High Respiratory Activity of Guard Cell Protoplasts from Vicia faba L.

The rate of O₂ uptake was about 29 times higher in guard cellprotoplasts (GCPs) than in mesophyll protoplasts (MGPs) on aChi basis. The O₂ uptake was inhibited by respiratory inhibitors,but stimulated by respiratory uncouplers. On a Chi basis, theactivities of Cyt c oxidase and NADH-Cyt c reductase, mitochondrialenzymes, were about 27 and 35 times higher in GCPs than in MCPs.On a Chi basis, the ATP content was about 9 times higher inGCPs. The amount of ATP in GCPs was decreased by respiratoryinhibitors, an energy transfer inhibitor, and uncouplers ofoxidative phosphorylation. On a volume basis, GCPs had 8- to10-fold higher respiratory activities than MCPs, but had a lowChi content and lacked the activity of NADP-glyceraldehyde-3-phosphatedehydrogenase (NADP-GAPD), the Calvin cycle enzyme. From theseresults, we concluded that oxidative phosphorylation plays amain role in ATP production in guard cells and that guard cellshave a heterotrophic feature. Salicylhydroxamic acid (SHAM)in combination with KCN or NaN₃ strongly inhibited O₂ uptake,indicating the presence of cyanide-resistant respiration inguard cells. Phenylmercuric acetate (PMA), a potent inhibitorof stomatal opening, reduced the ATP content of GCPs by about90%, whereas it had a relatively small effect on the ATP levelof MCPs. The specific effect of PMA on GCPs is discussed. (Received March 24, 1983; Accepted June 8, 1983)     

Participation of Hydrogen Peroxide in the Inactivation of Calvin-Cycle SH Enzymes in SO2-Fumigated Spinach Leaves

In SO₂-fumigated spinach leaves under light, chloroplast SHenzymes, glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPD)(EC 1.2.1.13 [EC] ), ribulose-5-phosphate kinase (Ru5PK) (EC 2.7.1.19 [EC] )and fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11 [EC] ) weremore remarkably inactivated than other chloroplast enzymes.Their activities recovered after removal of SO₂. The inactivationparalleled light-dependent CO₂-fixation in spinach leaves. Inilluminated chloroplasts isolated from SO₂-fumigated spinachleaves, NADP-GAPD and Ru5PK were more specifically in activatedthan other chloroplast enzymes. These two enzymes could be protectedfrom the inactivation by adding catalase. The NADP-GAPD inactivationwas suppressed by DCMU, cytochrome c or anaerobic conditions.By adding thiol compounds, the NADP-GAPD inactivation was dischargedand the activity increased. In chloroplasts or crude extractsfrom non-fumigated spinach leaves, NADP-GAPD and Ru5PK weremore strongly inhibited by externally added H₂O₂ than otherchloroplast enzymes. All results supported the idea that thesuppression of photosynthesis at the beginning of SO₂ fumigationwas caused by the reversible inhibition of chloroplast SH enzymewith H₂O₂. (Received October 7, 1981; Accepted June 16, 1982)     

HLA-DO is a lysosomal resident which requires association with HLA-DM for efficient intracellular transport.

The murine MHC class II molecule H2-O is expressed in B-cells and in thymic epithelium but the human equivalent, HLA-DO (DO), has not been detected, though the corresponding genes, HLA-DNA and HLA-DOB, are well known. Here we show DO to be a lysosomal resident in B-cells. Surprisingly, DO forms stable complexes with HLA-DM (DM), another lysosomal class II-like molecule which is important for class II-restricted antigen presentation. Association with DM is necessary for efficient exit of DO from the endoplasmic reticulum (ER) and thus for accumulation in lysosomes. The association is evolutionarily conserved and in mice lacking H2-M, the mouse equivalent of DM, the amount of intracellular H2-O is decreased and only minor amounts of H2-O appear to leave the ER. The DO-DM complexes survive in the lysosomal system suggesting that DO and DM functions may be intertwined.     

Isolation and characterization of a new acetate-requiring mutant strain, ace-9, of Neurospora crassa.

