R. Wayne Albers

Quantitative changes in cholinesterase activity of denervated muscle fibers and sole plates

Abstract Histochemical studies have indicated that normal muscle fibers exhibit little cholinesterase activity at regions other than the sole plate and that denervated muscle fibers manifest no decreased activity within 2 to 4 weeks. Because histochemical data may be quantitatively unreliable, it was decided to reinvestigate the distribution of cholinesterase with a microchemical modification of Ellmans quantitative cholinesterase method. Alternate frozen cross section (28 μ) from muscle stretched to constant length were examined histochemically and quantitatively. By counting the number of sole plates in the histochemical preparations it was possible to estimate the relative contributions of the sole-plate and the nonsole-plate tissue to the cholinesterase activity of the adjacent quantitatively analyzed tissue slice. Following denervation the sole-plate and non-sole-plate activity both decreased rapidly, reaching approximately 50 per cent of normal within 1 week. A very small additional loss in activity occurred during the following 7 weeks. The total protein content of the tissue slices declined more gradually, reaching 50 per cent of normal after 3 weeks. It is concluded that there is considerable cholinesterase activity not associated with sole plates and that denervation produces a very rapid decrease in cholinesterase activity. The neural influence on cholinesterase level is fairly specific inasmuch as denervation produces a more rapid decrease in cholinesterase activity than in total protein content whereas tenotomy produces less of a decrease in cholinesterase activity than in total protein content of the muscle.

Immunocytochemical localization of the sodium, potassium activated ATPase in knifefish brain

SummaryResults presented in this paper demonstrate the feasibility of using immunocytochemical methods to localize the (Na+ + K+)-ATPase, and its subunits, in the C.N.S. We have shown that in the Black Ghost knifefish,Sternarchus albifrons, the enzyme is located on the plasma membrane of the somata and dendrites of neurons and on the somata and cellular processes of glia. In myelinated axons the enzyme is restricted in localization to those portions of the axolemma not covered by the myelin sheath. The capacity of cell plasma membranes to restrict mobility of functionally important proteins should be considered in models of membrane structure in which lateral mobility of membrane components is considered a major characteristic.

The neural regulation of some phenotypic differences between the fiber types of mammalian skeletal muscle

Abstract The fibers of mammalian muscles differ qualitatively in the pH lability of their myosin ATPase. The ATPase of the α fibers is acid-labile, that of the β fibers is base-labile, and that of the αβ fibers is intermediate in pH lability. By using cross-reinnervation techniques, we sought to ascertain the role of the nerve in the regulation of these phenotypic differences between muscle fiber types. In cats and rats, the soleus muscle contains β fibers predominantly, whereas the flexor hallucis longus and medial gastrocnemius muscles contain all three fiber types. After reinnervation of the soleus by the nerve to the flexor hallucis longus or medial gastrocnemius, many clusters of α fibers were observed, and after crossreinnervation of the flexor hallucis longus many clusters of β fibers were found. We concluded that as a result of altered innervation there is a qualitative change in the type of ATPase in many muscle fibers; the αβ fibers can be converted to α or to β fibers and the α and β fibers can be interconverted. However, the α and β fibers do not appear to be convertible to αβ. Since the qualitative differences in pH lability probably reflect differences in protein composition of the enzyme, and since the type of protein manufactured by a cell is genetically controlled, the results are interpreted as indicating that regulation of gene expression is one of the “trophic” functions of the vertebrate neuron.

The neural regulation of gene expression in the muscle cell

Abstract Although it has long been known that the myosin adenosine triphosphatase (ATPase) activity of fast muscle is greater than that of slow muscle, qualitative differences between these myosins have only recently been demonstrated. The ATPase of slow myosin is relatively acid-stabile and alkali-labile whereas that of fast muscle is acid-labile and alkali-stabile. Moreover, the myosin from each of these types of muscle possesses specific closely associated proteins of low molecular weight (subunits) that are electrophoretically distinct. Thus, in these respects, the myosins of slow and fast muscles appear to be different proteins. After cross-reinnervation of cat slow and fast muscles, these qualitative properties of myosin become altered. The cross-reinnervated slow muscle does not merely develop increased myosin ATPase activity (as has been been previously reported), but the enzyme becomes more alkali-stabile and acid-labile; furthermore, the myosin loses its characteristic subunits and acquires those of fast-muscle myosin. The converse changes occur in cross-reinnervated fast muscle. Although the specific activity of enzymes is regulated in many ways (inhibition by metabolites, binding to intracellular constituents, changes in rate of protein degradation), the synthesis of proteins is qualitatively specified by gene action. Our observation that a qualitatively different myosin appears in cross-reinnervated muscle indicates that a new species of protein has been synthesized, and we therefore suggest that the nerve influences gene expression in the muscle cell.

Phenotypic differences between the actomyosin ATPase of the three fiber types of mammalian skeletal muscle

Abstract It is known that most mammalian hind limb muscles are composed of three types of muscle fibers when formalin-fixed tissue sections are stained for actomyosin ATPase activity. In the rat, the C fibers are high, the B fibers low, and the A fibers intermediate in enzyme activity. In addition to these quantitative differences in enzyme activity, we have observed that there are qualitative differences in the actomyosin ATPase of the A, B, and C fibers. Here we present our finding that the ATPase of the high-activity (C) fibers is labile in acid (pH 4.35) and the ATPase of the low-activity (B) fibers is labile in alkali (pH 10.4). The ATPase of the intermediate-activity (A) fibers is intermediate with respect to pH lability; its ATPase is more resistant to acid than is that of the C fibers and is more resistant to alkali than is that of the B fibers. Thus these muscle fibers are phenotypically different with respect to their actomyosin ATPase. The implications of these differences are noteworthy with reference to typing of muscle fibers in disease, neurotrophic influence on muscle fiber types, and genetic constitution of the skeletal muscle fiber.

