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Isoelectric focusing (IEF) of peroxidases of different organs and tissues of Nicotiana tabacum L. was performed on thin-layers of Sephadex and polyacrylamide. Isoelectric points (pI's) of peroxidase bands were measured by special electrodes. — The two types of layers showed very similar results. Reproductibility of pI's was better on polyacrylamide. This method is also easier to practise and requires less time than IEF on Sephadex (3 h versus 18). Thus for analytical purposes the acrylamide-technique is preferable, but if it is necessary to regain the separated enzymes it is better to perform IEF on Sephadex. — When IEF-patterns of peroxidase are compared with the disk electrophoresis (DE) patterns of the same tissue, important differences are observed. The 6 bands of GI (fast migrating, anodic group of DE) are reduced to 2 on the strongly acidic side of IEF (independent of the tissue studied). That means only 2 proteins in GI can be separated by pI's of the molecules. Maybe the heterogeneity of GI bands after DE indicates the presence of conformational isomers (conformers). — Because of the reduced number of bands in GI after IEF there is no difference in the patterns of many tissues (flowers, leaves, shoots, pith) as there is after disk electrophoresis. In the case of GII (slow migrating, anodic group of DE) on the other hand, there are always 4 different bands after isoelectric focusing in the lower acid region instead of 3 after disk electrophoresis. Disk electrophoresis of peroxidase-groups separated by isoelectric focusing shows the same patterns as direct disk electrophoresis of the extract. The methods produce no artifacts. —Comparison of these results with the peroxidase-patterns of tobacco found by other workers and by other techniques leads to the conclusion that there exist at least 4 “isoenzymes” of peroxidase corresponding to the 4 groups GI, GII, GIII, and GIV.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).

Mesophyll-protoplasts of tobacco show increasing peroxidase-activity immediately after isolation. This is due to an enhancement of activity of the constitutive isoenzymes of GIII (=slow migrating cathodic group) and to a new formation of GII-isoenzymes (=slow migrating anodic group). (GII is not present in intact leaves.) As both processes are inhibited by actinomycin and actidion it is assumed that there is a new synthesis of peroxidase enzymes —The peroxidase reaction is independent of the further development of the protoplasts, as was evidenced by culturing protoplasts in different media which regulate the development of the protoplasts. Peroxidase reaction is always the same whether or not there is cell-wall synthesis and cell division. This leads to the conclusion that peroxidases in this case have no relation to synthesis of primary cell-walls. On the other hand they could be related to the dedifferentiation processes that always take place in isolated protoplasts.

Peroxidase activity and isoenzyme pattern were studied during dedifferentiation of tobacco stem-sections leading to callus formation and during redifferentiation of tobacco callus leading to formation of shoots. These processes are both accompanied by an increase in total peroxidase activity and by characteristic changes in isoenzyme pattern. The isoenzyme pattern of tobacco callus differs from that of tobacco stem-tissue. The plantlets differentiated from the callus show the same pattern as seedlings do. During the differentiation process, before any buds are visible, the callus shows a peroxidase pattern that is determined by a reduction of fast-migrating anodic isoenzymes and by an increase of activity in all the other peroxidase isoenzymes. The formation of this pattern is independent of the growth regulators responsible for the differentiation: only the kind of differentiation itself determines the pattern. By artificial inhibition of callus growth it is possible to induce an isoenzyme pattern very similar to that of differentiation; the fast-migrating anodic enzymes are reduced in activity but the others are not increased as they are during differentiation. Therefore the question arises whether there are two independent processes taking place in differentiating callus. The one process, inhibition of growth in the cells that do not differentiate, is accompanied by a reduction of fast-migrating anodic isoenzymes. The other process, formation of meristemoids in the callus, is accompanied by a sharp rise in peroxidase activity of the other anodic and cathodic isoenzymes.

Upon disk-electrophoresis with guaiacol as a substrate the peroxidase-isoenzymes of Nicotiana tabacum (L.) were localized on the gels in two anodic and two cathodic groups. By preparation of protoplasts and isolation of cell walls it was possible to show that only cathodic enzymes are located in the protoplasts in measurable amounts, whereas all the isoenzymes, anodic and cathodic, can be found associated with cell walls. The different groups of isoenzymes are bound to the cell wall in different ways as evidenced by differences in their extration. It seems possible that different biological functions are associated with the different groups of isoenzymes.The isoenzyme patterns of different organs and tissues of tobacco show qualitative differences only in the anodic (i.e. wall located) isoenzymes. It is suggested that the ontogenetic change in peroxidase-patterns is direct evidence of biochemical differences in the cell walls of the different tissues and organs.

De-novo synthesis of acid and basic peroxidases has been studied in cell suspension cultures of tobacco by incorporation of (3)H- and (14)C-amino acids. Incorporation rates were found to be high for acid peroxidases and low for basic peroxidases. Synthesis of all peroxidases was inhibited by cycloheximide and actinomycin D. Subculturing of the cells increased the rates of radioactive amino-acid incorporation into all peroxidases within the first 24 h. This rise in peroxidase synthesis was correlated with the age of the transferred cells. The older the cells were the more pronounced was the effect. During the culture cycle the high rates of peroxidase synthesis at the second day dropped back to initial values. Peroxidase synthesis was thus inversely related to peroxidase accumulation which was very low at the beginning and increased continuously. By pulse-chase experiments it has been shown that newly synthesized acid peroxidases accumulated in the medium. This process was inhibited by monensin. Only the acid peroxidases were secreted into the cell wall and from there released. The basic peroxidases were not detectable in the medium.

Protein-body membranes (PBMs) were isolated from cotyledons of Phaseolus vulgaris L. by a procedure involving osmotic shock of purified protein bodies. The purified PBMs have a characteristic density of 1.16 g cm(-3). Treatment of the membranes with increasing concentrations of detergent (Triton X-100) or with a solution at pH 12.0 showed that the membranes contained a characteristic integral protein (IMP) with a relative molecular mass of 25,000. This IMP is not a glycoprotein. When developing cotyledons were labeled with (3)H-amino acids for 2-3 h, a radioactive polypeptide with the same mobility on denaturing polyacrylamide gels as IMP was found to be associated with the rough endoplasmic reticulum (ER). During a 24-h chase, a considerable portion of the radioactivity slowly transferred into the IMP associated with more rapidly sedimenting organelles, which sedimented in the same region of the sucrose gradients as the PBMs. Antibodies prepared against purified IMP crossreacted with an ER-associated protein which had the same mobility on denaturing acrylamide gels as authentic IMP. Synthesis of IMP occurred at all stages of cotyledon development examined, but not during seed germination. The results show that a newly synthesized protein of the PBM is associated with the rough ER, just like the soluble matrix proteins, phaseolin (R. Bollini, W. Van der Wilden and M.J. Chrispeels, 1983, J. Cell Biol. 96,999-1007) and phytohemagglutinin (M.J. Chrispeels and R. Bollini, 1982, Plant Physiol. 70, 1425-1428), but that the chase-out from the ER is much slower for IMP than for the matrix proteins.