Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Bromodeoxyuridine (BrdUrd) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and 3H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0.05% proteinase at 37 degrees C for 20 min and hydrolysis with 1N HCl at 37 degrees C for 20 min was sufficient to detect BrdUrd immunoreactivity in 3H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd immunohistochemistry in cell-kinetic studies.     

In vivo bromodeoxyuridine incorporation in human gastric cancer: A study on formalin-fixed and paraffin-embedded sections

Summary Bromodeoxyuridine (BUDR) is a non-radioactive thymidine analogue which is incorporated into the DNA of proliferating cells. This allows evaluation of the size of the S-phase as the BUDR labelling index (BUDR-LI) not onlyin vitro but alsoin vivo, since BUDR is not toxic at the doses needed to label cells. To ascertain whetherin vivo BUDR incorporation can be detected on routine histological material we tested several different procedures prior to immunoperoxidase staining, on formalin-fixed, paraffin-embedded sections from five patients with gastric cancer, who received BUDR (250 mg m^–2, intravenous) 4 h before surgery. To determine the optimal conditions for detecting BUDR in formalin-fixed tissues, immunohistochemical testing for BUDR was performed simultaneously on duplicate sections fixed with 70% ethanol. It was found that hydrolysis with 3N HCl at 37° C for 30 min and digestion with 0.5% in at 37° C for 30 min were sufficient to detect BUDR immunoreactivity in formalin-fixed sections.The method presented extends the range of applications of thein vivo BUDR technique for cell kinetics studies in human neoplasms because it can be used on routinely fixed archival material, with the advantage of correlating the kinetic data with histopathological characters.     

Einfluß der Fixierung und der Säurekonzentration auf die Feulgen-Hydrolyse bei 28° C

Zusammenfassung Es wurde eine für Routinezwecke geeignete Feulgenreaktion für die cytophotometrische DNS-Bestimmung ausgearbeitet, die gegen geringe Schwankungen der Temperatur, der Säurekonzentration und der Zeitdauer der HCl-Hydrolyse weniger empfindlich ist als die 60°-Standardhydrolyse. Dazu wurden die Feulgen-Hydrolysekurven von alkohol-, formalin- und methanol-formalin-eisessigfixierten Hühnererythrocyten nach Behandlung mit 5 N, 4 N und 2 N HCl bei 28° C, bzw. mit 1 N HCl bei 60° C geprüft und miteinander verglichen. Als besonders brauchbar erwies sich die Fixierung mit 70%igem Isopropylalkohol (20 min Hydrolyse in 5 N HCl oder 45 min in 4 N HCl) und die MethanolFormalin-Eisessig-Fixierung (105 min Hydrolyse in 4 N HCl). Reine Formalinfixierung erwies sich als ungeeignet, da eine starke Kernschrumpfung mit Extinktionen größer als 0,75 beobachtet wurden.An alkoholfixierten Leberzellausstrichen wurde ein gleichartiger Verlauf der Hydrolysekurven von di-, tetra- und oktoploiden Leberzellkernen festgestellt. Die relativen Farbstoffmengen (AE-Werte) dieser Kerne verhielten sich bei allen geprüften Hydrolysezeiten wie 124.

---

Influence of fixation and add concentration on the Feulgen hydrolysis at 28° C

Summary A routine Feulgen procedure for quantitative cytophotometric absorption measurements of DNA at the integrating microdensitometer should be established, which is less alterable by minor deviations in temperature, acid concentration and in duration of hydrolysis than is the 60° C standard treatment. Feulgen hydrolysis curves of alcohol-, formalin- and methanol-formalin-glacialacidicacid-fixed fowl erythrocytes have been examined after hydrolysis in 5 N, 4 N and 2 N HCl at 28° C, as well as in 1 N HCl at 60° C. Fixation in 70% isopropylalcohol and hydrolysis in 5 N HCl for 20 minutes or in 4 N HCl for 45 minutes proved to be particularly useful. Fixation in a mixture of 85% methanol, 10% formalin and 5% glacialacidicacid gave good results, too, but hydrolysis time had to be chosen considerably longer for maximum staining (105 minutes in 4 N HCl). Formalin fixation proved not to be suitable because of a considerable shrinkage of the nuclei resulting in extinctions well above 0.75.Identical hydrolysis curves have been obtained for di-, tetra- and octoploid liver cell nuclei from alcohol-fixed liver smears. The relative dye contents (AE-values) of these nuclei were in the ratio 124 at all hydrolysis times examined.

