Treating open lower limb fractures successfully; thoughts and current practice on therapy and centralization in The Netherlands.

INTRODUCTION: The British Orthopedic Association (BOA) and British Association of Plastic, Reconstructive and Aesthetic Surgeons (BAPRAS) updated the evidence-based guidelines for the treatment and care of open lower limb fractures (BOAST 4). Following this, a Dutch version has been developed. The main points are multidisciplinary care, planning, and treatment of these injuries. Early osteosynthesis (within 7-14 days) combined with soft-tissue coverage results in more efficient care and less complications. AIM: To study the variation in treatment and thoughts among trauma, orthopedic, and plastic surgeons. MATERIALS AND METHODS: In this cross-sectional study 94 surgeons (57 trauma, 23 plastic, and 14 orthopedic surgeons) working at 46 centers completed an online questionnaire, consisting of 5 demographic, 14 hospital-related, 8 BOAST 4-related, and 2 centralization-related questions. RESULTS: There was a strong agreement among surgeons about the best moment for multidisciplinary consultation, which was before initial debridement, while in practice, this often does not occur. All surgeons agreed that the initial debridement should be performed immediately by any surgeon, but not solely by trainees. Plastic surgeons responded that the definitive stabilization and wound cover should not exceed 7 days, while half of the trauma and orthopedic surgeons agreed that it should not exceed 14 days. Finally, most surgeons agreed that Gustilo 3 fractures should be centralized. However, there was disagreement on the need for centralization of Gustilo 2 fractures. DISCUSSION: Surgeons agree on better and earlier multidisciplinary treatment of open lower limb fractures and the centralization of Gustilo 3 fractures.

A prospective cohort study comparing the VAS spine score and Roland-Morris disability questionnaire in patients with a type A traumatic thoracolumbar spinal fracture.

The Roland Morris Disability Questionnaire (RMDQ-24) and the VAS spine score have been regularly used to measure functional outcome in patients with back pain. The RMDQ-24 is primarily used in degenerative disease of the spine and the VAS Spine is used in trauma patients. The aim of this study is to compare these scores and to see if there is a correlation in patients with a traumatic thoracolumbar spinal fracture. Prospective cohort study comparing the RMDQ-24 and the VAS spine score in patients with a traumatic type A fracture thoracolumbar spine fracture. Fifteen non-operatively patients (group one) completed 118 questionnaires and 17 operatively treated patients (group two) completed 140 questionnaires. Group one scored an average of 6.6 and 65.9 for the RMDQ-24 and VAS Spine, in group two this was 5.1 and 82.9. Spearman's correlation test showed a significant correlation, in group one 0.83 and for the second group 0.87. RMDQ-24 and VAS Spine have a strong positive correlation in measuring disability in a group of patients with back pain because of a spinal fracture. In both non-operatively and operatively treated groups this correlation is significant.

Changes in force, surface and motor unit EMG during post-exercise development of low frequency fatigue in vastus lateralis muscle.

We investigated the effects of low frequency fatigue (LFF) on post-exercise changes in rectified surface EMG (rsEMG) and single motor unit EMG (smuEMG) in vastus lateralis muscle (n = 9). On two experimental days the knee extensors were fatigued with a 60-s-isometric contraction (exercise) at 50% maximal force capacity (MFC). On the first day post-exercise (15 s, 3, 9, 15, 21 and 27 min) rsEMG and electrically-induced (surface stimulation) forces were investigated. SmuEMG was obtained on day two. During short ramp and hold (5 s) contractions at 50% MFC, motor unit discharges of the same units were followed over time. Post-exercise MFC and tetanic force (100 Hz stimulation) recovered to about 90% of the pre-exercise values, but recovery with 20 Hz stimulation was less complete: the 20-100 Hz force ratio (mean +/- SD) decreased from 0.65+/-0.06 (pre-exercise) to 0.56+/-0.04 at 27 min post-exercise (P<0.05), indicative of LFF. At 50% MFC, pre-exercise rsEMG (% pre-exercise maximum) and motor unit discharge rate were 51.1 +/- 12.7% and 14.1 +/- 3.7 (pulses per second; pps) respectively, 15 s post-exercise the respective values were 61.4 +/- 15.4% (P<0.05) and 13.2 +/- 5.6 pps (P>0.05). Thereafter, rsEMG (at 50% MFC) remained stable but motor unit discharge rate significantly increased to 17.7 +/- 3.9 pps 27 min post-exercise. The recruitment threshold decreased (P<0.05) from 27.7 +/- 6.6% MFC before exercise to 25.2 +/- 6.7% 27 min post-exercise. The increase in discharge rate was significantly greater than could be expected from the decrease in recruitment threshold. Thus, post-exercise LFF was compensated by increased motor unit discharge rates which could only partly be accounted for by the small decrease in motor unit recruitment threshold.

