The pKa of His-24 in the folding transition state of apomyoglobin.
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In native apomyoglobin, His-24 cannot be protonated, although at pH 4 the native protein forms a molten globule folding intermediate in which the histidine residues are readily protonated. The inability to protonate His-24 in the native protein dramatically affects the unfolding/refolding kinetics, as demonstrated by simulations for a simple model. Kinetic data for wild type and for a mutant lacking His-24 are analyzed. The pK(a) values of histidine residues in native apomyoglobin are known from earlier studies, and the average histidine pK(a) in the molten globule is determined from the pH dependence of the equilibrium between the native and molten globule forms. Analysis of the pH-dependent unfolding/refolding kinetics reveals that the average pK(a) of the histidine residues, including His-24, is closely similar in the folding transition state to the value found in the molten globule intermediate. Consequently, protonation of His-24 is not a barrier to refolding of the molten globule to the native protein. Instead, the normal pK(a) of His-24 in the transition state, coupled with its inaccessibility in the native state, promotes fast unfolding at low pH. The analysis of the wild-type results is confirmed and extended by using the wild-type parameters to fit the unfolding kinetics of a mutant lacking His-24. (+info)
A novel trypanoplasm-like flagellate Jarrellia atramenti n. g., n. sp. (Kinetoplastida: Bodonidae) and ciliates from the blowhole of a stranded pygmy sperm whale Kogia breviceps (Physeteridae): morphology, life cycle and potential pathogenicity.
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The successful 6 mo rehabilitation of a stranded juvenile pygmy sperm whale Kogia breviceps afforded the opportunity to study the poorly known protozoan fauna of the upper respiratory tract of cetaceans. Mucus samples were collected by holding either a petri dish or glass slides over the blowhole for 3 to 5 exhalations; preparations were examined as wet mounts, and then stained with Wrights-Giemsa or Gram stain. Blood smears were stained with Wrights-Giemsa. Unidentified spindle-shaped and unidentified broad ciliates, reported from the blowhole of the pygmy sperm whale for the first time, were seen only initially, while yeast-like organisms and bacteria were seen intermittently. Epithelial cells and white blood cells were often present in the blowhole mucus, but red blood cells were never seen. A novel trypanoplasm-like bodonid kinetoplastid biflagellate (Order Kinetoplastida) was commonly encountered in the blowhole mucus, but never in the blood. Both mature flagellates and those undergoing longitudinal binary fission were present. The elongate flagellate had a long whiplash anterior flagellum; the recurrent flagellum was attached along at least two-thirds of the body length, forming a prominent undulating membrane, and the trailing portion was short. The kinetoplast was irregularly fragmented. The flagellates were either free-swimming, or attached to host material via the free portion of the posterior flagellum. The prominent undulating membrane was characteristic of Trypanoplasma, while the fragmented kinetoplast was characteristic of some species of Cryptobia. For the novel bodonid kinetoplastid, with its unique combination of morphological features (prominent undulating membrane and fragmented kinetoplast), we propose the creation of a new genus Jarrellia. We believe this to be the first published description of a flagellate from a marine mammal, and among the first reports of a trypanoplasm-like flagellate from a warm-blooded host. We expect that a diversity of flagellates and ciliates are commonly present in the blowhole of cetaceans. Future studies on the identity of the protozoans and the health of their cetacean hosts, which are readily studied in captivity, are necessary to establish their status as commensals or parasites. (+info)
The size and detergent binding of membrane proteins.
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Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell. (+info)
Osmoregulation in marine mammals.
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Osmoregulation in marine mammals has been investigated for over a century; however, a review of recent advances in our understanding of water and electrolyte balance and of renal function in marine mammals is warranted. The following topics are discussed: (i) kidney structure and urine concentrating ability, (ii) sources of water, (iii) the effects of feeding, fasting and diving, (iv) the renal responses to infusions of varying salinity and (v) hormonal regulation. The kidneys of pinnipeds and cetaceans are reniculate in structure, unlike those of terrestrial mammals (except bears), but this difference does not confer any greater concentrating ability. Pinnipeds, cetaceans, manatees and sea otters can concentrate their urine above the concentration of sea water, but only pinnipeds and otters have been shown to produce urine concentrations of Na+ and Cl- that are similar to those in sea water. This could afford them the capacity to drink sea water and not lose fresh water. However, with few exceptions, drinking is not a common behavior in pinnipeds and cetaceans. Water balance is maintained in these animals via metabolic and dietary water, while incidental ingestion and dietary salt may help maintain electrolyte homeostasis. Unlike most other aquatic mammals, sea otters commonly drink sea water and manatees frequently drink fresh water. Among the various taxonomic groups of marine mammals, the sensitivity of the renin-angiotensin-aldosterone system appears to be influenced by the availability of Na+. The antidiuretic role of vasopressin remains inconclusive in marine mammals, while the natriuretic function of atrial natriuretic peptide has yet to be examined. Ideas on the direction of future studies are presented. (+info)
Prediction of peptide ion mobilities via a priori calculations from intrinsic size parameters of amino acid residues.
