The phenol oxidases of the ascomycete Podospora anserina

For the low molecular weight laccases II and III of Podospora anserina the kinetic parameters Michaelis constant (K _M) and maximum reaction velocity (V) were determined polarographically under pH optimum conditions for representative substrates of different substitution patterns.Laccase II showed two peaks in its pH optimum curve, each with a different substrate specificity, indicating structural differences to laccase III which exhibits only one broad peak.Under optimum conditions the affinities of various substrates are determined by their substitution patterns: high affinity for simple o-and p-diphenols, low affinity for m-phenols. The maximal velocity remains largely uninfluenced.This study of the effect of substitution on substrate utilization leads to the assumption that there is no specific reactive site for m-phenols in either laccase. Oxidation of m-phenols, however, takes only place at high pH values.     

The influence of temperature on catalytic efficiency of pyruvate kinase of crickets (Orthoptera: gryllidae)

Investigations have been conducted to determine the chemical nature of immediate temperature-regulatory mechanisms for enzyme activity, such as positive or negative temperature modulation and an adaptation-temperature dependence of the free energy of activation ΔG^≠. Three species of crickets have been selected for experiments in consideration of their different natural temperature demands: Gryllus campestris, Gryllus bimaculatus, and Acheta domesticus. Discontinuous Arrhenius plots (Fig. 1) show that all pyruvate kinases can exist in at least two temperature-dependent conformational states. Sizes of ΔH^≠-and ΔG^≠-values are correlated with the species' adaptation temperature (Table 1). Decreased barriers of ΔG^≠ after cold adaptation in G. campestris and A. domesticus are not sufficient for complete temperature compensation of the catalytic efficiency. Maximum enzyme-substrate affinity closely corresponds to the acclimation temperature of the crickets (Fig.2); K_m-values for PEP, however, are hardly influenced by experimental temperatures within the normal temperature range of the species. Data on enzyme function appear to corroborate the idea that optimal catalytic properties will be set according to the highest temperatures experienced respectively.     

The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2. I. Purification and characterization of the enzyme.

The deoxyribonuclease induced in KB cells by herpes simplex virus (HSV) type 1 and type 2 has been purified. Both enzymes are able to completely degrade single- and double-stranded DNA yielding 5'-monophosphonucleotides as the sole products. A divalent cation, either Mg2+ or Mn2+, is an absolute requirement for catalysis and a reducing agent is necessary for enzyme stability. The maximum rate of reaction is achieved with 5 mM MgCl2 for both HSV-1 and HSV-2 DNase. The optimum concentration for Mn2+ is 0.1 to 0.2 mM and no exonuclease activity is observed when the concentration of Mn2+ is greater than 1 mM. The rate of reaction at the optimal Mg2+ concentration is 3- to 5-fold greater than that at the optimal Mn2+ concentration. In the presence of Mg2+, the enzymes are inhibited upon the addition of Mn2+, Ca2+, and Zn2+. The enzymatic reaction is also inhibited by spermine and spermidine, but not by putrescine. Crude and purified HSV-1 and HSV-2 DNase can degrade both HSV-1 and HSV-2 DNA, but native HSV-1 DNA is hydrolyzed at only 22% of the rate and HSV-2 DNA at only 32% of the rate of Escherichia coli DNA. Although HSV-1 and HSV-2 DNase were similar, minor differences were observed in most other properties such as pH optimum, inhibition by high ionic strength, activation energy, and sedimentation coefficient. However, the enzymes differ immunologically.     

Determination of the rate of microthrombosis in experimentally induced disseminated intravascular coagulation (DIC)]

A procedure is described for the determination of the rate of microthrombosis in rats. It is based on the quantitative estimation of I125-fibrin deposits by measurement of the accumulated radioactivity in the organs after substitution of blood.     

Radioimmunoassays for free and conjugated trienbolone and for trienbolone acetate in bovine tissue and plasma samples.

A specific, sensitive, precise and accurate radioimmunoassay has been developed for the quantitation of the synthetic anabolic steroid trienbolone acetate (TBA) and its major metabolites, free and conjugated trienbolone (TBOH) in bovine tissues and plasma. With the extraction procedure described unspecific interference with the antigen-antibody reaction could be ruled out. The assay can significantly detect amounts of more than 40 pg TBOH and 70 pg TBA. 0.1 - 2.0 g tissue and 0.1 - 1 ml plasma are sufficient for 1 determination. Residues (range 0.1 -2.0 ng/g) were still present in calves 69 days after implantation of "Revalor" (20 mg estradiol-17beta and 140 mg TBA) with the highest concentrations found in liver and TBA could only be quantitated in fat.     

