Biomedical subjects

D L Mykles

Publications and source records attributed to D L Mykles.

At least 37 records · Page 2Linked to original sources

PubMed1996

Differential effects of bovine PA28 on six peptidase activities of the lobster muscle proteasome (multicatalytic proteinase).

PA28, an endogenous activator of the bovine proteasome, stimulated the branched-chain amino acid-preferring (BrAAP; 99-fold activation), small-neutral amino acid-preferring (11-fold), acidic chymotrypsin-like (26-fold), and peptidylglutamyl peptide hydrolase (14-fold) activities of the lobster muscle proteasome, while having little or no effect on the trypsin-like, neutral chymotrypsin-like, and caseinolytic activities. These results show that the BrAAP activity, which has been linked to the degradation of myofibrillar proteins by the heat-activated proteasome, is allosterically regulated. However, the activation by PA28 differs from that induced by heat treatment, since heat activation stimulated both the BrAAP and the proteolytic activities but not the other peptidase activities. PA28 shifted the pH optimum of the acidic chymotrypsin-like activity from pH 6-6.5 to pH 7-7.5, while stimulating the activity about 10-fold. These results suggest that PA28 is involved in the activation of the acidic chymotrypsin-like component at physiological pH.

Amino Acids↗

PubMed1996

Crustaceans as a model for microgravity-induced muscle atrophy.

Atrophy of skeletal muscles is a serious problem in a microgravity environment. It is hypothesized that the unloading of postural muscles, which no longer must resist gravity force, causes an accelerated breakdown of contractile proteins, resulting in a reduction in muscle mass and strength. A crustacean model using the land crab, Gecarcinus lateralis, to assess the effects of spaceflight on protein metabolism is presented. The model is compared to a developmentally-regulated atrophy in which a premolt reduction in muscle mass allows the withdrawal of the large claws at molt. The biochemical mechanisms underlying protein breakdown involves both Ca(2+)-dependent and multicatalytic proteolytic enzymes. Crustacean claw muscle can be used to determine the interactions between shortening and unloading at the molecular level.

Actin Cytoskeleton↗

PubMed1995

Polyubiquitin in crustacean striated muscle: increased expression and conjugation during molt-induced claw muscle atrophy.

The claw muscles of decapod crustaceans undergo a molt-induced atrophy to facilitate withdrawal of the claws at ecdysis. Polyubiquitin expression, as well as the levels of ubiquitin conjugates, a ubiquitin-conjugating enzyme involved in the ATP/ubiquitin-dependent proteolytic pathway (crustacean E2(16 kDa) homolog of Drosophila UbcD1), and proteasome, were examined to determine the role of ATP/ubiquitin-dependent proteolysis in the enhanced degradation of myofibrillar proteins during muscle atrophy. A partial-length clone (1.7 kb) of polyubiquitin was isolated from a lobster muscle cDNA library; the 5' end lacked the 5' untranslated region (UTR) and the beginning of the first ubiquitin monomer, while the 3' end contained the terminal ubiquitin monomer and 3' UTR. The deduced amino acid sequence was 100% identical with that from Manduca, Drosophila, and human. In land crab claw muscle, the polyubiquitin mRNA (2.7 kb) increased about 5-fold and ubiquitin-protein conjugates (> 200 kDa) increased about 8-fold during atrophy. In contrast, the level of a ubiquitin-conjugating enzyme (E2(16 kDa)) remained unchanged. The proteasome, which constitutes the catalytic core of the ATP/ubiquitin-dependent proteinase complex, increased about 2-fold during proecdysis, reaching its highest level immediately before ecdysis. These results suggest that the ATP/ubiquitin-dependent proteolytic pathway contributes to the changes in protein metabolism that occur during molt-induced muscle atrophy.

Amino Acid Sequence↗

PubMed1995

The multicatalytic proteinase (proteasome) of the hawkmoth, Manduca sexta: catalytic properties and immunological comparison with the lobster enzyme complex.