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Ion mobility spectrometry (IMS) has recently been established as a powerful tool to separate the protease digest mixtures and identify their peptide components. As accurate calculation of mobilities is critical for this technique, a new rapid method based on intrinsic size parameters (ISPs) of amino acid residues has been devised. However, those parameters had to be obtained by tedious statistical analysis of a large body of experimental data. Here we demonstrate that they can instead be derived a priori, based on the stoichiometry of a residue. Our main finding is that the ISP of a residue is essentially determined by its density, that is, the average mass/size ratio of its constituent atoms. This is in accordance with an interpretation in which peptides assume compact conformations in the gas phase dominated by the solvation of ionic charge. (+info)
Decrease in oxygen affinity of myoglobin by formylation of vinyl groups of heme.
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Three kinds of green synthetic myoglobin were prepared by recombination of horse heart apomyoglobin with spirographis (2-formyl-4-vinyl-), isospirographis (2-vinyl-4-formyl-), and 2,4-diformyldeuterohemins. The optical and oxygen binding properties of the reconstituted myoglobins containing two isomeric monoformyl-monovinylhemins were found to be different. The oxygen affinities (P50) of spirographis and 2,4-diformylmyoglobins are 2.7 and 2.8 mm Hg, respectively, at 25 degrees, and about 2.5 times lower than that of native protomyglobin, while that of isospirographis myoglobin is 1.0 mm Hg and is similar to native myoglobin. Spirographis oxymyoglobin has absorption maxima (alpha, beta, and Soret bands) at 601, 556.5, and 435 nm, isospirographis oxymyoglobin at 595, 550, 429 nm, and 2,4-diformyl oxymyoglobin at 603, 563.5, and 447 nm. The optical red shifts as well as the decrease in the oxygen affinities of these myoglobins are attributed mainly to the presence of strongly electron-attractive formyl side chains. Since the free isomers of monoformyl-monovinyl heme have similar properties, the differences observed after recombination with apoprotein must be caused by interactions with apomyoglobins. The degree of such a protein effect may be estimated by comparing the absorption spectra of heme before and after recombination and was found to differ among the various myoglobins. Comparison of the oxygen affinities of the myoglobins taking account of this protein factor showed that the increase in the P50 values are inversely related to that in the pK3 values of the free porphyrins. These results suggest the involvement of pi bonding in determining the oxygen-iron bond strength. (+info)
The Fe(2+)-His(F8) Raman band shape of deoxymyoglobin reveals taxonomic conformational substates of the proximal linkage.
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The band shape of the Raman line attributed to the Fe(2+)-N(epsilon)(His(F8)) stretching mode in deoxymyoglobin contains significant information on the nature of the Fe-His proximal linkage. Raman lines appearing close to it, however, obscure the true line profile. To isolate this from its accompanying lines we use its isotopic shift of approximately 1 cm(-1) when (56)Fe in natural-abundance deoxymyoglobin is substituted by (54)Fe. This enables us to isolate the true line shape. We have measured this line shape in sperm whale myoglobin dissolved in a 66% vol/vol glycerol/water solution for nine temperatures from 10 K to 300 K. The nu(Fe-His) band shows a complex temperature-dependent profile, with a shoulder on its high-frequency wing, which becomes more prominent with increasing temperature. Detailed analysis reveals that the band is composed of five distinct lines attributable to taxonomic conformational substates of the nu(Fe-His) linkage. These are in thermodynamic equilibrium above the glass transition temperature T(f) but freeze in into the thermodynamic distribution at T(f) for lower temperatures. Alternative models that try to explain the nu(Fe-His) band shape by either an anharmonic coupling of the nu(Fe-His) to a low-frequency heme doming mode or by conformational substates related to a Gaussian distribution of iron out-of-plane displacements are at variance with the distinct features observed experimentally. (+info)
Buoyant balaenids: the ups and downs of buoyancy in right whales.
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A variety of marine mammal species have been shown to conserve energy by using negative buoyancy to power prolonged descent glides during dives. A new non-invasive tag attached to North Atlantic right whales recorded swim stroke from changes in pitch angle derived from a three-axis accelerometer. These results show that right whales are positively buoyant near the surface, a finding that has significant implications for both energetics and management. Some of the most powerful fluke strokes observed in tagged right whales occur as they counteract this buoyancy as they start a dive. By contrast, right whales use positive buoyancy to power glides during ascent. Right whales appear to use their positive buoyancy for more efficient swimming and diving. However, this buoyancy may pose added risks of vessel collision. Such collisions are the primary source of anthropogenic mortality for North Atlantic right whales, whose population is critically endangered and declining. Buoyancy may impede diving responses to oncoming vessels and right whales may have a reduced ability to manoeuvre during free ascents. These risk factors can inform efforts to avoid collisions. (+info)