Voluntary drive-dependent changes in vastus lateralis motor unit firing rates during a sustained isometric contraction at 50% of maximum knee extension force.

The purpose of the present study was to relate the expected inter-subject variability in voluntary drive of the knee extensor muscles during a sustained isometric contraction to the changes in firing rates of single motor units. Voluntary activation, as established with superimposed electrical stimulation was high (range: 91-99%, n=8) during a short maximal contraction, but was lower (range: 69-100%) in most subjects at the point of force failure during a sustained (49.1+/-10.1 s) fatiguing contraction at 50% of maximum force. On a different experimental day the firing behaviour of 27 single motor units was recorded with wire electrodes in the vastus lateralis muscle, 24 of which could be monitored from the time of recruitment to the point of force failure (53.6+/-9.8 s). Motor unit firing behaviour differed considerably among subjects. During the second half of the sustained, fatiguing contraction the changes in firing rate firing rate variability of early recruited units ranged from -10% to +100% and from -50% to +160% respectively among subjects. There were significant positive linear relations between voluntary activation, on the one hand, and rectified surface electromyogram (rsEMG, r=0.82), the changes in motor unit firing rate ( r=0.49) and firing rate variability ( r=0.50) towards the point of force failure on the other. The present data suggest that differences in voluntary drive that appear among subjects during fatigue may be an important determinant of motor unit firing behaviour.

Two nonmuscle myosin II heavy chain isoforms expressed in rabbit brains: filament forming properties, the effects of phosphorylation by protein kinase C and casein kinase II, and location of the phosphorylation sites.

During the course of the expression of a 47-kDa COOH-terminal fragment of brain-type nonmuscle myosin heavy chain (MIIBF47), we found two closely related forms of MIIB, designated MIIB alpha and MIIB beta, in rabbit brains. The B alpha form corresponded to SMemb, described by Kuro-o et al. [(1991) J. Biol. Chem. 266, 3768] and was the more abundant form in rabbit brain, while the B beta form was novel. MIIB beta F47 differed from MIIB alpha F47 at six positions, three of which were within the carboxyl-terminal nonhelical domain; in MIIB beta F47, Ser, Pro, and Lys replaced Pro, Ser, and Glu, respectively. MIIB alpha F47 and MIIB beta F47 differed in filament assembly properties in the presence of various concentrations of salt, and a chimera containing the helical domain of MIIB beta F47 and the nonhelical domain of MIIB alpha F47 behaved very much like MIIB beta F47. Protein kinase C (PK C) incorporated 1 and 2 mol of phosphate/mol peptide of MIIB alpha F47 and MIIB beta F47, respectively, and caused similar levels of inhibition of assembly for both isoforms. Casein kinase II (CK II) incorporated 4 and 2 mol of phosphate/mol of MIIB alpha F47 and MIIB beta F47 peptides, respectively, and this caused strong inhibition of assembly for MIIB alpha F47 but only slight inhibition for MIIB beta F47. PK C sites in MIIB alpha F47 were localized within a region containing a cluster of Ser residues near the predicted junction of the helical and nonhelical domains: P-I-S(PO4)-F-S(PO4)-S(PO4)-S(PO4)-R-S(PO4)-. Out of the five potential PK C sites, only one site seemed to be phosphorylated per peptide. The PK C sites in MIIB beta F47 were localized as S(PO4)-I-S-F-S-S-(PO4)-R-S(PO4)-, with total incorporation of about 2 mol/mol of peptide. In addition, PK C phosphorylated a Ser within the predicted helical domain, E-V-S(PO4)-T-L, in both MIIB alpha F47 and MIIB beta F47. For CK II, five sites were identified within the COOH end of MIIB alpha F47: S(PO4)-L-E-L-S(PO4)-D-D-D-T(PO4)-E-S-K-T-S(PO4)-D-V-N-E-T-Q-P-P-Q-S(PO4) -E. The same sites were phosphorylated in MIIB beta F47 except for the first Ser, which was replaced by Pro in MIIB beta F47. An average of about two of the four potential sites were phosphorylated in MIIB beta F47, while in MIIB alpha F47 all five sites could be fully phosphorylated by CK II. Our results demonstrate that (1) the helical domains dictate the intrinsic salt dependence of assembly for nonmuscle myosin, (2) the isoforms are phosphorylatable by different kinases in an isoform specific manner mostly within the COOH-terminal nonhelical domain, and (3) the effects of the phosphorylation on assembly are isoform specific.