Measurements of fast light-induced light-scattering and -absorption changes in outer segments of vertebrate light sensitive rod cells

Flash-induced changes of light-absorption and of light-scattering of vertebrate rod outer segments (ROS) from frog and cattle in suspension were measured at 380 and 800 nm. The photometer used allows the observation of light intensity changes under well defined angles. We studied the successive decrease of the signal amplitude in series of flashes. One flash bleaches about 1% rhodopsin. The following results are discussed:

1. The signal at 380 nm is a superposition of the absorption change caused by formation of metarhodopsin II and of a biphasic additional signal. The latter exists only for the initial range of bleaching (15 to 25% rhodopsin).
2. At 800 nm three scattering signals are observed which are characterized by their successive amplitude decrease and time course:

N: A small signal with time course and successive amplitude decrease comparable to the metarhodopsin II absorption change, probably arising from a structural change within the disc membrane. Ni: A slow signal, disappearing with the first flash, which may be understood as an outer membrane effect. P: A biphasic signal with a successive decrease rate, by a factor of 10 to 20 higher than that of the metarhodopsin II signal. The two kinetically different components are separated by variation of the observation angle. Two regions of different extension appear to change structurally with different time course. “P” may reflect an influence of the light-induced transmitter release on disc shape and/or mass.     

Distribution of alpha-1-antitrypsin phenotypes in two Dutch population groups

Summary The ₁-antitrypsin phenotypes of two Dutch population groups (consisting of 672 and 802 individuals) were determined by the isoelectric focusing technique, which due to its recent development, has been used for the first time in large-scale phenotyping.As in other population studies on the ₁-antitrypsin phenotype distribution, Pi M is the most frequently occurring allele. The two investigated groups exhibit remarkable differences, both to other studied groups as well as to each other. The most interesting results are probably the high frequencies of the alleles Pi- and of the recently discovered Pi M_N. Comparison with phenotype studies carried out in other populations is also presented.     

Macrophage requirements of CR- and CR+ B lymphocytes for antibody production in vitro.

A Sephadex G-10 column coated with antigen-antibody complexes and complement retains complement receptor-bearing (CR+) mouse spleen cells. The effluent is rich in thymus-derived cells (T cells), and contains bone marrow-derived cells (B cells) which carry surface immunoglobulin (Ig), Ir-associated antigen (Ia), and Fc receptors, but no complement receptors (CR-). Although both unfractionated and CR- B cell populations are capable of producing antibody to red cell antigens, they differ in their requirements for the initiation of the response. Unfractionated B cells cooperate with primed as well as unprimed helper T cells; macrophages are required for this cooperation but can be replaced by 2-mercaptoethanol. CR- B cells cooperate with primed but not with unprimed T cells provided macrophages are added to cultures. After addition of culture supernatant from BCG-activated macrophages CR- B cells cooperate with both unprimed and primed T helper cells.     

Regulatory gene INO4 of yeast phospholipid biosynthesis is positively autoregulated and functions as a transactivator of fatty acid synthase genes FAS1 and FAS2 from Saccharomyces cerevisiae.

The sequence motif 5' TYTTCACATGY 3' functions as an upstream activation site common to both yeast fatty acid synthase genes, FAS1 and FAS2. In addition, this UASFAS element is shared by all so far characterized genes of yeast phospholipid biosynthesis. We have investigated the influence of a functional INO4 gene previously described as a regulator of inositol biosynthesis on the expression of FAS1 and FAS2. In a delta ino4 null allele strain, both genes are expressed at only 50% of wild type level. Using individual UASFAS sequence motifs inserted into a heterologous test system, a drastic decrease of reporter gene expression to 2-10% of the wild type reference was observed in the delta ino4 mutant. In gel retardation assays, the protein-DNA complex involving the previously described FAS binding factor 1, Fbf1, was absent when using a protein extract from the delta ino4 mutant. On the other hand, this signal was enhanced with an extract from cells grown under conditions of inositol/choline derepression. Subsequent experiments demonstrated that INO4 expression is itself affected by phospholipid precursors, mediated by an UASFAS element in the INO4 upstream region. Thus, in addition of being an activator of phospholipid biosynthetic genes, INO4 is also subject to a positive autoregulatory loop in its own biosynthesis.