The proteasome plays a central role in ubiquitin-dependent and -independent proteolysis in eukaryotic cells. The hawkmoth proteasome was purified from larval body wall and characterized with respect to substrate specificity, sensitivity to protease inhibitors, and cross-reactivity with monoclonal antibodies (mAbs) raised against human placenta proteasome. Leupeptin selectively inhibited the trypsin-like activity (T-L) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activities, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglutamyl peptide hydrolase, branched-chain amino acid preferring, and caseinolytic activities 20-, 18-, and 3.8-fold, respectively. All four peptidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimensional immunoblot analysis showed that the level and subunit composition of the proteasome varied between tissues. The relative levels of proteasome were high in intersegmental muscle and ovary, lower in Malpighian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body. The tissues differed in the relative amount of a 41-kDa doublet; a 22-kDa subunit was present only in the male accessory gland. Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, compared with 28 subunits in lobster. Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human zeta, C2, C3, and C8 alpha-type subunits in the hawkmoth and lobster enzymes. Two of the four mAbs reacted with three or more of the hawkmoth subunits and three of the mAbs reacted with two or more of the lobster subunits. In addition, two other mAbs that recognize epitopes shared by a number of alpha-type subunits indicated that at least 15 (lobster) or 16 (hawkmoth) subunits were alpha-type. These results suggest that much of the subunit complexity of the arthropod proteasomes is a consequence of extensive post-translational modifications.

Amino Acid Sequence↗

PubMed1995

Branched-chain-amino-acid-preferring peptidase activity of the lobster multicatalytic proteinase (proteasome) and the degradation of myofibrillar proteins.

The multicatalytic proteinase (MCP or proteasome) is a large proteolytic complex that contains at least five catalytic components: the trypsin-like, chymotrypsin-like, peptidylglutamyl-peptide hydrolase (PGPH), branched-chain-amino-acid-preferring (BrAAP) and small-neutral-amino-acid-preferring activities. We have shown that brief heating of the lobster muscle proteasome activates a proteolytic activity that degrades casein and myofibrillar proteins and is distinct from the trypsin-like, chymotrypsin-like and PGPH components. Here we identify the BrAAP activity as a catalytic component involved in the initial degradation of myofibrillar proteins in vitro. This conclusion is based on the following. (1) The BrAAP component was activated by heat-treatment, whereas the other four peptidase activities were not. (2) The BrAAP and proteolytic activities showed similar sensitivities to cations and protease inhibitors: both were inhibited by 3,4-dichloroisocoumarin, chymostatin, N-ethylmaleimide and Mg2+, but were not affected by leupeptin, phenylmethanesulphonyl fluoride or Li+. (3) The BrAAP activity was inhibited most strongly by casein substrates and troponin; conversely, the troponin-degrading activity was inhibited by the BrAAP substrate. Another significant finding was that incubation of the heat-activated MCP in the presence of chymostatin resulted in the limited cleavage of troponin-T2 (45 kDa) to two fragments of 41 and 42 kDa; this cleavage was completely suppressed by leupeptin. These results suggest that under certain conditions the trypsin-like component can cleave endogenous protein.

Amino Acids, Branched-Chain↗

PubMed1995

Changes in the structure and function of the multicatalytic proteinase (proteasome) during programmed cell death in the intersegmental muscles of the hawkmoth, Manduca sexta.

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta are a well-characterized model system for examining the biochemical changes that accompany programmed cell death during development. These giant muscles die during a 30-hr period in response to a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. When the ISMs become committed to die, there are dramatic increases in both ubiquitin expression and ubiquitin-dependent proteolysis. Since the multicatalytic proteinase (MCP) is responsible for ATP/ubiquitin-dependent proteolysis in cells, we examined its composition and properties. The purified enzyme from whole larval integumentary tissues resembles MCPs isolated from other species with respect to subunit composition and general catalytic properties. However, when MCP was isolated from condemned ISMs, we observed an approximately ninefold increase in proteinase activity compared to MCP from precommitment muscles. This increase in proteolytic activity was correlated with an approximately eightfold increase in the absolute amounts of MCP protein as determined by Western blotting and densitometry. When purified MCP from condemned muscles was examined by two-dimensional polyacrylamide gel electrophoresis, four new subunits that were not detected in the precommitment muscles were present. Correlated with the addition of these new subunits was a dramatic increase in the levels of immunodetectable MCP throughout the cytoplasm and within the nuclei of dying muscles. These changes in MCP were regulated by the same hormonal signals that mediate cell death. These data are consistent with the hypothesis that when the ISMs become committed to die, more MCP accumulates in cells and new subunits are synthesized that change both the enzymatic properties and the conformation of MCP, which in turn participates in the dramatic proteolysis that accompanies cell death.

