Klaus D. Linse

Stoichiometry of Subunits and Heme Content of Hemoglobin from the Earthworm Lumbricus terrestris

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major O2-binding chains, a, b, c (forming a disulfide-linked trimer), and d (“monomer”). Additional structural chains, “linkers,” are required for the assembly of the ∼200-polypeptide molecule. The proportion of linker chains had been reported to be one-third of the total mass on the basis of densitometry of Coomassie Blue-stained SDS-gels. Reverse-phase high performance liquid chromatography (HPLC), however, gave 16.3% linkers on the basis of both 220-nm absorbance and amino acid analysis (Ownby, D. W., Zhu, H., Schneider, K., Beavis, R. C., Chait, B. C., and Riggs, A. F. (1993) J. Biol. Chem. 268, 13539-13547). The subunit proportions have now been redetermined by SDS capillary electrophoresis as a test of the HPLC results. The electrophoresis, monitored at 214 nm, avoided the use of Coomassie Blue and provided results identical with those obtained by HPLC. Capillary electrophoresis monitored at both 214 and 415 nm was used to show that linker chains do not bind heme. Heme content has been found to be 2.9% by determination of hemin, amino acid analysis and dry weight. Measurement of the rate of hemin loss from oxidized L. terrestris Hb shows that high rates of loss can account for values of heme content significantly below 2.9% (or 0.26% iron).

Characterization of spherical amyloid protein from a prolactin-producing pituitary adenoma

Abstract Prolactin (PRL)-producing pituitary adenomas are in some cases associated with deposition of abundant spherical amyloid; however, the origin of the amyloid has not been established. In this report, a PRL-producing pituitary adenoma composed almost entirely of spherical amyloid was analyzed biochemically. The tumor was removed surgically from a 56-year-old man. Immunohistochemical analysis revealed that residual tumor cells were strongly positive for PRL, while the spherical amyloid was not. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a band of approximately 4 kDa associated with the amyloid, which was not present in a non-amyloid producing prolactinoma. The 4-kDa band is similar in size to other known amyloidogenic peptides. Immunoblot analysis of the tumor material using polyclonal anti-human PRL antibodies revealed a small amount of normal-sized PRL; however, the abundant 4-kDa band was nonimmunoreactive. Amino acid sequencing showed that this peptide represents the first 34 amino acids of the intact PRL protein with a predicted size of 4313 Da. The presence of a small amount of normal-sized PRL in this tumor, as well as elevated circulating levels of PRL implies that intact PRL is being abnormally processed in the formation of spherical amyloid.

Clostridium taeniosporum spore ribbon‐like appendage structure, composition and genes

Clostridium taeniosporum spores have about 12 large, flat, ribbon‐like appendages attached through a common trunk at one spore pole to a previously unknown surface layer outside the coat that is proposed to be called the ‘encasement’. Appendages are about 4.5 μm long, 0.5 μm wide and 30 nm thick and taper at the attachment end into a semicircle that is twisted relative to the flat ribbon. Individual fibrils, about 45 nm in length with spherical heads and long thin tails, form a hair‐like nap, visible along the appendage edge. Four appendage proteins have been detected: a paralogous pair of 29 kDa (designated P29a and P29b), a glycoprotein of about 37 kDa (designated GP85) and an orthologue of the Bacillus spore morphogenetic protein SpoVM. The P29 proteins consist of duplicated regions and each region includes a domain of unknown function 11. The GP85 glycoprotein contains a collagen‐like region. The genes for P29a and b, GP85 and possibly related proteins are closely linked on two small chromosome fragments. Putative σK‐dependent promoters upstream of the P29a and b genes indicate that they likely are expressed late in the mother cell, consistent with their deposition into the layer external to the coat.

