Substrate-concentration dependence of contraction parameters in glycerinated insect flight muscle fibers from Lethocerus derollei

The contraction characteristics of the dorsal longitudinal muscle of Lethocerus derollei were investigated by applying small sinusoidal length changes (+/- 1% of resting length) to glycerinated muscle bundles and studying the effect of varying the frequency from 0.1 to 10 Hz and the concentration of MgATP from 35 microM to 2.3 mM. The maximum work done by the muscle per cycle increased as the MgATP concentration was decreased from 2.3 mM to 52 microM. Between 52 and 35 microM, the maximum work suddenly changed from a positive to a negative value. The optimal frequency for maximal work shifted from low to high values with increase in the MgATP concentration. As the temperature was increased, the optimal work frequency in 2.3 mM MgATP solution shifted to a higher value. As the MgATP concentration was increased, the optimal frequency for maximal power increased. The maximal value of the power was an increasing function of the MgATP concentration, reaching a plateau above 52 microM MgATP. The muscle stiffness was a decreasing function of the MgATP concentration, and above 52 microM MgATP it reached a minimum of about 22% of that in the rigor solution. These results are discussed in relation to the crossbridge kinetics.     

Cell structure and the metabolism of insect flight muscle

The biochemical properties of insect flight muscle were investigated to ascertain the mechanisms whereby energy is made available for the contractile processes. It was found: 1. The endogenous respiration of muscle homogenates was diminished by starving the flies. The substrate for this respiration was probably glycogen. 2. To obtain the maximal rate of oxidation of glucose, the homogenate had to be fortified with inorganic phosphate, Mg ions, ATP, and cytochrome c. The nucleotides, AMP and ADP, were not as effective as ATP. The addition of DPN or TPN was not necessary for this system. 3. Flight muscle homogenates oxidized glycogen, some sugars, and amino acids, as well as the intermediates of the glycolytic and tricarboxylic acid cycles. Other evidence demonstrated the substrate specificity of the muscle. 4. By centrifugation, the muscle homogenate was divided into two fractions: one, a soluble fraction representing the sarcoplasm; the other, the particulate fraction which contained the fibrils and the sarcosomes. 5. The particulate fraction, alone, oxidized all the citric acid cycle intermediates, alpha-glycerophosphate, phosphopyruvate, and the amino acids, glutamic, proline, and cysteine. Regardless of the substrate, no oxygen uptake was found with the sarcoplasm by itself. 6. A recombination of the sarcoplasm and the particulate component was required for the oxidation of glycogen, the hexoses, and all the phosphorylated intermediates of glycolysis, except phosphopyruvate. 7. Isolated mitochondria accounted for all the enzymatic activity of the particulate fraction. These results demonstrate that the enzymes of intermediate metabolism are localized in the sarcoplasm or sarcosomes. The third cytological entity, the myofibrils, plays no role in the energy-providing scheme. From a functional viewpoint, the sarcoplasm and the mitochondria, in combination, furnish the energy for the actomyosin contraction. The results are discussed in relation to analogous findings in other insects and vertebrates.     

The paramyosin of insect flight muscle

Paramyosin has been extracted and purified from the flight muscle of the insects Lethocerus cordofanus, Lethocerus maximus (water bugs), Heliocopris japetus (dung beetle) and Pachnoda ephippiata (rosechafer beetle). The subunit molecular weight, estimated by sodium dodecyl sulphate electrophoresis, is 107,000 ± 6000. The intrinsic sedimentation constant is 3.17 S and circular dichroism measurements give about 87 % helix, showing that the molecule is likely to be a two-chain rod.The amino acid composition of insect paramyosins resembles that of molluscan and annelid paramyosins except that the Glu/Asp ratio is higher. The amino acid analysis of insect tropomyosin is also given. Electron microscopy of tactoids shows an axial periodicity of 732 ± 8 Å or 146 Å with fine structure differing from that of molluscan tactoids.The proportion of paramyosin in the myofibrils, estimated by densitometry of stained gels, is 6.3% in L. cordofanus and 9.5% in rosechafer, and the ratio of myosin to paramyosin in L. cordofanus is 8.2. The possibility that paramyosin is a core protein of the myosin filaments is discussed.     