Amino Acid Sequence↗

PubMed1994

Calpain inhibitors block Ca(2+)-induced suppression of neurite outgrowth in isolated hippocampal pyramidal neurons.

Ca2+ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these Ca(2+)-dependent effects is unclear. Since cytoskeletal proteins are rapidly degraded by Ca(2+)-dependent proteinases (calpains) in vitro and in vivo, we investigated whether Ca(2+)-induced pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal-like neurons were cultured and the ability of the membrane-permeable calpain inhibitors ethyl(+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butyl carbamoyl]-2 - oxiranecarboxylate (EST) and carbobenzoxyl-valyl-phenylalanyl-H (MDL 28170) to block the Ca2+ ionophore A23187-induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a significant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST (5 or 20 microM) to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 (20 microM) showed the same effect as EST in preventing ionophore-induced pruning of dendrites and axons at 100 and 300 nM concentrations, respectively, of A23187. EST (20 microM) did not block the A23187-induced rise of [Ca2+]i as measured with fura-2. These results suggest that calpains play a role in Ca(2+)-induced pruning of neurites in isolated hippocampal pyramidal neurons.

Animals↗

PubMed1994

Calcium-dependent proteinase activity in root cultures of Arabidopsis.

The presence of calcium-dependent proteinase in plants was investigated using soluble proteins from Arabidopsis root cultures. Calcium increased the proteolytic activity by two to three fold. Calcium-stimulated proteolytic activity was inhibited by different protease inhibitors. The highest inhibition (79%) of calcium-dependent proteolytic activity was observed in the presence of leupeptin. Thirty-eight percent of calcium-dependent proteolytic activity was inhibited by E-64, an inhibitor of cysteine proteinases. Calmodulin did not have any effect on calcium-dependent as well as calcium-independent proteolytic activity in the cellular extract. These results provide the first evidence for the presence of calcium activated proteinase in plants and open new avenues to investigate the mode of calcium action in plants.

Arabidopsis↗

PubMed1993

Cloning of a crustacean myosin heavy chain isoform: exclusive expression in fast muscle.

A clone of a fast isoform of myosin heavy chain (HC) gene was isolated from a cDNA1 expression library made from mRNA purified from the deep abdominal flexor muscle of the lobster, Homarus americanus. The cDNA (1.5 kb) contained the 3' untranslated region (UTR) and the coding sequence for the last 413 amino acid residues of the carboxyl terminus of the polypeptide. The deduced amino acid sequence showed high homology with that of myosin HCs from Drosophila (73% identity), nematode (57% identity), and vertebrates (49% identities). Hydropathy plots showed a 28-amino acid periodicity that is consistent with the alpha-helical coiled coil structure of the rod region of native myosin. Northern blot analysis and in situ hybridization showed that the fast myosin HC isoform was expressed only in fast fibers; the probes did not hybridize to mRNA from slow fibers. The message consisted of a single transcript of 6.6 kb. The intracellular localization of the fast myosin mRNA was not uniform. The mRNA was largely confined to the intermyofibrillar spaces and to the subsarcolemmal cytoplasm of the fiber periphery and large infoldings of the cell membrane. Within these regions the mRNA was concentrated in the cytoplasm immediately surrounding the nuclei. This constitutes the first report of the cloning and expression of a myosin HC gene from a crustacean species.

Amino Acid Sequence↗

PubMed1993

Lobster muscle proteasome and the degradation of myofibrillar proteins.

The lobster proteasome is primarily a cytosolic enzyme in crustacean striated muscles, although a small amount (< 1% of total) occurs in aggregates associated with invaginations of the cell membrane. The complex exists in vitro in three distinct catalytic states (basal, SDS-activated, and heat-activated forms) which have identical subunit compositions. This review summarizes recent results showing that the branched-chain amino acid-preferring (BrAAP) activity mediates the hydrolysis of myofibrillar proteins by the heat-activated proteasome: (a) only the BrAAP activity is stimulated by heat treatment; (b) the BrAAP activity is strongly inhibited by protein substrates, and (c) both the BrAAP and proteolytic activities show similar sensitivities to cations and protease inhibitors.

Amino Acid Sequence↗

PubMed1992

Immunocytochemical localization of the multicatalytic proteinase (proteasome) in crustacean striated muscles.