Intrastrand cross-linked actin between Gln-41 and Cys-374. I. Mapping of sites cross-linked in F-actin by N-(4-azido-2-nitrophenyl) putrescine.

A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40-50 (or 40-62 in the arginine limited digest) and residues 373-375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 A, constrains to that range the distance between the gamma-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin [Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44-49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836]. The effect of cross-linking on the function of actin is described in the companion papers.

Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends.

The beta-thymosins are a family of highly polar peptides which serve in vivo to maintain a reservoir of unpolymerized actin monomers. In vitro, beta-thymosins form 1:1 complexes with actin monomers and inhibit both polymerization and exchange of the bound nucleotide. Circular dichroism data indicate that free thymosin beta 4 is predominantly unstructured, containing at most six residues of alpha-helix, and that up to six additional residues may adopt an alpha-helical conformation upon binding actin. NMR data indicate that many parts of thymosin beta 4 are not in tight contact with actin. Contacts between specific residues in actin and thymosin beta 4 were identified by zero-length cross-linking followed by isolation and sequencing of cross-linked peptides. After carbodiimide-mediated cross-linking, Lys-3 of thymosin beta 4 was cross-linked to Glu-167 of actin, and Lys-18 of thymosin beta 4 was cross-linked to one of the the N-terminal acidic residues of actin (Asp-1-Glu-4); the cross-linked actin residues lie within subdomains 3 and 1, respectively. These two contacts flank the alpha-helical region of thymosin beta 4 and place it on the barbed end; thymosin beta 4 can thus block actin polymerization sterically. After transglutaminase-mediated cross-linking, Lys-38 of thymosin beta 4 was cross-linked to Gln-41 of actin, placing the C-terminal region of thymosin beta 4 in contact with subdomain 2 on the pointed end; thymosin beta 4 may sterically block actin polymerization at the pointed end as well as the barbed end of the monomer. The distance between the pointed-end and barbed-end contacts requires that the C-terminal half of thymosin beta 4 be in a predominantly extended conformation.

Phosphoinositide-dependent in vitro phosphorylation of profilin by protein kinase C. Phospholipid specificity and localization of the phosphorylation site.

Phosphoinositides bind to profilin and regulate actin-based cytoskeletal protein assembly. We report here that profilin is phosphorylated in vitro by protein kinase C (PKC) in the presence of phosphoinositides and micromolar concentrations of calcium. PKC-mediated phosphorylation of profilin was observed only in the presence of phosphoinositides; phosphatidylserine and diacylglycerol (known activators of PKC) and other lipids, including phosphatidic acid and phosphatidylglycerol phosphate, did not activate the phosphorylation. The activation of PKC-mediated phosphorylation of profilin by phosphoinositides was as follows: phosphatidylinositol (PI) 4-phosphate (K(m) = 18 microM) > PI 4,5-bisphosphate (K(m) = 30 microM) > PI (no activation). About 0.5 mol phosphate was incorporated per mol of profilin. Phosphorylation of profilin by PKC was not affected by the presence of various concentrations of actin. Phospho-amino acid analysis showed serine to be the only amino acid phosphorylated. The amino acid sequence of a phosphopeptide from CNBr-digested profilin corresponded to the COOH-terminal peptide of profilin (Ala-Ser-His-Leu-Arg-Ser-Gln-Tyr). Further digestion of this phosphopeptide by trypsin generated two phosphopeptides (Arg-Ser-Gln-Tyr and Ser-Gln-Tyr), thereby confirming that the phosphorylation site was the antepenultimate Ser (Ala-Ser-His-Leu-Arg-Arg-Ser(P)-Gln-Tyr).