     

6-Iodoacetamidofluorescein labelling to assess the state of sulphhydril groups after thermal stabilization of isolated nuclei

Summary Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37°C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry and confocal microscopy quantitative analysis did not reveal major differences in the fluorescence intensity of nuclei incubated at 37°C in comparison with those maintained at 0°C. Confocal scanning laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37°C exposure of nuclei. However, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix preparation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between heat-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37°C.     

Effects of temperature on ultrasound-assisted tryptic protein digestion

The effects of temperature on ultrasound-assisted tryptic protein digestion were comprehensively investigated using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Three standard proteins, cytochrome c, myoglobin, and bovine serum albumin, were digested at 4 °C (ice), room temperature (20–25), 37, and 55 °C for 0 s, 30 s, 1 min, and 5 min, in an ultrasonic bath. We found that the number of identified peptides generally increased with increasing temperature or digestion time. Compared with conventional overnight digestion at 37 °C without ultrasonication, digestions performed under ultrasonication generally produced more peptides under most of the above listed conditions, mainly due to miscleaved peptides. Tryptic digestions were also performed under all the conditions evaluated without using ultrasound, where the most significant improvement with the application of ultrasound in terms of sequence coverage and the number of identified peptides was observed at 4 °C, followed by room temperature, and 37 °C, while no improvement was observed at 55 °C with the application of ultrasound, which may be due to the fact that the current experiments were performed in an ultrasonic bath.     

The effects of varying key steps in the non-radioactive in situ hybridization protocol: a quantitative study

Summary In situ hybridization represents a major advance in the study of gene expression and, thus, in the evaluation of cellular function in histological sections. The availability of oligonucleotide probes labelled with biotin and sensitive immunohistochemical detection systems makes the study of different types of mRNA by in situ hybridization easier. However, a large number of protocols have been reported, which is sometimes confusing. The present study analyses quantitatively the influence of each important step of in situ hybridization on the staining intensity of rat proinsulin mRNA. The aim was to optimize technical conditions, to make the method sensitive and to evaluate its reproducibility for proinsulin mRNA detection and measurements. The duration of fixation and the digestion have an important impact on the results. The optimal digestion time depends on the fixation. With a digestion of 30 g ml^–1 proteinase K for 12 min at 37°C, the optimal fixation time was 24 h. Section thickness also influences the staining intensity. The intensity of the staining increases as the section thickness increases from 3 to 5 m before slowly decreasing. A weak paraformaldehyde post-fixation (0.4% for 20 min) gives best results in comparison to a stronger post-fixation (4% for 10 min). An increase of probe concentration leads to a higher specific labelling, reaching a plateau at 800 ng ml^–1. Hybridization temperature (37–42°C) exerts little influence. However, the temperature of the washes and the immunodetection system have a major effect on the intensity of labelling.Quantification has permitted the evaluation of the influence of each key for optimizing and standardizing the non-radioactive in situ hybridization protocol. In these well-defined conditions, the intra and inter-assay coefficient of variation remains lower than 6% and thus the method can be used to quantify the content of proinsulin mRNA or other specific mRNAs in experimental and pathological conditions.     