Three-dimensional image reconstruction of insect flight muscle. II. The rigor actin layer

The averaged structure of rigor cross-bridges in insect flight muscle is further revealed by three-dimensional reconstruction from 25-nm sections containing a single layer of thin filaments. These exhibit two thin filament orientations that differ by 60 degrees from each other and from myac layer filaments. Data from multiple tilt views (to +/- 60 degrees) was supplemented by data from thick sections (equivalent to 90 degrees tilts). In combination with the reconstruction from the myac layer (Taylor et al., 1989), the entire unit cell is reconstructed, giving the most complete view of in situ cross-bridges yet obtained. All our reconstructions show two classes of averaged rigor cross-bridges. Lead bridges have a triangular shape with leading edge angled at approximately 45 degrees and trailing edge angled at approximately 90 degrees to the filament axis. We propose that the lead bridge contains two myosin heads of differing conformation bound along one strand of F-actin. The lead bridge is associated with a region of the thin filament that is apparently untwisted. We suggest that the untwisting may reflect the distribution of strain between myosin and actin resulting from two-headed, single filament binding in the lead bridge. Rear bridges are oriented at approximately 90 degrees to the filament axis, and are smaller and more cylindrical, suggesting that they consist of single myosin heads. The rear bridge is associated with a region of apparently normal thin filament twist. We propose that differing myosin head angles and conformations consistently observed in rigor embody different stages of the power stroke which have been trapped by a temporal sequence of rigor cross-bridge formation under the constraints of the intact filament lattice.     

Three-dimensional image reconstruction of insect flight muscle. I. The rigor myac layer

We have obtained detailed three-dimensional images of in situ cross-bridge structure in insect flight muscle by electron microscopy of multiple tilt views of single filament layers in ultrathin sections, supplemented with data from thick sections. In this report, we describe the images obtained of the myac layer, a 25-nm longitudinal section containing a single layer of alternating myosin and actin filaments. The reconstruction reveals averaged rigor cross-bridges that clearly separate into two classes constituting lead and rear chevrons within each 38.7-nm axial repeat. These two classes differ in tilt angle, size and shape, density, and slew. This new reconstruction confirms our earlier interpretation of the lead bridge as a two-headed cross-bridge and the rear bridge as a single-headed cross-bridge. The importance of complementing tilt series with additional projections outside the goniometer tilt range is demonstrated by comparison with our earlier myac layer reconstruction. Incorporation of this additional data reveals new details of rigor cross-bridge structure in situ which include clear delineation of (a) a triangular shape for the lead bridge, (b) a smaller size for the rear bridge, and (c) density continuity across the thin filament in the lead bridge. Within actin's regular 38.7-nm helical repeat, local twist variations in the thin filament that correlate with the two cross-bridge classes persist in this new reconstruction. These observations show that in situ rigor cross-bridges are not uniform, and suggest three different myosin head conformations in rigor.     

The influence of flight on the phospholipid metabolism in insect flight muscle

Males of the American cockroach, Periplaneta americana, received an injection of 32P-orthophosphates and the specific activity of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) was determined after 120 min of in vivo incorporation. If the insects were forced to fly for 10 min immediately before the end of the experiment, the specific activity (S.A.) of PC and PE was lowered by 34.3 and 31.0%, respectively, that of PI by 17.5%. If the animals were allowed to rest for 10 min after cessation of flight, the S.A. of PC and PE did not differ significantly from the controls, whereas that of PI rose by 91.0% above the control value. These effects cannot be due to changes in precursor labelling (glycerophosphate and phosphoarginine were measured) and reflect changes in the rate of phospholipid biosynthesis. The possibility is discussed that mechanisms regulating the rate of phospholipid biosynthesis are involved.     