Phospholipid binding, phosphorylation by protein kinase C, and filament assembly of the COOH terminal heavy chain fragments of nonmuscle myosin II isoforms MIIA and MIIB.

Previously, we showed that myosin II heavy chains bind to phosphatidylserine (PS) liposomes via their COOH terminal regions and that protein kinase C (PK C) phosphorylates the PS-bound heavy chains [Murakami et al. (1994) J. Biol. Chem. 269, 16082-16090]. In this report, we studied the phospholipid binding, the kinetics of phosphorylation by PK C, and the effect of PK C-mediated phosphorylation on assembly using 46-47 kDa fragments from the COOH termini of macrophage (MIIAF46) and brain type (MIIBF47) heavy chain isoforms. Binding of the fragments to PS or phosphatidylinositol liposomes increased turbidity, but MIIAF46 gave higher turbidity than MIIBF47. Both fragments were sedimented similarly by ultracentrifugation in PS concentration and mole percent of PS dependent manners. With mixed PS/phosphatidylcholine (PC) liposomes, at least 70 mol % PS was required for heavy chain binding. A similar level of PS was required for phosphorylation of fragments by PK C, indicating that binding of tail regions to PS is a prerequisite for phosphorylation by PK C. PK C phosphorylated MIIBF47 with Vmax values 4-5 times higher than those of MIIAF46, but the Km values for the two substrates were similar. The apparent Km values for PS liposomes (Klipid) were also similar for phosphorylation of both isoforms. Mixing PS with PC increased the Klipid and reduced the Vmax values but did not alter the Km values for the substrates. Assembly of MIIBF47, but not MIIAF46, was significantly inhibited by the phosphorylation, indicating that nonmuscle myosin assembly can be regulated, in an isoform specific manner, via phosphorylation of heavy chains by PK C.

Direct binding of myosin II to phospholipid vesicles via tail regions and phosphorylation of the heavy chains by protein kinase C.

Recent cloning and sequencing studies suggest that heavy chains of all non-muscle myosins II have a protein kinase C (PKC) phosphorylation site within their tail regions. A fragment of human macrophage myosin heavy chain, encompassing its COOH-terminal 396 amino acids (MIIAF46), was expressed in Escherichia coli to provide a model system for study of PKC-mediated phosphorylation. PKC phosphorylated this fragment when phosphatidylserine (PS) liposomes were present, but not when liposomes made from PS/phosphatidylcholine (PC) were used. The reaction required Ca2+, but not other activators such as diacylglycerol (DG) or phosphatidylinositol 4,5-bisphosphate. Phosphorylation of MIIAF46 was not observed in the presence of micelles of PS or PS/DG. Similar results were obtained using native myosin II purified from bovine brain and chicken intestine brush border. Phosphorylation of light chains, in contrast, occurred even with PS/PC liposomes if DG was present. Addition of the PS and PS/DG liposomes significantly increased the turbidities at 340 nm of MIIAF46 and native myosin II, and the extent of increase depended upon the type of myosin used. Also, PS and PS/DG liposomes shifted the gel filtration elution positions of MIIAF46 and myosin II. In contrast, liposomes of PS/PC and PS/PC/DG gave only a slight increase in turbidity with all myosins and fragments and did not noticeably shift their gel filtration elution positions. These results suggest that myosins II bind to PS liposomes via the COOH-terminal regions of their heavy chains with affinities specific to each myosin isoform, that the binding is dependent upon the PS composition, and that PKC phosphorylates the PS-bound heavy chains.

Primary structure of the large subunit of trifunctional beta-oxidation complex from pig heart mitochondria.

