Infrared spectroscopy of proteins

This review discusses the application of infrared spectroscopy to the study of proteins. The focus is on the mid-infrared spectral region and the study of protein reactions by reaction-induced infrared difference spectroscopy.     

Evidence of increased synthesis of δ-aminolevulinic acid dehydratase in experimental lead-poisoned rats

δ-Aminolevulinic acid dehydratase (porphobilinogen synthase; 5-aminolevulinate hydro-lyase, EC 4.2.1.24) was purified from rat and rabbit erythrocytes to a homogeneous state. Specific activities were 26.0 and 26.6 units/mg protein for the rat and rabbit enzymes, respectively, and their estimated molecular weight was 280 000, each consisting of 8 subunits of M_r 35 000. In order to quantitate rat δ-aminolevulinic acid dehydratase at several stages of lead-poisoning, a radioimmunoassay technique using goat antiserum against the rat enzyme was developed for the first time. This technique was specific, reproducible and high sensitive allowing determination of 1 ng enzyme. When drinking water containing 25 mM lead acetate was given daily to rats ad lib. the δ-aminolevulinic acid dehydratase activity in the blood, assayed without any pretreatment, decreased to 8% of the control level on the next day. On the contrary, the restored enzyme activity, assayed in the presence of Zn²⁺ and dithiothreitol, was greater than normal by the fourth day of lead administration in bone-marrow cells and by the ninth day in the peripheral blood. The increased activity level stayed the same from the ninth day onward. The enzyme content as determined directly by the radioimmunoassay technique at this stage was about 2-fold above that the control. There was no significant difference in the number of reticulocytes and the distribution profile of different types of reticulocytes between the lead-exposed and non-exposed rats. Therefore, the increase in the amount of δ-aminolevulinic acid dehydratase in erythrocytes of lead-poisoned rats was suggested to be due to an increased rate of synthesis in the bone-marrow cells.     

Constituents of the essential oil of Lavandula latifolia

Thirty components were identified in Lavandula latifolia essential oil (spike oil). One of the compounds, espliegol (δ-terpineol), is a new natural product.     

Photodynamic herbicides: 1. Concept and phenomenology

A new approach to the design of conceptually and phenomenologically new herbicides is described. It involves the joint utilization of tetrapyrrole precursors, such as δ-aminolaevulinic acid (a biodegradable amino acid) and activators of the chlorophyll biosynthetic pathway, such as 2,2′-dipyridyl, in order to induce treated plants to biosynthesize and accumulate massive amounts of tetrapyrrole intermediates of the chlorophyll biosynthetic pathway in the dark (i.e. at night). During the subsequent light period (daylight) the accumulated tetrapyrroles act as potent photodynamic sensitiziers, which in turn result in the death of susceptible plants in a matter of hours. We have therefore proposed to name herbicides that act via this mechanism as photodynamic herbicides, or more pictorially as laser herbicides. From a limited survey of agricultural plant and weed species it appears that photodynamic herbicides exhibit a very pronounced organ, age and species-dependent selectivity. For example, dicotyledonous weeds such as mustard, red-root pigweed, common purslane and lambsquarter are very susceptible while monocotyledonous plants such as corn, wheat, barley and oats are not. The biochemical basis of this selectivity seems to lie, among other things, in the rates of tetrapyrrole turnover and in a differential enhancement by the applied chemicals of the monovinyl and divinyl tetrapyrrole biosynthetic pathways in the various species. A survey of various groups of chemicals (herbicides and other selected biochemicals) that are likely to exhibit photodynamic herbicidal properties is currently under investigation.     

Plasmid vectors designed for high-efficiency expression controlled by the portable recA promoter-operator of Escherichia coli

A novel expression vector using the 236-bp promoter-operator fragment of the recA gene (recApo) of Escherichia coli has been constructed. This DNA fragment contains complete signals for the initiation of RNA synthesis, as well as for regulation by the lexA product, but lacks the coding sequence for the RecA protein. The strength of the recA promoter was examined by assaying beta-galactosidase activity expressed from a cro-lacZ fused gene placed downstream of the promoter. Under noninducing conditions, the promoter was regulated by the LexA protein, and the fused gene was expressed only weakly. Upon induction by nalidixic acid in a recA+ strain, high expression was observed for an extended period. After 5 h under inducing conditions, as much as 11% of the total cellular protein was cro-lacZ product. The expression level was higher than that from promoters of lac, trp, and lambda early genes.     

Sesquiterpenes of thalloid liverworts of the genera Conocephalum,Lunularia, Metzgeria and Riccardia

Thalloid liverworts of orders Metzgeriales and Marchantiales elaborate essential oils distinguishable from those of the Jungermanniales by the absence of β-barbatene and anastreptene. Riccardia sinuata elaborates a novel tricyclic exomethylene sesquiterpene of as yet undetermined structure. Conocephalum conicum elaborates cadinene-type sesquiterpenes. β-Cadinene from the latter species is clearly enantiomeric to the same product from vascular plants.     

Interactions between the tobacco budworm,Heliothis virescens,and the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki: Relationship between length of exposure to the toxin and survival

Larvae of the tobacco budworm, Heliothis virescens, which were fed ad libitum for 24, 48, and 72 hr on a diet treated with various levels of the δ-endotoxin produced by the HD-1 isolate of Bacillus thuringiensis var. kurstaki and then transferred to an untreated diet, showed an unexpected capacity to recover from the effects of the toxin, although, as the length of exposure increased, the capacity decreased. Observations on larvae held to emergence indicated that recovery from the toxin was complete. X-ray studies using Ba²⁺ incorporated into the diet showed that, although the toxin paralyzed the midgut of the treated animals, many animals recovered after the toxin was removed, with food once again passing through the gut.     

The effect of haem on chlorophyll synthesis in barley leaves

Exogenously supplied bovine haemin, fed to etiolated barley leaves, inhibited chlorophyll synthesis in leaves exposed to light. Haemin inhibited the regeneration of protochlorophyllide (P650) and the conversion of exogenously supplied δ-aminolaevulinate (ALA) to protochlorophyll (P630). The effect of haemin on chlorophyll production was overcome by incubating the leaves in water in the dark before light treatment, suggesting the operation of a rapid haem destruction mechanism in leaves. Protohaem turnover in dark-grown leaves was between 8 and 9 hr, based on the rate of degradation of erogenous haemin and the rate of protohaem breakdown in laevulinic acid (LA) treated leaves. The rate constant for haem destruction was 85 pmol/nmol/hr in the dark and 45 pmol/nmol/hr after 4 hr light. There was no evidence that light affects the synthesis of protohaem. It appears that the regulation of endogenous levels of protohaem is by breakdown and it is this mechanism which is under light control. Haem considerably decreased the incorporation of radioactivity from glycollate-[¹⁴C], glycine-[¹⁴C] and glutamate-[¹⁴C] into accumulated ALA in the presence of LA.     

Chlorophyllide b

Chlorophylls a,b and chlorophyllides a,b were isolated from pea chloroplasts as pheophorbides a,b following the administration of $\text{[}{}^{14}\text{C]δ-aminolevulinic}$ acid. Relative pool sizes suggest that chlorophyllide b precedes chlorophyll b and does not arise from the latter by the action of chlorophyllase.     

Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K⁺ concentration of hemolymph. Thereafter, the rise in K⁺ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K⁺ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K⁺ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.