Die Bedeutung der Hydrolyse-Art in der Feulgen-Cytophotometrie von Kernen mit unterschiedlichen Ploidiegraden

Zusammenfassung An Ausstrichen von Leberparenchymzellen der weißen Maus (Fixierung nach Lillie; Äthanol, Pormol, Eisessig) wurde Feulgen-cytophotometrisch die DNS bestimmt, und zwar vergleichsweise nach der klassischen Hitzehydrolyse (n HCl, 60°C) sowie nach der Langzeit-Hydrolyse mit 5n HCl bei 20°C. Bei der Hitzehydrolyse liegt das Hydrolyse-Optimum zwischen 8 und 10 min, und zwar einheitlich bei Kernen aller Ploidiestufen (2n, 4n, 8n, 16n). Die Bildung reaktiver Aldehydgruppen sowie die spätere Extraktion von hydrolysierter DNS bei Überhydrolyse sind also unabhängig von der unterschiedlichen Struktur bzw. Chromatindichte der Kerne verschiedener Ploidiegrade. Bei der Langzeithydrolyse findet sich bei allen Kerntypen ein breites Plateau zwischen 30 und 60 min. Karyogramme lassen bei beiden Hydrolyse-Methoden exakt die verschiedenen Ploidiestufen der Kerne erkennen. Jedoch liegen die Meßwerte nach der Langzeithydrolyse um 20–30% höher als nach der Hitzehydrolyse. Offenbar überlappen sich bei letzterer die Prozesse Hydrolyse und Extraktion. Daher gebührt der Langzeithydrolyse für quantitative DNS-Bestimmungen der Vorzug. Besonders beim Vergleich verschiedener Gewebe ist von Wichtigkeit, daß die Lage des Hydrolyse-Optimums nicht kritisch ist.

---

Importance of the method of hydrolysis in Feulgen-cytophotometry of nuclei with different stages of ploidy

Summary In smears of liver parenchyme cells of the white mouse (fixation with Lillie's fluid; ethanol, formalin, acetic acid) DNA was determined Feulgen-cytophotometrically, and that comparatively after the conventional heat hydrolysis (N HCl, 60°C) as well as the longtime hydrolysis with 5N HCl at 20°C. With the heat hydrolysis the optimum was found between 8 and 10 min, and this uniformly in nuclei of all stages of ploidy (2n, 4n, 8n, 16n). Thus, the formation of reactive aldehyde groups as well as the subsequent extraction of hydrolized DNA are independent of the different structure and/or the concentration of the chromatin of the nuclei with different ploidy. With the long-time hydrolysis in all types of nuclei a wide plateau between 30 and 60 min was found. With both methods of hydrolysis karyograms exactly reveal the different stages of ploidy. However, the measuring values after long-time hydrolysis are 20–30% higher than those after heat hydrolysis. In the latter case it should be clear that the processes of hydrolysis and of extraction overlap. Therefore, in cases of quantitative DNA determinations the long-time hydrolysis should be prefered. Especially, when comparing different tissues, it is important that the position of the optimum of hydrolysis is not critical.

     

Purification and Characterization of Alkaline Proteinase from AlkalophilicBacillussp.

Alkaline proteinase was purified from Bacillussp. isolated from soil. The pH optimum was 11.5 at 37°C. Calcium divalent cation was effective in stabilizing the enzyme, especially at higher temperatures. The proteolytic activity was inhibited by the specific serine proteinase inhibitor PMSF (phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, and Hg. The enzyme was stable in the presence of detergents, such as Triton-X100, Tween-80, SDS (sodium dodecyl sulfate), and EDTA (ethylenediaminetetraacetic acid), at pH 11.5 and 37°C for 30 min. The optimum pH was 11.5 at 37°C, and the optimum temperature was 62°C at pH 11.5.     

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated wheter or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M_r 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0° C or 37° C. The matrix fraction retained 20–40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37° C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.     

