Ian P. Trayer

A new method of preparation of troponin I (inhibitory protein) using affinity chromatography. Evidence for three different forms of troponin I in striated muscle

The methods currently in use for the preparation of the components of the troponin complex involve the preliminary isolation of the troponin complex and its subsequent fractionation into individual components. The procedure is satisfactory for rabbit skeletal muscle andoften can be modified for other muscles. In some cases, however, the troponin preparations particularly from smooth muscle usually contain proteins in addition to the troponin I, troponin C and troponin T which leads to difficulties in the isolation of pure samples of the individual components. The relatively long period of isolation required by these procedures often leads .LO some degradation of the components, especially of troponin I which is particularly susceptible to breakdown by endogenous cathepsins. The report that troponin C and troponin I form, in the presence of Ca’*, a specific complex that is stable in high urea concentrations [l-3] and which can be dissociated by ethanedeoxybis(ethylamine) tetra-acetic acid (EGTA), presents an ideal system for the application of affinity chromatography for the isolation of these two components. A method using troponin C linked to Sepharose is described for the rapid isolation of pure troponin I by a one-stage procedure from a few grams of whole muscle. Application of the procedure to the striated muscle of rabbit has indicated that there are at least three forms of troponin I present in this tissue, each form being specific for the type of muscle from which it is isolated.

STRUCTURE AND FUNCTION OF X-PRO DIPEPTIDE REPEATS IN THE TONB PROTEINS OF SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI

The TonB protein is required for several outer membrane transport processes in bacteria. A short 33-residue peptide segment of TonB has been studied by 1H and 13C nuclear magnetic resonance spectroscopy. The sequence of this peptide segment contains multiple Glu-Pro and Lys-Pro dipeptide repeats that maintain rigid, elongated structures and flank a short connecting segment that adopts a beta-strand configuration. This TonB peptide is shown to interact specifically with the FhuA protein, the outer membrane receptor for ferrichrome-iron, providing the first direct evidence that the TonB protein interacts with outer membrane receptors. Interaction with the FhuA protein involves the extended structural element containing positively charged Lys-Pro repeats, and suggests a functional role for this segment of the TonB protein. As TonB is anchored in the cytoplasmic membrane the protein must, uniquely, span the periplasm. These data, together with studies described in the accompanying paper, suggest a model by which TonB serves to transduce conformational information over extended distances, from the cytoplasmic membrane to the outer membrane.

Sequence-imposed structural constraints in the TonB protein of E. coli

The solution conformation of a 33‐residue peptide segment derived from the TonB protein which is implicated in bacterial membrane transport processes, has been investigated using high‐resolution proton magnetic resonance techniques. This proline‐rich peptide possesses sequence‐imposed sections of elongated secondary structure that must be retained in the native protein configuration. These structural constraints provide elements of stiffness that imply a purely structural role for TonB and are relevant to the subcellular location and biological role of the protein. On the basis of these data we suggest that this protein spans the periplasmic space linking the inner and outer membrane components of TonB‐dependent transport systems.

Determination of the Structure of the N-terminal Splice Region of the Cyclic AMP-specific Phosphodiesterase RD1 (RNPDE4A1) by 1H NMR and Identification of the Membrane Association Domain Using Chimeric Constructs

