Mark A. Stahmann

Studies on the Laccase of Lentinus edodes: Specificity, Localization and Association with the Development of Fruiting Bodies

The phenol oxidases from mature fruiting bodies of Lentinus edodes (Berk.) Sing., a commercially cultivated mushroom, were studied. The major phenol oxidase was a laccase with a pH optimum near 4.0 and an apparent molecular weight of 100000. Catechol oxidase and tyrosinase were also present. The laccase investigated was primarily extracellular; the highest activity was in the pigmented rind of the pileus and in the stipe. Increased laccase activity was associated with rapid growth of non-pigmented aerial mycelium and formation of pigmented primordia and fruiting bodies. Possible functions of the laccase and its regulation during the development of fruiting bodies are discussed.

Soluble proteins and multiple enzyme forms in early growth of wheat

Abstract Changes in the soluble proteins and enzymes of wheat ( Triticum aestivun L. ), var. Lee, in relation to germination and early plant development were studied. Extracts of dormant seed, germinated seed, etiolated coleoptile, and first green leaf were analyzed by polyacrylamide disc electrophoresis. Patterns of soluble proteins and the isoenzyme bands of the following enzymes were studied: glucose-6-phosphate, 6-phosphogluconate, malate, isocitrate, glutamate, and alcohol dehydrogenases; peroxidase; catalase; acid and alkaline phosphatases; esterase; and amylase. Photographs and densitometer tracings of gels showing changes during germination and growth are presented.

Ethylene production by detached leaves infected with tobacco mosaic virus

Abstract Ethylene production by detached leaves infected with tobacco mosaic virus (TMV) with or without local lesions and the effect of exogenously introduced ethylene on TMV multiplication and lesion formation were investigated. Ethylene production was enhanced concurrently with local lesion development, but not with systemic infection. Increase in the rate of ethylene production was proportional to the number and size of lesions. This enhanced ethylene production reflects necrotization of the cells invaded by the virus; ethylene production was also enhanced by senescence and by chemical injury of leaves. Chemicals causing mild cell necrosis, such as Ni, Hg, and Cu salts, remarkably increased ethylene production with the development of toxic symptoms. Perchloric acid and trichloroacetic acid were less effective in increasing the rate of ethylene production. Ethylene production was inhibited by disodium ethylenediaminetetraacetate and by the protein synthesis inhibitors blasticidin S and puromycin. Exogenously introduced ethylene stimulated chlorophyll degradation and slightly inhibited peroxidase activity, but did not inhibit TMV multiplication or lesion formation.

Multiple molecular forms of enzymes in barley leaves infected with Erysiphe graminis f.sp. hordei

Abstract Multiple molecular forms of 14 enzymes (isoenzymes) from near-isogenic barley lines inoculated with powdery mildew were studied by polyacrylamide disk electrophoresis. Following inoculation of the susceptible line (ml-g) the number of isozyme bands of acetylesterase, acid phosphatase, malate dehydrogenase (NADP), succinate dehydrogenase and peroxidase increased. These new bands were not detected in extracts of healthy tissue or mycelium and conidia from infected leaves; their intensity was not reduced by removing mycelium and conidia before preparing extracts. Only the new peroxidase band appeared within one day after inoculation. Eleven enzymes from inoculated susceptible lines showed an increase in intensity of one or more bands within 7 days; four enzymes showed a decrease. A hypersensitive line (Ml-k) showed changes after inoculation that were similar to those of the susceptible line. With a resistant line (Ml-g) there was an intensity increase in one or more bands of seven enzymes within 7 days after inoculation. Such changes in the multiple molecular forms and activity of enzymes are biochemical symptoms of disease, may be a basis for the increased metabolism associated with infection and suggest profound interactions between parasite and host.

A simple procedure for basic hydrolysis of proteins and rapid determination of tryptophan using a starch column

Abstract The use of an alkali resistant centrifuge tube as a liner for an evacuated glass apparatus for the hydrolysis of proteins by sodium hydroxide is deseribed. The analysis of tryptophan is made with an automatic amino acid analyzer in which the medium-length resin column is replaced by a potato starch column. Tryptophan in a basic hydrolyzate can be determined within 2 hr. This procedure gave high recoveries of free tryptophan and of tryptophan added to proteins. Some proteins and food stuffs were analyzed by this method to show its applicability. This method can be used for milligram quantities of protein.

Antigens in moldy hay as the cause of farmer's lung.