Ca-activation and stretch-activation in insect flight muscle

Asynchronous insect flight muscle is specialized for myogenic oscillatory work, but can also produce isometric tetanic contraction. In skinned insect flight muscle fibers from Lethocerus, with sarcomere length monitored by a striation follower, we determined the relation between isometric force (F(0)) at serial increments of [Ca(2+)] and the additional active force recruited at each [Ca(2+)] by a stretch of approximately 12 nm per half-sarcomere (F(SA)). The isometric force-pCa relation shows that 1.5-2 units of pCa are necessary to raise isometric force from its threshold (pCa approximately 6.5) to its maximum (F(0,max)). The amplitude of F(SA) depends only on the preceding baseline level of isometric force, which must reach at least 0.05 F(0,max) to enable stretch-activation. F(SA) rises very steeply to its maximum as F(0) reaches approximately 0.2 F(0,max), then decreases as F(0) increases so as to produce a constant sum (F(0) + F(SA)) = F(max). Thus Ca- and stretch-activation are complementary pathways that trigger a common process of cross-bridge attachment and force production. We suggest that stretch-induced distortion of attached cross-bridges relieves the steric blocking by tropomyosin of additional binding sites on actin, thereby enabling maximum force even at low [Ca(2+)].     

On the contractile mechanism of insect fibrillar flight muscle

Zusammenfassung Als bestimmend für die Dynamik des Langzeitvorganges des Spannungsabfalles im Muskel nach aufgebrachten stufen- und rampenförmigen Längenänderungen ist das Übertragungsverhalten eines s ^k -Elementes mit einer elastischen Komponente ermittelt worden. Vergleichende Betrachtungen über die Veränderung der H-Zonenlänge und des Exponenten k haben zu einer Lokalisierung der Übertragungseigenschaften in der in der Mitte des Sarkomers befindlichen H-Zone geführt. Es wird vorgeschlagen, daß die elastische Rückstellkraft des Muskels in den parallel zur H-Zone befindlichen m-Filamenten generiert wird; diese Strukturen stellen zusätzliche Verbindungen zwischen den Myosinfilamenten in den beiden Halbsarkomeren dar. Aus der Modellierung mit einem Analogrechner folgt, daß das s ^k -Element physikalisch einer Kombination einer stark nichtlinearen Feder mit einem konventionellen Dämpfertopf entspricht. Durch vektorielle Subtraktion des s ^k -Anteiles mit elastischer Komponente von der Ortskurve der Dehnbarkeit des entspannten Insektenflugmuskels ist mit sehr guter Genauigkeit die Übertragungsfunktion von zwei in Serie befindlichen viskoelastischen Elementen des Maxwell-Typs ermittelt worden. Durch die Annahme, daß in einem der drei bereits früher postulierten Maxwell-Elemente, die als konzentriert in den dominanten passiven Strukturen — den Verbindungsfilamenten, den Myosinfilamenten und der H-Zone — angenommen wurden, die Feder in Serie mit dem Dämpfertopf eine stark nichtlineare Kraft-Längen-Charakteristik hat, wird es möglich, die Gültigkeit eines schon früher formulierten Modelles auf Langzeitveränderungen ausdehnen.Die Möglichkeit der Beschreibung von Spannungsabfällen, gemessen für verschiedene andere Muskeln, durch die Übertragungseigenschaften eines s ^k -Gliedes läßt eine Allgemeingültigkeit der am Insektenmuskel erzielten Ergebnisse als wahrscheinlich erscheinen. Die Möglichkeit ist diskutiert, daß die für Dehnungsreceptoren abgeleitete Übertragungsfunktion das im Muskel lokalisierte s ^k -Glied mit einbezieht.     