A 25-residue peptide representing the membrane targeting N-terminal splice region of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1) was synthesized, and its structure was determined by 1H NMR. Two independently folding helical regions were identified, separated by a highly mobile “hinge” region. The first helical region was formed by an N-terminal amphipathic α-helix, and the second consisted of multiple overlapping turns and contained a distinct compact, hydrophobic, tryptophan-rich domain (residues 14-20). Chimeric molecules, formed between the N-terminal region of RD1 and the soluble bacterial protein chloramphenicol acetyltransferase, were used in an in vitro system to determine the features within the splice region that were required for membrane association. The ability of RD1-chloramphenicol acetyltransferase chimera to become membrane-associated was not affected by deletion of any of the following regions: the apolar section (residues 2-7) of the first helical region, the polar part of this region together with the hinge region (residues 8-13), or the polar end of the C-terminal helical region (residues 21-25). In marked contrast, deletion of the compact, hydrophobic tryptophan-rich domain (residues 14-20) found in the second helical region obliterated membrane association. Replacement of this domain with a hydrophobic cassette of seven alanine residues also abolished membrane association, indicating that membrane-association occurred by virtue of specific hydrophobic interactions with residues within the compact, tryptophan-rich domain. The structure of this domain is well defined in the peptide, and although the region is helical, both the backbone and the distribution of side chains are somewhat distorted as compared with an ideal α-helix. Hydrophobic interactions, such as the “stacked” rings of residues Pro14 and Trp15, stabilize this domain with the side chain of residue Leu16 adopting a central position, interacting with the side chains of all three tryptophan residues 15, 19, and 20. These bulky side chains thus form a hydrophobic cluster. In contrast, the side chain of residue Val17 is relatively exposed, pointing out from the opposite “face” of the peptide. Although it appears that this compact, tryptophan-rich domain is responsible for membrane association, at present the target site and hence the specific interactions involved in membrane targeting by the RD1 splice region remain unidentified.

Purification and properties of rat skeletal muscle hexokinase.

Rat tissues contain four hexokinase isoenzymes which are present in differing proportions in the various tissues. Each of the low-KM hexokinase types (EC 2.7.1 .l), designated I, II and III, has been partially purified from one or more rat tissues [ 1 ] but of these only type I from rat brain has been purified to homogeneity [2]. Recently a low yield preparation has been reported of rat hepatic glucokinase (EC 2.7.1.2) sometimes also called type IV hexokinase, which exhibits a high KM for glucose and a narrower substrate specificity than the other hexokinases [3]. The purification of hexokinase types II-IV by conventional methods has proved to be difficult due to the small qumtities available from the tissues and their inherent instability. A high yield preparation of homogeneous glucokinase from rat liver has recently been achieved by exploiting affinity chromatography on an agaroseN-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose matrix [4-61. In this report we describe how such an affinity chromatographic method can be utilized along with more conventional techniques in order to prepare homogeneous type II hexokinase from rat skeletal muscle in high yield. The enzyme has a polypeptide chain mol. wt of 96 000. Amino acid analysis reveals similarities between the compositions of the hexokinase isozymes types 1, II and IV (glucokinase) which may have evolutionary significance.

Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin‐I mutants and troponin‐C

Troponin C (TnC) could be extracted from skinned porcine cardiac muscle fibres and their Ca2+ sensitivity restored by reconstitution with recombinant human cardiac TnC. After extraction of troponin I (TnI) and TnC using the vanadate treatment method of Strauss et al. [Strauss, J. D., Zeugner, C., Van Eyk, J.E., Bletz, C., Troschka, M. and Rüegg, J.C. (1992) FEBS Lett. 310, 229–234], skinned porcine cardiac muscle fibres were reconstituted with wild‐type recombinant human cardiac TnC and either wild‐type cardiac TnI or several mutant isoforms of human TnI. Reconstitution with wild‐type proteins restored the Ca2+ sensitivity of the tissue and phosphorylation of the TnI with the catalytic subunit of protein kinase A reduced the Ca2+ sensitivity (i.e. ‐log[Ca2+] for 50% of maximal force) as has been shown by others. However, reconstitution with the TnI mutant Ser‐23Asp/Ser‐24Asp mimicking the phosphorylated form of cardiac TnI, led to a reduced Ca2+ sensitivity compared with reconstitution with wild‐type TnI, whereas the mutant Ser‐23Ala/Ser‐24Ala behaved as the dephosphorylated form of TnI. These data confirm the importance of negative charge in this region of the TnI molecule in altering the Ca2+ responsiveness in this system.

