Proteinases and the instability of isocitrate lyase in extracts of developing flax seedlings

In extracts of flax seedlings 4 days after imbibition, isocitrate lyase activity is unstable in comparison to that in extracts from 2.5-day seedlings or to malate syntheses analysed at several stages of development. This instability in extracts of 4-day seedlings is especially pronounced when a large number of seedlings is homogenized per unit volume of Tris-Mg²⁺-EDTA-dithioerythritol buffer. However, isocitrate lyase can be stabilized when the resultant homogenate is diluted soon after seedling breakage. The pronounced instability in more concentrated extracts is not due to inadequate buffering by the homogenization medium, nor can it be due to polyphenols because added polyvinylpyrrolidone has no effect. Mixing of a heated supernatant from concentrated extract with dilute unheated extract yields the units of stable isocitrate lyase expected in the dilute extract, ruling out stoichiometric inactivation by a heat-stable component. The pronounced instability is attributed to the action of proteinases. A theoretical model assuming a decay process that is first order in isocitrate lyase and first-order in one or more proteinases is in reasonable agreement with the results. Malate synthase and NADP⁺-isocitrate dehydrogenase are much more stable in concentrated extracts prepared from 4-day flax seedlings. Isocitrate lyase is stable in concentrated extracts of 5-day watermelon seedlings, which is a developmental stage analogous to that for 4-day flax seedlings.     

Nachweis der Phytochrom-induzierten de novo-Synthese von Phenylalaninammoniumlyase (PAL,E.C.4.3.1.5) in Keimlingen von Sinapis alba L. durch Dichtemarkierung mit Deuterium

Summary Using the in vivo density labeling technique with deuterium oxide it is confirmed that during phytochrome mediated photomorphogenesis in mustard seedlings a true de novo synthesis of phenylalanine ammonia-lyase is induced by active phytochrome (P _fr).     

Fluorescence immunolocalization of bound atrazine residues in plant tissue

Bound atrazine was detected inElodea canadensis by an improved immunohistochemical fluorescence procedure using anti-triazine antibodies from rabbits, biotin-labelled anti-rabbit immunoglobulin G and streptavidin-phycoerythrin conjugate. Whereas no labelling was found in control plants grown in charcoal-filtered, atrazine-free water, the labelling of plants obtained from their natural habitat and grown in tap water was sometimes nearly as high as in samples loaded with atrazine. The efficiency of the immunofluorescence procedure was compared using several antisera obtained by immunizing with different hapten conjugates and purified by various purification methods. The best results were observed with the atrazine analogue ametryn sulfoxide, which was coupled to bovine serum albumin for immunization and to Sepharose for immunoaffinity chromatography. The procedure described in this paper may serve as a general tool for detecting bound pesticide residues in plant material. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Highly fluorochrome labeled gene probes for quantitative tracing of RNA in individual cells by in situ hybridization.

A new method is presented for preparing highly fluorochrome labeled gene probes suitable for in situ hybridization. For this purpose fluorochromes were attached to a synthetic polypeptide, which was then coupled covalently to various gene probes. The advantage of the reported method is its high labeling efficiency and the easy coupling procedure. The method allows rapid and quantitative detection of homologous RNA at the single cell level. Optimal conditions for the hybridization of fluorochrome-labeled gene probes were established microfluorimetrically, and the specificity and sensitivity of the method were tested. Quantitation of the RNA with a fluorochrome-labeled gene probe in situ in individual cells allows determination of the degree of gene activation in individual cells and may thus provide a new tool for investigation of normal and malignant cells with respect to activation of genes controlling differentiation and proliferation.     

Proteolytic inactivation of alpha 1-proteinase inhibitor in vivo: detection, characterization and quantitation of the main fragment excreted in the urine of leukemia patients.

During the search for a therapy response parameter in patients with acute myeloid leukemia, we observed the appearance of a 41 kDa glycoprotein band in the urines of these patients under therapy. To investigate the nature of this molecule and to develop a specific detection system, the protein was isolated and antibodies were raised. Urines and sera of patients and healthy subjects were screened for crossreacting proteins by immunoblotting. Only the leukemia patients showed the urinary 41 kDa protein plus a 53 kDa band. In all sera, including those from healthy donors, a 53 kDa protein was intensely stained. Isolation of the plasma protein and sequence analysis of the urinary protein revealed that alpha 1-proteinase inhibitor is the crossreacting plasma protein and that the 41 kDa molecule is proteolytically modified alpha 1-PI, which has lost its antitryptic activity. Cleavage occurred in the N-terminal part as well as in the reactive site loop of the inhibitor. The 41 kDa truncated inhibitor was also found in the leukemic blast cells. A densitometric method is described for the quantitation of the molecule in the nanomolar range.     

Multiplication of Coxsackie B1 Virus in Synchronized HeLa Cells

A higher yield of Coxsackie B(1) virus was obtained when HeLa cells were infected late during S phase as compared to the amount produced by random cultures.