Summary Sera from farmers lung patients and non-farmers lung control individuals were reacted with trichloroacetic acid extracts of moldy hays. All the sera from patients with acute symptoms and two-thirds of the total which included recovered farmers lung patients formed specific precipitin lines with the moldy hay extracts. No precipitin lines were obtained with sera from healthy farmers who had no history of farmers lung or with extracts of good hay. Evidence is presented to show that farmers lung is associated with the presence of specific antibodies in the patients“sera against antigens found in moldy hays and that these antigens are the cause of farmers lung.

The stabilization of extracts of cabbage leaf proteins by polyhydroxy compounds for electrophoretic and immunological studies.

Abstract Addition of polyhydroxy compounds, such as sugars, sugar alcohols, or glycerol, prevented precipitation and hydrolysis of cabbage-leaf proteins during storage at low temperatures. These compounds increased the number of protein components detectable by starch electrophoresis and facilitated immunochemical studies. Evidence that polyhydroxy compounds prevent or reverse aggregation of proteins is presented.

Effect of light and aeration on fruiting of Lentinula edodes

Adequate light and aeration are essential for the fruiting of Lentinula edodes (syn. Lentinus edodes ). The stimulatory wavelengths were dependent on the composition of the growth medium. Red wavelengths (620–680 nm) stimulated and blue wavelengths (400–500 nm) inhibited fruiting on low calcium ( 130 p.p.m.). Addition of oak bark extract to the medium permitted otherwise suboptimal light intensities to stimulate fruiting. Cultures were receptive to light stimulation during vegetative growth and showed memory of the exposure, fruiting later in the dark. Exposure, especially to blue light, elicited pigmentation of primordia. Only pigmented primordia were capable of expansion. Inadequate aeration (probably resulting in CO 2 accumulation) near the time of fruiting resulted in failure of the primordia to expand. The practical implications of these findings are discussed.

Peroxidase and ethylene production by barley leaves infected with Erysiphe graminis f. sp. hordei

Abstract Inoculation of primary leaves of barley of differing compatibilities (types 0, 0–1, 2, 2H and 4) to race 3 of Erysiphe graminis f. sp. hordei resulted in increased peroxidase activity in all cases. An increase was usually evident within 24 h of inoculation and was accompanied by the appearance of one new major isoenzyme band after gel electrophoresis. This band was not seen in extracts of conidia of the pathogen, which contained little peroxidase. It was concluded that the increase in peroxidase was a host response to infection. Treatment of leaves with maleic hydrazide and 2-chloroethyltrimethylammonium chloride also increased peroxidase activity and stimulated the appearance of the same isoenzyme band seen after infection. Plants incubated in an ethylene-containing atmosphere, and plants sprayed with 2-chloroethane phosphonic acid showed neither quantitative nor qualitative changes in peroxidase. There was some indication that maleic hydrazide increased the resistance of susceptible plants to infection. Detached inoculated leaves containing the Ml-a and Ml-g genes for resistance (types 0 and 0–1) showed a marked peak in ethylene evolution within 24 h after inoculation, which however, was not always reproducible. Lines containing the ml-a and ml-g alleles for susceptibility did not show this peak. Later, ethylene evolution from the incompatible combinations declined to a low level while increasingly large amounts were produced by the compatible combinations. Extracts of infected leaves containing the Ml-g gene did not produce ethylene in vitro , and usually inhibited the production of ethylene from a methional/horse-radish peroxidase system. The inhibitor(s), of a low molecular weight, were heat stable but decreased in activity with time. Some evidence indicated that the inhibitor(s) may be phenols and that their changing levels might parallel the pattern of ethylene evolution from intact leaves.

The potential for protein production from green plants.

Data on protein yields show that forage crops, particularly alfalfa, produce several times more protein per acre than do seed crops. Amino acid analyses and estimations of biological values by enzymatic hydrolysis and feeding trials indicate that protein concentrates from green plants have high nutritive value. The protein concentrates from 10 plant species had a similar amino acid composition and biological value which indicates that good protein might be obtained from many plant species. It is suggested that the use of the fibrous residue as a feed for ruminants and the use of the protein concentrate as a high protein feed or base for processing into new protein foods may make it possible for the production of protein from green plants to compete with other sources of protein. This would markedly increase protein production per acre and allow the use of new plant species in our agriculture. The need for more research on protein production from different types of green plants and on ways to harvest, concentrate and process their proteins into edible forms is discussed.