Isolation and Characterization of CvIV4: A Pain Inducing α- Scorpion Toxin

Background Among scorpion species, the Buthidae produce the most deadly and painful venoms. However, little is known regarding the venom components that cause pain and their mechanism of action. Using a paw-licking assay (Mus musculus), this study compared the pain-inducing capabilities of venoms from two species of New World scorpion (Centruroides vittatus, C. exilicauda) belonging to the neurotoxin-producing family Buthidae with one species of non-neurotoxin producing scorpion (Vaejovis spinigerus) in the family Vaejovidae. A pain-inducing α-toxin (CvIV4) was isolated from the venom of C. vittatus and tested on five Na+ channel isoforms. Principal Findings C. vittatus and C. exilicauda venoms produced significantly more paw licking in Mus than V. spinigerus venom. CvIV4 produced paw licking in Mus equivalent to the effects of whole venom. CvIV4 slowed the fast inactivation of Nav1.7, a Na+ channel expressed in peripheral pain-pathway neurons (nociceptors), but did not affect the Nav1.8-based sodium currents of these neurons. CvIV4 also slowed the fast inactivation of Nav1.2, Nav1.3 and Nav1.4. The effects of CvIV4 are similar to Old World α-toxins that target Nav1.7 (AahII, BmK MI, LqhIII, OD1), however the primary structure of CvIV4 is not similar to these toxins. Mutant Nav1.7 channels (D1586A and E1589Q, DIV S3–S4 linker) reduced but did not abolish the effects of CvIV4. Conclusions This study: 1) agrees with anecdotal evidence suggesting that buthid venom is significantly more painful than non-neurotoxic venom; 2) demonstrates that New World buthids inflict painful stings via toxins that modulate Na+ channels expressed in nociceptors; 3) reveals that Old and New World buthids employ similar mechanisms to produce pain. Old and New World α-toxins that target Nav1.7 have diverged in sequence, but the activity of these toxins is similar. Pain-inducing toxins may have evolved in a common ancestor. Alternatively, these toxins may be the product of convergent evolution.

Biochemistry of the Growth Hormone-Releasing Peptides, Secretagogues and Ghrelin

A general chronology of growth hormone-releasing peptide (GHRP) and ghrelin and their shared receptor is shown in Table 1 [1] [3]. The discovery of the natural hormone ghrelin appears to be the exciting beginning of a new unusual hormone system. Initially, in 1980, GH-releasing activity of the unnatural GHRPs was thought to represent the activit y of the as-yet unidentified natural hypophysiotrophic hormone GH-releasing hormone (GHRH). However, in 1982, GHRH was isolated from a pancreatic tumour and the hypothalamus and chemically identified as 44 and 40 amino acid linear peptides. Although the GH-releasing activity of GHRPs and GHRH are similar, it is also apparent that they are definitely chemically and functionally different because of the uniqueness of the action of GHRP on GH secretion in vitro as well as in vivo in a variety of animal species. In 1984, it was postulated that GHRP reflected the activity of another new hypothalamic hormone involved in the increased secretion of GH and thus was in need of isolation.

Development of a method for analysis of free amino acids from physiological samples using a 420A ABI/PE amino acid analysis

Publisher Summary In recent years, there has been a recurring interest and demand for the quantitation of free amino acids in physiological fluids, for example, urine, blood serum, tissue cultures, as well as in hydrolysates. Amino acid analysis is a well established technique for the quantitation of free amino acids found in either hydrolysates or physiological fluids. Because of such increased interest in analysis of physiological samples, several studies establishes analyzer methods that would allow choosing between the standard protocol for protein and peptide hydrolysates and a separate protocol for an expanded number of amino acids. This is to include the most important free amino acids found in physiological samples. A separation protocol has been developed that allows for the separation and quantitation of up to 30 amino acids using the 420H system. While this separation protocol does not allow for the separation of all possible free amino acids found in physiological samples, it enables to identify and quantify those most commonly requested. The major difference in separation conditions among the standard versus the physiological method is the addition of an extra step at six minutes into the gradient. This step decreases the steepness of the slope of the gradient development, thus allowing for the separation of citrulline, taurine, and arginine.