On the contractile mechanism of insect fibrillar flight muscle

Zusammenfassung Aus sinusoidalen Analysen im Frequenzbereich von 0,01–70 Hz ist es gelungen, das dynamische Verhalten des passiven Muskels durch eine Serienschaltung dreier Maxwell-Elemente zu approximieren. Die MaxwellElemente werden den im entspannten Zustand bestimmenden morphologischen Strukturen — Verbindungsfilament, Myosinfilament und H-Zone — zugeordnet. Der passive Muskel kann als ein lineares System mit konzentrierten Parametern aufgefaßt werden, da viscose Zwischenwirkungen zwischen den Actinfilamenten und den dominanten passiven Elementen vernachlässigbar klein sind. Über die aus elektronenmikroskopischen Untersuchungen und Röntgenstrukturanalysen bekannten Dehnbarkeiten der einzelnen Filamentstrukturen des Muskels ist es möglich, Steifigkeitswerte für das Verbindungsfilament (1,4 (g/m), das Myosinfilament (34,2 g/m) und die H-Zone (4,6 g/m) zu bestimmen. Der elastische Modul des Myosinfilamentes, 1,5×10¹⁰ dyn/cm² ist vergleichbar mit den in der Literatur für andere natürliche Polymere angegebenen Elastizitätswerten.Für den Muskel im Zustand der Totenstarre, wo alle Myosinbrücken am Actinfilament festhalten, wird die Dehnbarkeit der H-Zone zum bestimmenden Faktor.Die Dynamik des passiven Muskels ist im beträchtlichen Maße abhängig von der Verstärkung der Restaktivität bei sehr niedrigen Ca⁺⁺-Konzentrationen. Bei zunehmender Dehnbarkeit des Myosinfilamentes wird dieser Verstärkungsfaktor größer und die resultierende Phasennacheilung wird dominant über die durch die passiven Strukturen hervorgerufene Phasenvoreilung. Bei hoher Ionenstärke wird das Myosinfilament so weich, daß die vorhandenen niedrigen Ca⁺⁺-Konzentrationen von 10^–9M, bei denen der Muskel sich normalerweise im entspannten Zustand befindet, für eine Aktivierung ausreichen; der Muskel leistet oszillatorische Arbeit.     

On the contractile mechanism of insect fibrillar flight muscle

Zusammenfassung Zur Erfassung der dynamischen Vorgänge bei der Muskelkontraktion wurde das System des fibrillären Insektenflugmuskels unter Bedingungen, die eine Linearisierung zulassen, untersucht. Um die Frequenzantwort als das Systemverhalten im sinusförmigen stationären Zustand zu erhalten, wurden die durch aufgeprägte sinusförmige Längenänderungen erzeugten Spannungsänderungen gemessen. Die Frequenzganganalysen beziehen sich auf die Annahme eines zeitinvarianten Systems mit konzentrierten Elementen. Die dominanten passiven Strukturen des Muskels, die Verbindungs-filamente und die Myosinbrücken, können in dem Frequenzbereich, der den Arbeitsfrequenzen des Insektenflugmuskels entspricht, durch ein aus drei Elementen bestehendes viscoelastisches System des Maxwell-Typs mit Zeitkonstanten vergleichbarer Größe beschrieben werden. Für die Frequenzantwort des aktivierten Muskels wurde mit hinreichender Genauigkeit die Übertragungsfunktion eines Phasenminimum-systems ermittelt. Eine theoretische Übertragungsfunktion für aktive Seite der Kontraktion ist durch Differenzbildung experimentell erhaltener Ortskurven bestätigt worden. Auf der Grundlage dieser theoretischen Differenzkurve wurde eine Beziehung zwischen oscillatorischer Arbeit und enzymatischer Hydrolyse des Adenosintriphosphates (ATP) als Energiequelle hergestellt. Bis zur optimalen Frequenz der Oscillation unter den benutzten Bedingungen wird eine Proportionalität zwischen diesen Größen festgestellt, die durch einen konstanten mechanochemischen Kopplungsfaktor bestimmt ist.     

Immunolocalization of ubiquitin in degenerating insect flight muscle

Summary Ubiquitin was localized by immunofluorescence microscopy during post-mating histolysis of fibrillar flight muscle in female fire ants, Solenopsis spp. Normal muscles, as well as histolysing muscles from artificially inseminated and haemolymph-injected females contained ubiquitin in association with nuclei, Z-lines, myofilaments and mitochondria. However, the density of the ubiquitin immunoreaction was markedly increased in the nuclei, Z-lines and mitochondria of degenerating tissues 6, 12 and 24 h post treatment. At these times the heaviest immunoreactivity for ubiquitin was seen in association with the nuclei, Z-lines and mitochondria. Immuno-controls, incubated in the absence of the primary antibody, showed no similar immunostaining. When insemination was preceded by the injection of actinomycin D, muscle degradation was significantly depressed after a 24-h period. Also, ubiquitin immunofluorescence was markedly reduced in tissues pre-treated with actinomycin D. These observations suggest that insemination increases the ubiquitination of specific myofibrillar proteins destined for degradation.     