The amino-terminal and internal sequences of the isolated large subunit of trifunctional beta-oxidation complex from pig heart mitochondria were determined by Edman degradation. The results demonstrated that the sequence of this novel beta-oxidation enzyme is identical with the sequence recently reported for a porcine gastrin binding protein that serves as the gastrin receptor on parietal cell surfaces. Evidence is provided to show that it is unlikely that the porcine gastrin binding protein has such a sequence. The data lead us to conclude that the mature large subunit of porcine trifunctional beta-oxidation complex is composed of 727 amino acid residues with a calculated molecular weight of 79,113, while the precursor of this long-chain fatty acid oxidation enzyme has a mitochondrial presequence consisting of 36 residues and a calculated M(r) of 83,099.

Changes in expression of nonmuscle myosin heavy chain isoforms during muscle and nonmuscle tissue development.

Anti-human platelet myosin antibodies and two anti-peptide antibodies, anti-peptide IIA and anti-peptide IIB, which recognize macrophage-type (MIIA) and brain-type (MIIB) isoforms of nonmuscle myosin heavy chain, respectively, were used to study expression of nonmuscle myosin isoforms in various tissues of mice during development. Tissue-specific changes in the relative isoform concentrations were observed by performing immunoblots of crude myosin extracts from nonmuscle and muscle tissues. In fetal and neonatal mouse tissues, the anti-peptide IIB antibodies stained a single band, called MIIB2, while the anti-peptide IIA and anti-platelet myosin antibodies stained a band that migrated faster than MIIB2. In brain, a slower moving band, MIIB1, started to appear at 2 weeks after birth, and in the adult cerebellum it was at least as abundant as MIIB2. In thymus, MIIB2 decreased selectively shortly after birth, while in liver both MIIB2 and MIIA rapidly disappeared, but the isoform(s) detected by anti-platelet myosin antibodies (MIIApla) remained constant. The MIIB2 and MIIA as well as MIIApla found in striated muscles from fetal and neonatal mice decreased to levels that were below the limit of detection by 3 weeks of age. In cryosections of skeletal and cardiac muscles, MIIB2 was localized within the muscle cells, while MIIA and MIIApla were primarily in the blood vessels and capillaries.

Association of both enoyl coenzyme A hydratase and 3-hydroxyacyl coenzyme A epimerase with an active site in the amino-terminal domain of the multifunctional fatty acid oxidation protein from Escherichia coli.

An Escherichia coli mutant multienzyme complex of fatty acid oxidation, composed of two 41-kDa beta-subunits and two 79-kDa mutant alpha-subunits with the alpha/Gly116-->Phe substitution, has been overproduced and purified. The catalytic properties of 3-ketoacyl-coenzyme A (CoA) thiolase and L-3-hydroxyacyl-CoA dehydrogenase were found to be virtually identical with those of the wild type, whereas both enoyl-CoA hydratase and 3-hydroxyacyl-CoA epimerase activities were eliminated by the alpha/Gly116-->Phe mutation. delta 3-cis-delta 2-trans-Enoyl-CoA isomerase was only slightly affected by the mutation. The results of this study, together with the sequence analysis of the large alpha-subunit of the E. coli complex (Yang, X.-Y. H., Schulz, H., Elzinga, M., and Yang, S.-Y. (1991) Biochemistry 30, 6788-6795) and a demonstration of the epimerization of D-3-hydroxyacyl-CoAs in E. coli via a dehydration/hydration mechanism (Smeland, T. E., Cuebas, D., and Schulz, H. (1991) J. Biol. Chem. 266, 23904-23908), lead to the conclusion that enoyl-CoA hydratase and 3-hydroxyacyl-CoA epimerase are associated with a common active site in the amino-terminal domain of the multifunctional fatty acid oxidation protein. Thus the E. coli hydratase and epimerase activities represent two functions of a unique crotonase that converts both L- and D-3-hydroxyacyl-CoAs to 2-trans-enoyl-CoAs. Moreover, the results suggest that the amino-terminal domain of the large alpha-subunit is also involved in the isomerase activity but the key residue(s) required for catalyzing the isomerization is distinct from the crotonase.

Localization of myosin IIB at the leading edge of growth cones from rat dorsal root ganglionic cells.