A new acetate-requiring mutant strain of Neurospora crassa, ace-9, has been isolated. The mutant gene was mapped between nuc-2 and arg-12 on the right arm of the second linkage group. The ace-9 mutant strain shows very weak activity of pyruvate dehydrogenase complex (PDHC). Three strains that show no activity of PDHC had already been found, i.e., ace-2, ace-3, and ace-4. Thus the ace-9 is the fourth gene that causes the deficiency in PDHC activity by a mutation. Deficiency of PDHC activity in ace-9 strain seems to be due to defective E1 component, because (1) the activity of E1 component enzyme is very weak in ace-9 mutant strain, and (2) normal PDHC activity was resumed when a preparation of ace-9 was mixed with E1-E2 fraction of wild type or with E1 component of wild type E. coli. Difference in thermostability of both E1 component enzyme and PDHC between ace-9 and the wild type strains supports this conclusion.     

Computer-assisted indirect coulometric titrations of biological redox components

The optically coupled, indirect coulometric titration (ICT) method has been applied to assist in the characterization of several redox biocomponents such as heme proteins. This method has provided rapid and repetitive evaluation of redox stoichiometry and energeties with a high degree of precision. The usual manual ICT procedure requires operator control of the repetitive incremental additions of electrochemical charge, the on/off of the magnetic stirrer between such additions, and the recording of optical spectra after each addition. The sequence and timing of events in the above procedure can be fixed which then lends itself ideally to computer control. In this paper, a computer-controlled ICT intsrumentation is described. The hardware and software developed for the computerization permit versatility of displaying ICT data in various formats, i.e., conventional absorbance or derivative spectra and dual wavelength or differences between dual-wavelength spectra. Also, absorbance at a given wavelength could be obtained from the spectra as a function of the incremental addition of charge. The three-dimensional plots of spectral absorbance as a function of wavelength and incremental charge could also easily be obtained. To demonstrate the capability and versatility of the computer-controlled instrumentation, the ICT of the redox components, cytochrome c and cytochrome c oxidase, and of intact mitochondria is presented.     

Existence of Two Types of Electrostatic Interaction in Reactions Catalyzed by Ferredoxin-NADP$ Oxidoreductase

Effects of salt and pH on diaphorase and NADP^$ photoreductionactivities were studied with broken spinach chloroplasts andpurified ferredoxin-NADP^$ oxidoreductase. Two types of electrostatic interactions, which regulate thereaction rate, were observed. One is the long-range electrostaticinteraction which determines the local concentrations of reactantsin the surface-mediated processes due to the change in the surfacepotential. In addition to the salt-induced change in the reactionrate, the pH optimum shift by salt was also remarkable: theoptimum pH in the diaphorase activity of chloroplasts shiftedto the more acidic pH region with an increase of salt concentration,while that of the membrane-free enzyme was not affected by salts. A more specific, short-range electrostatic interaction in reactionsbetween NADP(H) and ferredoxin-NADP^$ oxidoreductase was observed.This interaction became clearer when fixed charges on the membranesurface were masked by an addition of salts. Complete dissociationof the 2'-phosphate group of NADP(H) was necessary for its associationwith the enzyme. The eletrostatic attraction between the negativelychargedpart of NADPH and the positively-charged part of the enzyme(probably lysyl and arginyl residues) may play a role in theshort-range interaction. ¹Present address: Department of Agronomy, University of Kentucky,Lexington, Kentucky 40546, U.S.A. ²Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received February 21, 1983; Accepted March 17, 1984)     

Organ specific expression of ras oncoproteins during growth and development of the rat

Summary Expression of proteins encoded by the ras proto-oncogenes was examined in extracts from normal rat organs using anti-ras p21 antibodies generated against synthetic peptides. The highest level of ras p21 was found in brain (cerebrum) and was predominantly of c-Ha-ras origin. Levels of brain ras p21 did not vary from the newborn period of 3 months of age. Moderate levels of ras p21s were detected in lung, spleen and thymus. In contrast to the p21 in brain, these levels varied with the age of the rats and were encoded by other members of ras proto-oncogene family (Ki-ras or N-ras). This organ specific expression of different ras genes might be related to developmental control of gene expressions.