On the role of arginine kinase in insect flight muscle

In the flight muscle of Locusta migratoria L., arginine kinase activity increased 10-fold when 5th instar larvae and adult animals were compared. During the onset of flight, ATP decreased slightly with the amount of phospho-l-arginine remaining constant. Thus, high arginine kinase activity characterizes the adult muscle, giving rise to the speculation that the phospho-l-arginine/l-arginine kinase system does not act only as a buffer system for high-energy phosphate but also as a shuttle mechanism for high-energy phosphate between mitochondria and myofibrils. Judged from electrophoretic mobility, only one isoenzyme exists that is not bound to subcellular structures. Calculations of the diffusive fluxes of ATP, ADP, phosphate, phospho-l-arginine and l-arginine between the sites of ATP-consumption and production, respectively, can be interpreted in such a way, that the low concentration of ADP_free might limit ATP-turnover during flight. Judging from the high arginine kinase activity, the major acceptor for high-energy phosphate at the mitochondria could be l-arginine, while phospho-l-arginine is transphosphorylated to ATP at the myofibrils, thus presumably serving as an energy shuttle.     

Calcium binding and the activation of fibrillar insect flight muscle

A new method has been used to measure calcium binding in intact glycerol extracted muscle fibres; results with rabbit psoas muscle are in agreement with previous work. Lethocerus cordofanus flight muscle bound up to 140 μM calcium at high affinity in the presence of ATP; removal of the ATP increased the maximum amount bound to 210 μM and the affinity approx. 3-fold. Calcium binding in the presence of ATP correlated with calcium activation of the ATPase but no changes in calcium binding occurred when the muscle was further activated by stretching.     

A proline shuttle in insect flight muscle.

A proline shuttle for oxidation of extramitochondrial NADH was reconstituted from soluble and mitochondrial fractions of blowfly ($\text{Phormia}$$\text{regina}$) flight muscle. The soluble fraction catalyzed reduction of Δ′-pyrroline-5-carboxylate to proline via the action of Δ′-pyrroline-5-carboxylate reductase (EC 1.5.1.2). The reaction required NADH as hydrogen donor, NAD (P) H being ineffective in this regard. Mitochondria catalyzed regeneration of Δ′-pyrroline-5-carboxylate from proline via action of proline oxidase. The capacity of the shuttle to operate under conditions of possible competition for Δ′-pyrroline-5-carboxylate between Δ′-pyrroline-5-carboxylate reductase and Δ′-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was incestigated. Results of these investigations indicate that dehydrogenase activity does not significantly interfere with shuttle activity.     

The contractile and regulatory proteins of insect flight muscle

1. Myosin, actin and the regulatory proteins were prepared from insect flight muscle. 2. The light subunit composition of the myosin differed from that of vertebrate muscle myosin. The ionic strength and pH dependence of the myosin adenosine triphosphatase (ATPase) were measured. 3. Actin was associated with a protein of subunit molecular weight 55000 and was purified by gel filtration. Impure actin had protein bound at a periodicity of about 40nm. 4. Regulatory protein extracts had tropomyosin and troponin components of subunit molecular weight 18000, 27000 and 30000. Crude extracts of regulatory proteins inhibited the ATPase activity of desensitized or synthetic actomyosin; this inhibition was relatively insensitive to high Ca(2+) concentrations. Purified insect regulatory protein produced as much sensitivity to Ca(2+) as did the rabbit troponin-tropomyosin complex. 5. Synthetic actomyosins were made from rabbit and insect proteins. Actomyosins containing insect myosin had a low ATPase activity that was activated by tropomyosin. The Ca(2+) sensitivity of actomyosins containing insect myosin or actin, with added troponin-tropomyosin complex from rabbit, was comparable with that of rabbit actomyosin.