Primary cultures of rat dorsal root ganglionic (DRG) cells were stained with isoform-specific antibodies against non-muscle myosin II. Antibodies against the brain type myosin (MIIB) stained the peripheries of growth cones and non-neuronal cells. Double staining of the cells with the anti-myosin antibodies and rhodamine-phalloidin or anti-actin antibodies indicated that MIIB co-exists, with F-actin, at the leading edge. Antibodies against platelet myosin stained neither leading edges nor neurites, but stained the cell bodies of neurons and the stress fibers of non-neuronal cells. These results suggest that MIIB functions in the motility of the leading edge of DRG cells.

Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta.

The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels.

Gln-41 is intermolecularly cross-linked to Lys-113 in F-actin by N-(4-azidobenzoyl)-putrescine.

The bifunctional reagent N-(4-azidobenzoyl)-putrescine was synthesized and covalently bound to rabbit skeletal muscle actin. The incorporation was mediated by guinea pig liver transglutaminase under conditions similar to those described by Takashi (1988, Biochemistry 27, 938-943); up to 0.5 M/M were incorporated into G-actin, whereas F-actin was refractory to incorporation. Peptide fractionation showed that at least 90% of the label was bound to Gln-41. The labeled G-actin was polymerized, and irradiation of the F-actin led to covalent intermolecular cross-linking. A cross-linked peptide complex was isolated from a tryptic digest of the cross-linked actin in which digestion was limited to arginine; sequence analysis as well as mass spectrometry indicated that the linked peptides contained residues 40-62 and residues 96-116, and that the actual cross-link was between Gln-41 and Lys-113. Thus the gamma-carboxyl group of Gln-41 must be within 10.7 A of the side chain (probably the amino group) of Lys-113 in an adjacent actin monomer. In the atomic model for F-actin proposed by Holmes et al. (1990, Nature 347, 44-49), the alpha-carbons of these residues in adjacent monomers along the two-start helices are sufficiently close to permit cross-linking of their side chains, and, pending atomic resolution of the side chains, the results presented here seem to support the proposed model.

Nucleotide sequence of the promoter and fadB gene of the fadBA operon and primary structure of the multifunctional fatty acid oxidation protein from Escherichia coli.

The primary structure of a multifunctional protein, the large alpha-subunit of the Escherichia coli fatty acid oxidation complex, was determined by sequencing the fadB region of the fadBA operon. The amino-terminal sequence of this protein had been established by Edman degradation. The transcription start site of the fadBA operon was located 42 nucleotides upstream of the initiator codon of the fadB gene by primer extension analysis. Sequences of -10 and -35 regions of the promoter responsible for interaction with RNA polymerase were found to be CACACT and TTTGCA, respectively. The location of the promoter of the fadBA operon was defined, and the transcription direction of this operon, from fadB to fadA, as previously proposed [Yang, S.-Y., et al. (1990) J. Biol. Chem. 265, 10424-10429], was corroborated. The multifunctional protein is composed of 729 amino acid residues and has a calculated Mr of 79,593. A putative NAD-binding beta alpha beta-fold necessary for L-3-hydroxyacyl-CoA dehydrogenase function was found in the central region of the fadB gene product. Sequence analyses suggest that the functional domains of the multifunctional protein are arranged in the order enoyl-CoA hydratase:L-3-hydroxyacyl-CoA dehydrogenase: delta 3-cis-delta 2-trans-enoyl-CoA isomerase and suggest that the genes of the E. coli multifunctional protein and rat peroxisomal trifunctional beta-oxidation enzyme evolved from a common ancestral gene.

Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable.

At least 50% of the actin in resting human platelets is unpolymerized, and the bulk of this unpolymerized actin is complexed with a recently identified acidic, heat-stable 5-kDa peptide, named "Fx." Purified Fx binds stoichiometrically to muscle G-actin, forming a complex identifiable by nondenaturing polyacrylamide gel electrophoresis. Formation of the complex inhibits salt-induced polymerization of G-actin. Here we report that Fx is indistinguishable from thymosin beta 4, an acidic, heat-stable 5-kDa peptide first isolated from calf thymus and thought to be a thymic hormone. The complete amino acid sequence of Fx was determined and was found to be identical with that of thymosin beta 4. Authentic thymosin beta 4 is functionally equivalent to Fx, forming a 1:1 complex with actin monomers and inhibiting polymerization. The widespread distribution and high intracellular concentration of thymosin beta 4 (Fx) strongly suggest that it plays a significant role in regulating actin polymerization in many cell types.