The compartmentation of glycolytic and gluconeogenic enzymes in rat kidney and liver and its significance to renal and hepatic metabolism

SummaryAn indirect immunoperoxidase procedure has been used to demonstrate sites of glycolysis and gluconeogenesis in normal rat kidney and liver. In kidney, the gluconeogenic enzyme fructose 1,6-biphosphatase was restricted to the proximal tubular epithelium, while the glycolytic enzyme hexokinase predominated in more distal segments. Intense staining for the biphosphatase in proximal convoluted tubular brush borders suggests that reabsorbed substrates may be used directly at this site in renal gluconeogenesis. In view of the high phosphofructokinase and pyruvate kinase activities present in collecting ducts, their relatively low hexokinase activities and their relatively pale immunostaining for hexokinase indicate that glycolytic substrates which feed into the pathway subsequent to the initial phosphorylation step, rather than glucose, may be the major energy source for the rat renal papilla.Immunostaining in the liver was consistent with the metabolic zonation of liver parenchyma, in that glucokinase occurred mainly in perivenous regions and fructose 1,6-bisphosphatase in periportal areas. The presence of such metabolic zonation is difficult to reconcile with the widely held view that the majority of hepatic glucogen is derived directly from glucose. A model for hepatic glycogen synthesis is proposed which links the concept of parenchymal zonal heterogeneity with recent biochemical evidence concerning the ‘glucose paradox’ and with microscopical studies on the dynamics of glycogen deposition after refeeding.

Size and charge requirements for kinetic modulation and actin binding by alkali 1-type myosin essential light chains.

The alkali 1-type isoforms of myosin essential light chains from vertebrate striated muscles have an additional 40 or so amino acids at their N terminus compared with the alkali 2-type. Consequently two light chain isoenzymes of myosin subfragment-1 can be isolated. Using synthesized peptide mimics of the N-terminal region of alkali 1-type essential light chains, we have found by1H NMR that the major actin binding region occurred in the N-terminal four residues, APKK. . . . . These results were confirmed by mutating this region of the human atrial essential light chain, resulting in altered actin-activated MgATPase kinetics when the recombinant light chains were hybridized into rabbit skeletal subfragment 1. Substitution of either Lys3 or Lys4 with Ala resulted in increased K m and k cat and decreased actin binding (as judged by chemical cross-linking). Replacement of Lys4 with Asp reduced actin binding and increased K m andk cat still further. Alteration of Ala1 to Val did not alter the kinetic parameters of the hybrid subfragment 1 or the essential light chain’s ability to bind actin. Furthermore, we found a significant correlation between the apparent K m for actin and thek cat for MgATP turnover for each mutant hybrid, strengthening our belief that the binding of actin by alkali 1-type essential light chains results directly in modulation of the myosin motor.

Role of the myosin light chains in binding to actin

Myosin from the fast twitch muscles of rabbit is hexameric and comprises two polypeptide chains of approx. mol. wt 200 000 (heavy chains) and 4 mol (per mol myosin) of light chains: two identical phosphorylatable polypeptides of mol. wt 18 000 (the P-LC) and two polypeptides of molecular weight 22 000 and 16 000 (the so-called alkali light chains, Al and A2 respectively (see review [l] and references therein). The four light chain components reside in the globular ‘heads’ of the myosin molecule, the subfragment 1 (S-l) regions, which possess both the actin binding and ATPase activities of the myosin [ 1 ] . Densitometric and radiochemical methods have shown that there is an unequal distribution of the two alkali light chains which supports the hypothesis that myosin isoenzymes exist [2,3]. Myosin from chicken breast-muscle also contains three species of light chain and it is likely that this myosin is similar to that of the rabbit in overall structure and design. Chymotryptic digestion of insoluble myosin filaments from rabbit fast-twitch muscle produces S-l species without the P-LC and such fragments have been separated into two species by ion-exchange chromatography, each of which contains a single type of alkali light chain [4]. The precise role of the myosin light chains is still

The occurrence of α‐N‐trimethylalanine as the N‐terminal amino acid of some myosin light chains

The alkali light chains are small subunits of myosin; one is associated with each head region. Rabbit fast-twitch skeletal muscle myosin contains two types, A 1 (LC 1) and A2 (LC3), which occur in a ratio of 2: 1 [ 1,2]. These differ from one another primarily in that Al possesses a 41 residue N-terminal ‘tail’ rich in proline, lysine and alanine residues, although they are otherwise almost identical 131. ‘H-NMR spectrum of calmodulin 171. However, subsequent analyses confirmed that the unknown signal arises not from the heavy chain, but from the associated alkali light chain, A 1, which contains no trimethyllysine.