Neural regulation of the structure of myosin

Abstract Mammalian skeletal muscle responds metabolically both to neural influences and to the demands of work. For example, the soleus exhibits increased speed of contraction when reinnervated by the peroneal nerve that normally supplies fast muscles, but this change can also be brought about merely by peroneal transection (a procedure which denervates the muscles antagonistic to soleus). We have investigated the effects of these two operative procedures on soleus myosin. Seven to eight months after reinnervation of rat soleus with peroneal nerve, the myosin Ca 2+ -ATPase had increased in specific activity, alkaline stability, and susceptibility to tryptic digestion. Tryptic digestion (25 C, 10 min, myosin: trypsin = 200:1) followed by SDS-gel electrophoresis produces seven distinct peptide bands in the case of normal soleus myosin. An additional peptide band (88,000 daltons) is characteristic of fast muscle myosin. This 88,000 dalton band is demonstrable in myosin prepared from cross-reinnervated soleus muscles. Qualitatively identical changes were produced in all of these properties of soleus myosin by transecting the peroneal nerves 7–8 months previously. However, the magnitudes of the changes were less than those occurring after cross-reinnervation. Cross-reinnervation experiments have been used as evidence for neurotrophic control of skeletal muscle myosin; however, the conversion of slow myosin to fast myosin when the peroneal nerve is merely transected renders this argument questionable. Since fast and slow muscle myosin heavy chains differ in primary or tertiary structure (or both), their changes after cross-reinnervation or after inactivation of antagonistic muscles must result from a qualitative transformation of myosin which is presumably accomplished by the regulation of myosin biosynthesis.

Transamination reactions of rat brain

Abstract The spectrum of transamination reactions of rat brain involving α-ketoglutarate and pyruvate have been quantitatively estimated employing an extremely sensitive, fast and simple technique of assay. Evidence is presented in support of the separate identities of enzymes catalyzing the transaminations from phenylalanine, tyrosine, 3,4-dihydroxyphenylalanine, tryptophan and 5-hydroxytryptophan to α-ketoglutarate.

Complex Kinetic Behavior in the Na,K‐and Ca‐ATPases. Evidence for Subunit‐Subunit Interactions and Energy Conservation during Catalysis

A recurrent problem in analysis of Na,K-ATPase partial reactions is the failure of linear consecutive schemes (such as the Albers-Post mechanism) to account for the presence of complex kinetic behavior in time-resolved measurements. Examples of this behavior include the multiphasic patterns of phosphorylation and dephosphorylation catalyzed by the electric organ+ and mammalian Na,K-ATPases5 and the inability of rate constants measured in the pre-steady state to account for results in the steady To resolve these questions, alternatives to the Albers-Post model have been proposed involving parallel independent pathways for ATP h y d r ~ l y s i s ~ . ~ , ~ or a unique initiation sequence leading to the steady-state reaction cycle.6 We summarize here kinetic evidence that requires for explanation elements of both of these models and, in addition, suggests that these complexities arise from homologous interactions between a subunits in an oligomer. In essence, the oligomer is a parallel pathway model with conformational coupling between the catalytic subunits, which prevents

Evidence for Parallel Pathways of Phosphoenzyme Formation in the Mechanism of ATP Hydrolysis by Electrophorus Na,K-ATPase

Publisher Summary In a paper presented at the Second International Conference on the Properties and Function of the Na, K–ATPase, Klodos and Norby showed that the measured rate of ATP hydrolysis in Na, K–ATPase from ox brain was faster than the expected rate calculated from the amount of K + -sensitive phosphoenzyme (E 2 -P) and its turnover rate. An underestimation of the rate of dephosphorylation, and hence ATP hydrolysis might result from the presence of tightly bound ATP that continues to produce phosphoenzyme after rephosphorylation of the enzyme is prevented by the dilution of the labeled free substrate. The chapter discusses the measurements of P i release under conditions preventing rephosphorylation of the enzyme. The results show that when EDTA or excess unlabeled ATP is added to the phosphoenzyme, P i release exceeds the amount of E–P decay supporting the existence of a pool of bound nucleotide that does not rapidly exchange with ATP in the medium. A comparison of the observed pattern of E–P decay versus time with the predicted patterns for enzyme mechanisms containing more than one phosphoenzyme intermediate revealed that the mechanism of ATP hydrolysis in eel Na, K–ATPase very likely involves more than one pathway for phosphoenzyme formation.

Cold resistance of the brain during hibernation: Temperature sensitivity of the partial reactions of the Na+,K+-ATPase☆

Brain Na+,K+-ATPase from the hibernating hamster was shown to have a lower temperature coefficient than that from the warm-adapted hamster. However, the temperature coefficients of the partial reactions associated with the Na+,K+-ATPase were similar in both the warm-adapted and hibernating hamster. The steady-state level of phosphorylated enzyme was also similar in both physiological states. This type of acclimation in hibernation is apparently one in which a qualitative change in the enzyme has occurred. It was concluded that these temperature sensitive sites are associated with conformational processes that occur within the enzyme and further are dependent upon the lipid environment of the enzyme.