Multicatalytic proteinase (MCP) is thought to play a central role in the processing and turnover of intracellular proteins in eukaryotic cells. Immunocytochemistry was used to determine the intracellular distribution of the MCP in the claw muscles of the land crab, Gecarcinus lateralis, and the claw and abdominal muscles of the American lobster, Homarus americanus. Cryosections were stained with an affinity-purified polyclonal antibody to lobster MCP that cross-reacted with the land crab enzyme. Two types of staining were observed: a diffuse cytoplasmic staining, and a dense aggregate staining primarily associated with invaginations of the cell membrane. The cytoplasmic staining appeared reticulated in favorable transverse sections due to a preferential localization of MCP to the intermyofibrillar space. The aggregate staining was associated with neither nuclei nor mitochondria, since stains specific for these organelles (Hoechst stain and nicotinamide adenine dinucleotide diaphorase histochemistry, respectively) did not colocalize with the aggregates. Trypsinlike peptidase activities of isolated microsomal and postmicrosomal fractions indicated that less than 1% of the total MCP was associated with the microsomal fraction. Immunoprecipitation of the same fractions confirmed the presence of MCP in the microsomes as well as in the cytosol. These results suggest that the MCP is primarily associated with cytoplasmic components; the aggregate staining may result from the association of the MCP with cellular membrane systems.

Animals↗

PubMed1991

Sodium dodecyl sulfate and heat induce two distinct forms of lobster muscle multicatalytic proteinase: the heat-activated form degrades myofibrillar proteins.

A multicatalytic proteinase (MCP) purified from lobster claw and abdominal muscles degrades a variety of peptide and protein substrates. The enzyme is activated by low concentrations (0.03%) of sodium dodecyl sulfate (SDS) and brief (1 min) heating at 60 degrees C. The lobster MCP can assume three stable and functionally distinct states in vitro; these are classified as the basal, heat-activated, and SDS-activated forms. The basal MCP possessed high trypsin-like peptidase activity and low chymotrypsin-like peptidase, peptidylglutamyl-peptide hydrolase, and caseinolytic activities; incubation of the basal form with SDS stimulated the peptidylglutamyl-hydrolase activity about 30-fold and inhibited the other three activities 80% to 100%. Heating the basal form stimulated caseinolytic activity about 6-fold with little effect on the peptidase activities. The heat-activated enzyme also degraded myosin, tropomyosin, troponin, and actin depolymerizing factor; alpha-actinin was resistant to proteolysis. Incubation of the heat-activated MCP with SDS inhibited the trypsin-like, chymotrypsin-like, and proteinase activities 95 to 100% and stimulated the peptidylglutamyl-hydrolase activity about 16-fold. Incubation of myosin with either the basal or the heat-activated forms in the presence of SDS generated identical proteolytic fragments of the myosin heavy chain, suggesting that SDS induced a third form that can be produced from either the basal or the heat-activated forms. The heat-activated form produced proteolytic fragments of myosin heavy chain different from those generated by either basal or heat-activated enzymes in the presence of SDS. Furthermore, 100 mM KCl stimulated the caseinolytic activity of the heat-activated form 24% and inhibited the trypsin-like and peptidylglutamyl-hydrolase activities 56 and 20%, respectively. These results, though indirect, suggest that heating induced a proteinase activity that was distinct from the three peptidase activities. Activation of the basal form with SDS was reversible, since precipitation of dodecyl sulfate with 100 mM KCl restored trypsin-like activity and inhibited peptidylglutamyl-hydrolase activity. In contrast, removal of dodecyl sulfate from the SDS-activated form that was derived from the heat-activated MCP induced its conversion to the basal form. Thus, although heat-activation was irreversible, the heat-activated form was converted back to the basal form via the SDS-activated form.

Amino Acid Sequence↗

PubMed1991

Differential effects of oleic acid, sodium dodecyl sulfate, and protease inhibitors on the endopeptidase activities of the lobster multicatalytic proteinase.

