive rubidium uptake was rapidly regulated by a calcium ionophore, the phorbol ester PDBu, forskolin, and adenosine. These data demonstrate an Na/K/Cl cotransport system in the A6 cell which will serve as a useful model for studying cotransporter regulation by endogenous signaling pathways.