We describe an alternate terminal oxidase found in the plasma membrane of Thermus thermophilus and designate it cytochrome ba3. The enzyme consists of a single -:35-kDa polypeptide that binds one heme B molecule, one heme A molecule, and two Cu ions. Optical spectra suggest the presence of cytochrome b, cytochrome a3, and CUA in this protein. Quantitative EPR and Mdssbauer studies of the oxidized protein indicate the presence of one low-spin ferric heme, which is assigned to cytochrome b. Mossbauer studies of the reduced protein show the presence of one low-spin ferrous heme, assigned to cytochrome b, and a predominant high-spin ferrous heme that reacts quantitatively with CO to yield an additional low-spin ferrous heme. The latter Fe atom is associated with the heme A and is designated cytochrome a3. The EPR spectrum of the oxidized protein also reveals the presence of a CuA-type center that accounts for half the total Cu. The remainder of the Cu would appear to be present as CUB that is magnetically coupled to the heme A. Amino acid analyses of cytochrome ba3 show the presence of eight to nine histidine residues and one cysteine residue.

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Examples of a variety of approaches for studying the mechanism and regulation of cellulose biosynthesis are presented. Attempts to demonstrate conclusively a cellulose synthase activity using membrane preparations derived from higher plants have not been successful; the predominant UDP-glucose: /3-glucan-/3-glucosyltransferase detected in these preparations is a /3-(l->3)-glucan synthase that is dependent upon Ca2+ and a /3-glucobiose, such as laminaribiose or cellobiose, for activity. Xyloglucan glucosyltransferase activity is detected in all plant preparations examined and, in cotton fibres, the activity of this enzyme correlates well with the level of xyloglucan found in the fibre. Low activity for a Mg2+-dependent /3-(l-»4)-glucan synthase is found in extracts from soy beans and mung beans, but not cotton fibres; this enzyme could represent either a dissociated form of xyloglucan glucosyltransferase or a partially latent form of cellulose synthase. In vitro translation of RNA from developing cotton fibres shows notable increases in the level of several relatively abundant translatable mRNAs associated with the time of onset of secondary-wall cellulose synthesis. In order to determine whether these apparent changes in gene expression represent enhanced expression of specific genes required for cellulose synthesis, several strategies are being developed for identification of specific polypeptides required for this process. One strategy involves the successful development of a technique for detection of glucan synthase activity in acrylamide gels, a technique that should prove useful for characterization of the polypeptide composition of such enzymes. We have also synthesized a photoaffinity analogue of 2,6-dichlorobenzonitrile (DCB), a potent and specific inhibitor of cellulose synthesis. The analogue is also an effective inhibitor in vivo, and upon ultraviolet irradiation of extracts in the presence of the radioactive analogue, we observe a relatively specific labelling of a polypeptide of 18 X 103 Mr. Finally, we have studied the spatial regulation and structural requirements for cellulose synthesis in internode cells of the alga Chara corallina. Cellulose deposition in vivo shows spatial localization, which correlates with acid and base bands along the cell. Using internodes perfused with solutions containing UDP-[14C]glucose and subsequently ligated, we were able to demonstrate synthesis of a highly insoluble cell-wall-localized glucan, thus offering hope that Chara can be developed as another useful system for studying the mechanism and regulation of cellulose synthesis. I N T R O D U C T I O NSince microfibrillar cellulose is a major structural component of the cell walls of higher plants, and many algae and fungi, regulation of the extent and pattern of deposition of this polymer must certainly be involved in the overall regulation of growth and development in these organisms. Thus, it is regrettable that biochemical studies on the mechanism of synthesis of cellulose have proven so difficult; our lack o...

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values for Whatman 41 filter, Whatman GF/A filter, glass AE filter and ceramic filter is poor. Also the (\kW)l/'/ (\klo)1/2 ratios for these filters are all greater than 1.3. ConclusionsOn the basis of results obtained with oleic acid aerosols, Millipore AA and VC filters, Selas silver membrane filter, and stainless steel substrates appear to be the most suitable collection substrates of those tested. Fiber-filter substrates exhibited poor collection efficiency curves when (\kw)1/2/(\klo)1/2 and (\k60)1/2 values were used as the determining factors. In using any substrate it is important that the cascade impactor be carefully calibrated with the specific substrate. S. J. Rothenberg for technical review of the manuscript and E. E. Goff for illustrations. Literature Cited(1) Rao, A. K.; Whitby, K. T.rn The equilibrium constant for the extraction of nitric acid from water by a fatty amine in a water-immiscible liquid (eq 1) is increased by a factor of lo2 when the solvent is changed from benzene to trioctyl phosphate. This permits the H+ and NO, to be coextracted from a near-neutral solution. The amine solution in trioctyl phosphate can be carried on a thin porous plastic support and used as a membrane. Such a membrane has been mounted in a modified dialysis cell, with loading carried out on one side and stripping with base on the other. This device is effective in removing nitrate from water.

Summary:reversible inhibitor of in vivo cellulose biosynthesis in higher plants and algae. We now report the synthesis of 2,6-dichlorophenylazide (DCPA) with a nonexchangeable tritium label of high specific activity. We have shown this compound to compete with DCB in binding t o cotton fiber extracts, and to specifically label a low molecularweight protein in this extract on irradiation with UV light.The compound 2,6-dichlorobenzonitrile (dichlobenil, DCB) is known t o be a Introduction:Although cellulose is one of the world's most abundant natural products, the enzymes catalyzing its biosynthesis are poorly characterized. In vitro cellulose biosynthesis was carried out effectively for the first time very recently (1).The compound 2,6-dichlorobenzonitrile (DCB, dichlobenil), long used as a commercial herbicide, has been shown to inhibit cellulose biosynthesis (2). The initial target of DCB is unknown, but it must play a key role in the polymerization of glucose in vivo. Identification of the DCB receptor and characterization of its normal function in biosynthesis would be useful in more fully understanding this system.For this reason, we undertook to label and isolate the DCB receptor. Examining the structure of DCB, it was clear that the benzonitrile moiety was similar in terms of steric and electronic factors to an aryl azide. As aryl azides are also well known to be useful as photoaffinity labels, we proposed that 2,6-dichlorophenylazide (DCPA) should be an inhibitor of DCB bindin competitive in the dark and irreversible after UV irradiation. It also appeared possgle that DCPA might itself block cellulose biosynthesis. Synthesizing this compound with a non-exchangeable radiolabel should then allow us to detect and purify the covalently modified receptor after p hotoaffinity la belling.The biological results, which are described elsewhere (31, confirm that DCPA is a useful photoaffinity analog of DCB, specifically labelling a polypeptide with a

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