RESEARCH
Structural Protein Isolated from Mitochondria Wisconsin findings help explain protein organization and stabilization of multienzyme transducing systems A big step has been taken toward explaining how protein is organized in multienzyme transducing systems. In research on mitochondria, biochemists at the University of Wisconsin and its Institute for Enzyme Research have isolated and characterized a protein which behaves as a structural protein. Their findings [Biochemistry, 1, 827 (1962)] shed light on the makeup of the mitochondrion, as well as on the nature of the various components which stabilize the system. Mitochondria, the minute, semifluid globules found in cells, are the "powerhouses" of living systems. Like the chloroplast, the cell membrane, and the kidney tubule, they belong to the class of primary transducers. Found in all kinds of aerobic cells, they release energy by oxidation, conserve it in the form of bond energy of adenosine triphosphate (ATP). ATP is the chemical key for all the major synthetic processes of living systems. A typical mitochondrion looks like a fat sausage, says Dr. David E. Green, co-director of the institute. It has an outer and inner wall and internal structures (cristae) which radiate inward from the sides. The structural elements in both the external structure and the cristae contain the thousands of units which carry out the transduction process. Each of these units consists of proteins, lipids, and coenzymes. Usually the materials oxidized by these units are substrates of the citric acid cycle. Structural Concept. Biochemists have long agreed that the transduction process depends on this array of enzymes. But the question is just how are the enzymes organized? According to Dr. Green, studies have pointed to the theory of a structural protein. This concept holds that enzymes involved in the oxidation are organized not only in an enzymatic (catalytic) way, but also in a structural or patterned manner. The mitochondrion can be pictured as a structural mosaic containing: 40
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These observations led the Wisconsin group to believe that mitochondrion contains a structural protein. So they set out to isolate it and to answer: • How are the proteins of the mitochondrion ordered into stable molecular networks? • What is the nature of the forces which stabilize the interaction between the structural protein and the cytochromes? YIELD. Dr. D. E. Green (left) and Dr. R. M. Bock find high protein yield
• Elementary particles ( containing cytochromes a, b, cl9 and c) which make up the electron transfer chain. • An insoluble protein network. • A group of readily soluble dehydrogenase systems and auxiliary enzymes. The dehydrogenase systems generate diphosphopyridine nucleotide ( D P N H ) from a number of oxidizable materials, while the auxiliary enzymes catalyze ATP-dependent syntheses. Even with this picture, though, the Wisconsin research workers couldn't begin to put the theory of a structural protein to a test until relatively recently, when a program for systematically isolating the protein components of the mitochondrion finally reached completion. In adding up the isolated components ( DPNH dehydrogenase, succinic dehydrogenase, and the cytochromes), the biochemists find that the components don't account for the total protein of the mitochondrion. In fact, they amount to only about 2 1 % of the total. Other evidence also pointed to a structural protein, Dr. Green says. Chief contaminant in attempts to isolate cytochromes a, b, and cx is an insoluble, colorless protein which doesn't have a redox group. The contaminant forms tight complexes with each of the cytochromes; thus, they appear to be homogeneous.
Protein Isolation. Most of the physical studies of structural protein were carried out in the biochemistry department by Dr. Richard S. Criddle (now at the University of California, Davis) and Dr. Robert M. Bock. For the studies, they used mitochondria extracted from beef hearts. The structural protein is isolated from the mitochondria by treating it in a sucrose suspension with a mixture of deoxycholate, cholate, and sodium dodecyl sulfate (SDS). The resulting solution is clarified and reduced to give a white precipitate. After washing and extracting the precipitate, the yield is about 3 3 % of total protein. The supernatant liquid contains the four cytochromes. The insoluble protein can also be isolated from particulate fractions or from preparations of purified cytochromes with similar methods. In fact, Dr. Green points out, first indication that the material was a structural protein came from the latter approach. Preparations of structural protein made from cytochromes b, ci9 and a (each of which occupies different positions in the electron transfer sequence) are similar in sedimentation, molecular weight, electrophoretic migration, and solubility. The biochemists also isolated and studied the properties of each of the cytochromes. The first one studied was Cj. Its molecular weight is calculated to be 360,000—nearly six times the minimal molecular weight of the cytochrome. So the compound obtained must be the hexamer, which
is apparently the most stable polymer configuration. To depolymerize it, the Wisconsin team tried various reagents, found that low concentrations of anionic detergent (0.0005M SDS) or thioglycolate with a nonionic detergent would do the job. They theorize that SDS does this by competing with the cytochrome ct molecules for the individual hydrophobic bonding sites. Another factor needed for depolymerizing these compounds is charge repulsion. For this reason, nonionic detergents won't generate the monomer. But in combination with thioglycolate (which can put a charged group into a few key positions on the molecule), nonionic detergents can give the monomer. In isolating cytochrome a, the Wisconsin group finds that a tends to form higher polymers than c^ Again SDS breaks down the polymer into the monomer. And like cl9 a won't depolymerize with thioglycolate, deoxycholate, 6M urea, or nonionic detergent. The only form of cytochrome b which can be isolated is a high molecular weight complex, which can also be depolymerized with lipophilic agents. Most effective is the cationic detergent, cetyldiethylmethyl ammonium bromide. Molecular weight of the monomer is 28,000. In sharp contrast to the others, cytochrome c doesn't form a polymer and its monomer is easily isolated. Molecular weight of c is about 13,000. Physical
Properties.
Structural
protein has a variety of unusual properties. For instance, it is almost completely insoluble in water at neutral pH. But it dissolves readily in aqueous solutions containing reagents which induce charge repulsions between the molecules or which attach hydrophobic bonds. The best reagents are those used to depolymerize the cytochrome polymers. Nonionic detergents and organic solvents, the biochemists note, aren't effective in dissolving the protein. Molecular weight of the structural protein isn't easy to determine, Dr. Criddle and his co-workers found. First, the protein has a strong tendency to form high polymers. Second, dissolving it is a big problem since large amounts of solvents are needed; such large amounts introduce variables. When the research workers finally found a mild solvent, it gave two ranges in sedimentation studies—a lower one with a molecular weight of about 22,000, and a higher one of about 45,000. The second figure suggested a dimer, Dr. Criddle notes, and later work proved this to be the case. In another approach to the molecular weight problem, the Wisconsin biochemists decided to take advantage of the protein's strong binding equimolar properties. They added an equimolar amount of myoglobin to a solution of the protein in 0 . 1 % SDS at p H 10.5 to form a 1:1 complex. The complex gives a single, symmetrical, sedimenting boundary. With this approach, Dr. Criddle reasoned, they can get around all the shortcomings of the
conventional method, since the protein could be dissolved at neutral p H in aqueous solution that is low in detergent and with no evidence of high polymer formation. Subtracting the molecular weight of myoglobin, he calculates the protein's molecular weight to be about 20,000—a figure close to the earlier measurement. Interaction Complexes. If structural protein in mitochondrion is to function as it does in other systems, Dr. Bock points out, it must interact with itself, and interact with the cytochromes. Obviously it reacts with itself, he adds, since it is very highly insoluble. And experimental evidence shows that it reacts with the cytochromes, too. The best example of this is action It is easily isowith cytochrome cv lated to produce a product that behaves like a single species. But c1 can be further purified to give what still acts like a homogeneous species. So in the initial preparation, c x must exist as part of a complex containing structural protein. Sedimentation studies support this conclusion. More evidence for the interaction includes the 1:1 molecular complex formed when equimolar amounts of structural protein and hexameric ct are mixed. Apparently, the protein monomer competes with c x subunits for the same binding sites which stabilize the hexamer. The result is that the
