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j.rushbrook

Julie Rushbrook

Professor of Biochemistry; Director, Protein Structure
Core Laboratory
Ph.D. degree from Rutgers, the State University of New Jersey




Exploration of protein structure/function, focusing currently on myosin isoforms and NAD+-dependent isocitrate dehydrogenase

A. Myosin
Myosin, a major player in muscle contraction, contains two heavy chains of about 2000 residues which intertwine in an alpha-helical coiled coil over their C-terminal halves and then separate to form elongated globular structures. Each globular "head" binds two light chains of about 200 amino acids, one termed the regulatory light chain (LC2), the other the essential light chain (LC1/3) .

The avian adult pectoralis major, the larger of the breast muscles, is a fast white (glycolytic) muscle involved in flight. Throughout most of its fibers there is a single myosin molecule with two heavy chains which are the product of a single gene. The regulatory and essential light chains are generated from separate genes. The essential light chain has two forms differing at their N-termini, LC1 and LC3, resulting from different transcription start sites and alternate splicing. A single myosin molecule contains LC1 on one head, LC3 on the other.

During development there is a progression of six fast-type heavy heavy chains, the products of unique genes. Four heavy chains are present at 16 days in ovo, HCs II, IV, VI and VII. One, HC IV, becomes the predominant form at 19 days in ovo. Hatching occurs at embryonic day 21. Between post-hatch days 3 and 5 there is a rapid transition to HC V, the post-hatch heavy chain. HC V persists for 90 days, during which it is gradually replaced by the adult isoform, HC II. While LC2 and the essential LC1 are present in the earliest myosin forms, LC3 does not appear until after hatching.

Study of additional fast contracting adult muscles revealed a surprising number of unique heavy chain forms distributed in a muscle specific fashion. In general, HCs I, III and V were favored in fast white (glycolytic) fibers, while heavy chains II and IV were characteristic of fast red (aerobic) fibers. The forms found include some also present during development in the pectoralis major muscle (II, IV and V).

The heavy chain of adult fast myosin from several species is known to be methylated at four positions. A lysine residue near the N-terminus, Lys-35, is partially monomethylated, Lys-130 and Lys-550 are trimethylated, and His-754 is monomethylated.

Current research addresses the requirement for multiple fast myosin heavy chains in developing and adult muscles, and the function of methylated residues.

B. NAD+-dependent isocitrate dehydrogenase
Mammalian mitochondrial NAD+-dependent isocitrate dehydrogenase, a focal point in aerobic metabolism, is subject to exquisite control. The enzyme displays homotropic allosteric behaviour for substrates isocitrate and NAD+, and reacts in a heterotropic fashion positively to ADP and negatively to ATP, NADH and NADPH.

The basic unit is a tetramer containing two regulatory subunits, IDH-1 and IDH-2, the products of distinct genes, and two catalytic subunits, IDH-3 and IDH-4, the products of a single gene but differing in some as yet unknown way at the protein level. The regulatory subunits possess 3'-alternatively spliced forms with tissue-specific expression, one form predominating in heart muscle. The two protein products from alternative splicing for IDH-1 differ markedly in charge at their C-termini.

The issues for this enzyme are, first, how do the subunits combine to produce the complex allosteric effects, and, second, how do the various tissue-specific versions of the subunits modulate behavior of the enzyme?

 

PUBLICATIONS:

Bovine NAD+-Dependent Isocitrate Dehydrogenase: Alternative Splicing And Tissue Dependent Expression of Subunit 1. Weiss, C., Zeng, Y., Huang, J., Sobocka, S.B., Rushbrook, J.I. (2000) Biochemistry 39, 1807-1816.

Protein and mRNA Analysis of Myosin Heavy Chains in the Developing Pectoralis Major Muscle. Rushbrook, J.I., Huang, J., Weiss, C., Yao, T.-T., Siconolfi-Baez, L., Becker, E. (1998) J. Muscle Res.& Cell Motil. 19, 157-168.

Characterization of the Myosin Heavy Chains of Avian Adult Fast Muscles at the Protein and mRNA Levels. Rushbrook, J.I., Huang, J., Weiss, C., Siconolfi-Baez, L., Becker, E., Yao, T.-T., Feuerman, M.H. (1997) J. Muscle. Res. Cell Motil. 18, 1-15.

Isocitrate Dehydrogenase from Beef Heart: Primary Structure of Subunit 3/4. Zeng, Y., Weiss, C., Yao, T.-T., Huang, J., Siconolfi-Baez, L., Hsu, P., Rushbrook, J.I. (1995) Biochem. J. 310, 507-516.

Developmental Myosin Heavy Chain Species in the Avian Pectoralis Major Muscle. Rushbrook, J.I., Weiss, C., Yao, T.-T (1991) J. Muscle Res. and Cell Motility, 12, 281-291.

Comparison of Adult, Embryonic and Dystrophic Chicken Muscle Myosin by SDS-Gel --Electrophoresis and Peptide Mapping. Rushbrook, J.I., Stracher, A. (1979) Proc. Nat. Acad. Sci. 76, 433l-4334.

Nicotinamide Adenine Dinucleotide Dependent Isocitrate Dehydrogenase from --Beef Heart: Subunit Heterogeneity and Enzyme Dissociation. Rushbrook,J.I., Harvey, R.A. (1978) Biochemistry 17, 5339-5346.