Molecular mechanism of actomyosin-based motility

Sophisticated molecular genetic, biochemical and biophysical studies have been used to probe the molecular mechanism of actomyosin-based motility. Recent solution measurements, high-resolution structures of recombinant myosin motor domains, and lower resolution structures of the complex formed by filamentous actin and the myosin motor domain provide detailed insights into the mechanism of chemomechanical coupling in the actomyosin system. They show how small conformational changes are amplified by a lever-arm mechanism to a working stroke of several nanometres, explain the mechanism that governs the directionality of actin-based movement, and reveal a communication pathway between the nucleotide binding pocket and the actin-binding region that explains the reciprocal relationship between actin and nucleotide affinity. Here we focus on the interacting elements in the actomyosin system and the communication pathways in the myosin motor domain that respond to actin binding.Received 12 January 2005; received after revision 4 March 2005; accepted 23 March 2005     

Dynamics of proteins in Golgi membranes: comparisons between mammalian and plant cells highlighted by photobleaching techniques

In less than a decade the green fluorescent protein (GFP) has become one of the most popular tools for cell biologists for the study of dynamic processes in vivo. GFP has revolutionised the scientific approach for the study of vital organelles, such as the Golgi apparatus. As Golgi proteins can be tagged with GFP, in most cases without altering their targeting and function, it is a great substitute to conventional dyes used in the past to highlight this compartment. In this review, we cover the application of GFP and its spectral derivatives in the study of Golgi dynamics in mammalian and plant cells. In particular, we focus on the technique of selective photobleaching known as fluorescence recovery after photobleaching, which has successfully shed light on essential differences in the biology of the Golgi apparatus in mammalian and plant cells.     

Molecular mechanism for the production of multiple forms of MM creatine kinase

Incubation of human, canine or rabbit MM creatine kinase with carboxypeptidase-N or B resulted in the production of 2 additional enzyme forms with increased anodal migration on polyacrylamide gels. The C-terminal amino acid of tissue MM creatine kinase from all 3 species was shown to be lysine, a specific substrate for carboxypeptidase-N and B.     

Molecular mechanism for the production of multiple form of MM creatine kinase

Summary Incubation of human, canine or rabbit MM creatine kinase with carboxypeptidase-N or B resulted in the production of 2 additional enzyme forms with increased anodal migration on polyacrylamide gels. The C-terminal amino acid of tissue MM creatine kinase from all 3 species was shown to be lysine, a specific substrate for carboxypeptidase-N and B.     

Molecular mechanism on the energy transfer and conversion in the photosynthetic system

Zusammenfassung Auf molekularer Basis wird die Energieübertragung und die Energieumsetzung in einem primären photosynthetischen System untersucht. Die Energieübertragung in den Pigmenten und im Chlorophyll-A wird mit der Energieübergangswahrscheinlichkeit und dem Diffusionsmodell eines intramolekularen «Exitons» mit Dipol-Dipol-Wechselwirkung behandelt. Bei einer Chlorophyllkonzentration von 0.1M/1 wird die Diffusionskonstante des «Exitons»D _e 1.1 × 10^–3 cm²/sec und seine Diffusionslängel _e 250 Å. Als Energieumsetzung wird die Anregung eines – -Tripletts des Chlorophyll-A mit Hilfe eines paramagnetischen Metallenzyms vorgeschlagen und diskutiert.

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This work was supported by grants from the Japanese Society for the Promote of Science. The author wishes to express his gratitude to ProfessorM. Kotani of Tokyo University and ProfessorK. Oomori and ProfessorG. Tomita for their encouragement.     

Association of Golgi vesicles containing acid phosphatase with the chromatoid body of rat spermatids

The relationship between the Golgi apparatus and the small vesicles associated with the chromatoid body was investigated using a cytochemical technique. It was observed that in early spermatids, when the chromatoid body appears in close contact with the Golgi complex, and all through its migration to the caudal pole of the nucleus, some of the vesicles that accompany the organelle display acid phosphatase activity. It is concluded that these smooth vesicles originate in the Golgi apparatus.     

Analysis of a sub-proteome which co-purifies with and is phosphorylated by the Golgi casein kinase

In an attempt to gain information about the identity of the Golgi apparatus casein kinase(s) (G-CK), responsible for the phosphorylation of caseins in lactating mammary gland, the proteins present in fractions enriched in G-CK activity eluted from DEAE-Sepharose and heparin-Sepharose columns were resolved by two-dimensional electrophoresis and analyzed by mass spectrometry. This led to the identification of 47 proteins altogether, none of which is a bona fide protein kinase. At least 9 of the identified proteins however, are readily phosphorylated by co-purifying G-CK activity, and 7 are physically associated with it to give supramolecular complex(es) of about 500 kDa as judged from Superdex S200 gel fitration and glycerol gradient ultracentrifugation experiments. In contrast, the apparent molecular weight of G-CK estimated from an in gel activity assay after SDSPAGE and renaturation is about 41 kDa. Many of the proteins phosphorylated by and/or associated with G-CK belong to the category of chaperonines, including HSP90, GRP-94 and −78, and various isoforms of protein disulfide isomerases, suggesting a global role of this kinase in the modulation of protein folding. Received 21 October 2005; received after revision 30 November 2005; accepted 6 December 2005 †These authors contributed equally to this work.     

Localization of cholesterol in the Golgi apparatus of cardiac muscle cells

Summary Filipin (a polyene) interacts with cholesterol in membranes, producing distinctive deformations that can be detected by freeze-fracture. The distribution of filipin-induced deformations in the Golgi apparatus of cardiac myocytes suggests a role for this organelle in the transformation of cholesterol-poor membrane to cholesterol-rich membrane.Acknowledgments. This work was supported by a grant (No. 779) from the British Heart Foundation. I thank Dr E. Massey and Mr A. Slade for their help. Filipin was generously donated by Upjohn Ltd, Crawley, Sussex, U.K.