The effect of cyclic adenosine 3′,5′-monophosphate on the uptake of estradiol by the neonatal mouse uterus

Summary Uterine tissue from neonatal mice was incubated in vitro at 0° C for 1 h in a medium containing 1×10^-8 M ³H-estradiol with or without 1×10^-4M dibutyryl cyclic AMP. In some incubations the temperature was raised to 37° C for 15 min after incubation in the cold, in others the temperature was kept at 0° C during this 15 min period. The tissue was frozen in liquid propane cooled in liquid nitrogen, sectioned at 2 or 4 microns, and autoradiograms prepared according to the dry-mount procedure. cAMP increases the cellular uptake of ³H-estradiol in uterine tissue. After rising the temperature to 37° C, grains appeared over the nuclei. cAMP at low temperature increased the cellular uptake of ³H-estradiol, but the grains were not associated with the nuclei. In the autoradiograms the grain number above the epithelium was markedly less than above the stroma.This work has been supported by grants from the Norwegian Research Council for Science and the Humanities, and from the Norwegian Cancer Society (Landsforeningen mot Kreft)     

Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion

Summary A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37° C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.     

Optimization of furfural production from hemicellulose extracted from delignified palm pressed fiber using a two-stage process

This study aims to optimize the conditions for furfural production from hemicellulose extracted from delignified palm pressed fiber (dPPF) via two-stage process: acid hydrolysis followed by dehydration, using response surface methodology (RSM). The extracted hemicellulose contained 80.8% xylose. In order to convert hemicellulose to xylose in the acid hydrolysis step, there were four important parameters consisting of reaction temperature (100–150 °C), sulfuric acid concentration (1–10% v/v), ratio of sulfuric acid to hemicellulose (L/S ratio) (10, 9, and 8 v/w), and reaction time (30–120 min). The maximum xylose production (12.58 g/L) was achieved at 125 °C, 5.5% sulfuric acid, L/S ratio of 9 mL/g for 30 min with the determination coefficient (R²) value of 0.90. For the dehydration process, two parameters; reaction temperature (120–160 °C) and reaction time (30–150 min), were optimized. The maximum furfural production (8.67 g/L) was achieved at a reaction temperature of 140 °C for 90 min with the determination coefficient (R²) value of 0.93.     

A neutral proteinase produced by Bacillus cereus with high sequence homology to thermolysin: Production,isolation and characterization

Summary Extracellular neutral proteinase was produced in 10 l and 240 l batch cultivations of Bacillus isolate X-3, identified as B. cereus and deposited as DSM 3101. The enzyme concentration was about 37–47 mg/l in the fermentation broth. The enzyme was extracted from the medium by adsorption chromatography with Amberlite XAD-7-resin, and further purified by acetone precipitation and affinity chromatography. The mol. wt. is 35 000 Da. The enzyme is thermostabilized by calcium, inhibited by EDTA and o-phenanthrolin and has its pH-optimum at pH 6.8. The specific activity is 4.36·10^-4 kat·mg^-1 at 35°C and the k _cat/K _m on FAGLA (furylacryloyl-glyleu-NH₂) is 2.25·10⁴ M^-1 s^-1 at 30°C, pH 6.8. The proteinase is stable up to 60°C. The N-terminal amino acid sequence exhibits a high sequence homology (63%) to thermolysin and a low homology (18%) to B. subtilis neutral protease A. The enzyme may therefore be suitable for structural comparison with thermolysin in order to study factors affecting thermostability.     

Pellet forming and fragmentation in liquid culture of Streptomyces griseus

Pellets of Streptomyces griseus formed during submerged liquid culture were effectively dispersed in Tris/HCl buffer at 37 °C. However, inclusion of Tris/HCl in the culture medium did not lead to dispersed growth. In order to decrease the pellet formation in batch culture, a large inoculum of spores was required and dispersed growth was further favoured by culture at 37 °C. This method was also used to achieve dispersed culture of S. coelicolor A3(2). Autolytic enzyme activity was presumed to be responsible for fragmentation of pellets.     