1. Lobster muscles contain a latent multicatalytic proteinase; heating at 60 degrees C for 1-2 min converts the latent form to a heat-activated form with enhanced proteolytic activity. Both forms have three endopeptidase activities, which are classified as the trypsin-like, chymotrypsin-like, and peptidylglutamylpeptide bond hydrolyzing activities. 2. Sulfhydryl reagents (mersalyl acid, N-ethylmaleimide, hemin, iodoacetamide, and p-chloromercurisulfonic acid), benzamidine, and chloromethyl ketones inhibited all three activities of the heat-activated form. Leupeptin and antipain inhibited only the trypsin-like activity, while the chymotrypsin-like activity was the most sensitive to diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, aprotinin, and soybean trypsin inhibitor. Pepstatin and L-trans-epoxysuccinylpeptides had little effect on the peptidase activities. 3. Sodium dodecyl sulfate and oleic acid preferentially activated the peptidylglutamyl-peptide hydrolyzing activity of the latent form, whereas N-ethylmaleimide stimulated both the trypsin-like and peptidylglutamyl-peptide hydrolases. These results suggest that the lobster enzyme is an atypical serine proteinase.

Amino Acid Sequence↗

PubMed1989

High-molecular-weight serine proteinase from lobster muscle that degrades myofibrillar proteins.

A latent alkaline serine proteinase (ASP) has been extracted from the soluble fraction of lobster claw and abdominal muscles. The enzyme, which was irreversibly activated 30- to 40-fold by brief (2-3 min) heating at 60 degrees C, had an optimal caseinolytic activity at pH 7.75. Its molecular weight was estimated to be 740,000 by gel filtration chromatography. Serine protease inhibitors (diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, benzamidine, and chloromethyl ketones) suppressed ASP activity 22 to 70%. In addition, sulfhydryl-blocking reagents and hemin inhibited activity 69 to 100%; leupeptin and E-64, however, did not. Pepstatin A, ethylenediaminetetraacetate, and adenosine triphosphate were without effect. These results suggest that the lobster ASP is a serine proteinase that contains one or more sulfhydryl groups essential for catalysis. ASP was stimulated by dithiothreitol and inhibited by CaCl2 and oleic and linoleic acids. The enzyme was partially activated by low concentrations of sodium dodecyl sulfate; 0.05% produced activities 13% of that of preparations heated at 60 degrees C. Neither poly-L-lysine, urea, dimethylsulfoxide, oleic acid, linoleic acid, nor N-ethylmaleimide activated the enzyme. The ASP degraded most myofibrillar proteins, but showed a preferential hydrolysis of paramyosin, troponin-I and -C, and myosin alpha light chain.

Actomyosin↗

PubMed1989

Purification and characterization of a multicatalytic proteinase from crustacean muscle: comparison of latent and heat-activated forms.

A high-molecular-weight (Mr 740,000) multicatalytic proteinase (MCP) was purified over 3100-fold from soluble extracts of lobster claw and abdominal muscles. The enzyme was extracted from muscle in a latent state; brief (3 min) heating of an ammonium sulfate fraction (45-65% saturation) at 60 degrees C irreversibly activated the proteinase while denaturing about 55% of the protein. MCP was further purified by chromatography on two sequential arginine-Sepharose columns and a Mono Q column with a yield of 60%. About 1.12 mg MCP was obtained for every 100 g tissue. In addition to [14C]methylcasein, the MCP hydrolyzed synthetic peptide substrates of trypsin and chymotrypsin at pH 7.75. Serine protease inhibitors (diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, aprotinin, benzamidine, soybean trypsin inhibitor, chloromethyl ketones), leupeptin, antipain, hemin, sulfhydryl-blocking reagents (N-ethylmaleimide, mersalyl acid, p-chloromercurisulfonic acid, iodoacetamide) suppressed activity while Ep-475, a specific inhibitor of cysteine proteinases, had no effect, suggesting the MCP is a serine proteinase with one or more cysteine residues indirectly involved in catalysis. The latent MCP was purified using the same procedure as that for the active form, except that thermal activation was omitted. The elution characteristics of latent MCP from the arginine-Sepharose and Mono Q columns were identical to those of active MCP. Since the purified latent form could still be activated by heating, activation did not involve denaturation of an endogenous inhibitor or substrate. Subunit compositions of both forms were identical in two-dimensional polyacrylamide gels; each was composed of eight polypeptides with molecular weights between 25,000 and 32,500 and a ninth polypeptide with a molecular weight of 41,000. Electron microscopy of negatively stained material showed that each form was a cylinder-shaped particle (approximately 10 x 15 nm) consisting of a stack of four rings with a hollow center; no differences in shape, dimensions, or submolecular structure were observed. These results suggest that activation probably involved small conformational changes rather than covalent modifications or rearrangement of subunits within the complex.

Animals↗

PubMed1988

Histochemical and biochemical characterization of two slow fiber types in decapod crustacean muscles.

Myofibrillar proteins in muscles of the claws and abdomen of lobster, Homarus americanus, and the claws of fiddler crab, Uca pugnax, and land crab, Gecarcinus lateralis, have been analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fibers contained numerous isoforms of structural and regulatory proteins in assemblages correlated with fiber type. One fast (F) and two slow (S1 and S2) fibers were identified. All F fibers possessed two isoforms of paramyosin (P1 and P2), while all slow fibers, with the exception of Uca major claw, contained only the P2 variant. S1 and S2 fibers were distinguished by the distribution of a large isoform of troponin-T (T1; Mr = 55,000); S2 fibers in all three species contained T1 in addition to one or two smaller-molecular-weight variants usually associated with S1 fibers. In order to determine whether the slow fibers differed in histochemical properties, land crab claw closer muscle was cryosectioned and stained for myofibrillar ATPase and NADH diaphorase activities. Most S2 fibers had lower ATPase and higher NADH diaphorase activities than S1 fibers, which indicated that S2 fibers had a lower rate of contraction and were more fatigue-resistant than S1 fibers. It is proposed that the S1 and S2 fibers defined by biochemical and histochemical criteria are identical to the slow-twitch and tonic fibers, respectively characterized physiologically.

Adenosine Triphosphatases↗

PubMed1986

Four Ca2+-dependent proteinase activities isolated from crustacean muscle differ in size, net charge, and sensitivity to Ca2+ and inhibitors.

Four Ca2+-dependent proteinase activities in lobster claw and abdominal muscle have been resolved by high-performance liquid chromatography on gel filtration and ion-exchange columns. These activities, which do not appear to be generated by autolytic or other degradative processes, differed from each other in molecular weight (peak I, Mr = 310,000; peak IIa, Mr = 125,000; peak IIb, Mr = 195,000; peak III, Mr = 59,000) and net charge, as indicated by elution from an ion-exchange column with a NaCl gradient. Although optimum activity occurred at 5-10 mM Ca2+ at pH 6.8, the enzymes differed in activation at lower Ca2+ concentrations. The concentrations required for half-maximal activation were 0.6 mM for peak III, 1 mM for peak I, 1.5 mM for peak IIa, and 2 mM for peak IIb. Only the peak III proteinase was active at 100 microM Ca2+; none were active at 10 microM and below. Although the lobster Ca2+-dependent proteinases were all inhibited, from 75 to 98%, by the cysteine proteinase inhibitors leupeptin, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine, and iodoacetamide, they showed differential responses to the aspartic proteinase inhibitor pepstatin and the serine proteinase inhibitor phenylmethanesulfonyl fluoride. Peak I was moderately (26%) inhibited by phenylmethanesulfonyl fluoride, whereas peaks IIb and III were inhibited 26 and 90%, respectively, by pepstatin. This is the first description of multiple forms of Ca2+-dependent proteinase that require Ca2+ at millimolar levels in any tissue, either vertebrate or invertebrate.

Animals↗

PubMed1985

Heterogeneity of myofibrillar proteins in lobster fast and slow muscles: variants of troponin, paramyosin, and myosin light chains comprise four distinct protein assemblages.

Fast and slow muscles from the claws and abdomen of the American lobster Homarus americanus were examined for adenosine triphosphatase (ATPase) activity and for differences in myofibrillar proteins. Both myosin and actomyosin ATPase were correlated with fiber composition and contractile speed. Four distinct patterns of myofibrillar proteins observed in sodium dodecyl sulfate-polyacrylamide gels were distinguished by different assemblages of regulatory and contractile protein variants. A total of three species of troponin-T, five species of troponin-I, and three species of troponin-C were observed. Lobster myosins contained two groups of light chains (LC), termed "alpha" and "beta." There were three alpha-LC variants and two beta-LC variants. There were no apparent differences in myosin heavy chain, actin, and tropomyosin. Only paramyosin showed a pattern completely consistent with muscle fiber type: slow fibers contained a species (105 kD) slightly smaller than the principle variant (110 kD) in fast fibers. It is proposed that the type of paramyosin present could provide a biochemical marker to identify the fiber composition of muscles that have not been fully characterized. The diversity of troponin and myosin LC variants suggests that subtle differences in physiological performance exist within the broader categories of fast- and slow-twitch muscles.

Actomyosin↗