An effect of corticosteroids on thymocytes not mediated by macromolecule synthesis

Rat thymocytes were incubated for 2 min at 37°C and the cells then broken by osmotic shock in 1.5 mM MgCl₂ and the nuclei harvested. Treatment with 50 nM dexamethasone for 2 min resulted in about one third of nuclei showing abnormalities in appearance, in shape and density. This was not prevented by prior incubation for 10 min with actinomycin D and cycloheximide, but was when nuclei were isolated in the presence of anions larger than F^– and Cl^–, including I^–, Br^–, SO = ₄ and citrate . Subsequent addition of Cl^– ion, however, resulted in development of abnormalities in steroid-treated nuclei. It is concluded that the steroid induces a mechanism resulting in influx of chloride ion leading to nuclear edema, which is not mediated by processes involving synthesis of macromolecules.     

The effect of hypothermic treatment on the repair or expression of X-ray damage measured in split dose experiments on L5178Y-S cells

Summary The nature of the post-irradiation lesions and processes leading to cellular reproductive death or survival were investigated in mouse lymphoblastic leukemia L5178Y-S (LY-S) cells. Post-(x-)irradiation incubation at 25° C protects LY-S cells against the fixation of biologically expressed damage which takes place at 37° C. An optimal condition for the repair of damage, assayed in split-dose experiments as split-dose recovery (SDR), is 1 h at 37° C followed by 4 h holding at 25° C prior to the second half of a split dose, or 5 h holding at 25° C without a 37° C incubation during the interval between doses. Longer incubations at 37° C resulted in progressively decreased survivals. Postirradiation inhibition of DNA synthesis at 37° C was observed only during the first 30 min; thereafter,³H-dThdR incorporation washigher than in unirradiated controls. Theexcess synthesis effect was removed by shifting irradiated cells to 25° C holding. The inhibition observed at 25° C was reversed by shifting to 37° C. Thus the degree of postirradiation DNA synthesis is inversely related to SDR. DNA filter elution shows complete strand break repair by 20 min at 37° C, and by 3 h at 25° C; DNA double-strand break (DSB) repair plateaus at 80% (37° C) and 60% (25° C) after 90 min. An inverse correlation was found between total strand break repair rate, as assayed by filter elution methods, and cell survival. This work was supported by a grant from The Mathers Charitable Foundation.A preliminary report of this work was presented at the 35th Annual Meeting of the Radiation Research Society, Atlanta, GA 1987, USA     

Effects of temperature and holding time on production of renewable fuels from pyrolysis of Chlorella protothecoides

To investigate the influence of temperature andholding time on the pyrolyzate yields of Chlorella protothecoides, the microalgal cells weresubjected to pyrolysis at 200, 300, 400, 500 and 600 ^°Cfor 5, 20, 60 and 120 min, separately. High oil yields above 40% dry weight cells wereobtained both at relatively low temperature (300 ^°C)with relatively long holding times (20–120min) and relatively high temperatures (400–500 ^°C)with relatively short holding times (5–20min). The maximum oil yield of 52.0% was achieved at500 ^°C for 5 min. The gas yield was generallyincreased with the increasing temperature and holdingtime. It could reach 63.3–76.0% at 600 ^°C.High pyrolytic rates of 72–87% were obtained at allexperiments except at 200 ^°C for 5–20 min or300 ^°C for 5 min. Thermogravimetric analysisindicated that the main thermal degradation of thismicroalga occurred at 200–520 ^°C. The resultsimply that C. protothecoides is a good candidatefor the production of renewable fuels by pyrolysis.     

Improved Histology for the Chick-Quail Chimera

A simple modification of nuclear staining after acid hydrolysis has been made which provides easy identification of quail nuclear markings in a chick-quail chimera. This method also improves the histologic detail normally seen with hematoxylin and eosin when compared to the more commonly used Feulgen reaction. Embryonic tissues can be fixed in Zenker's or Helly's solution and the sections obtained are hydrolyzed in acid (3.5 N HCl at 37 C for 40-50 min). After acid hydrolysis the sections are stained with hematoxylin and eosin rather than Schiff reagent and fast green. The interphase nuclei of chick cells show homogeneous or mottled purplish blue staining, while quail nuclei contain a dark blue spot. This staining corresponds to the reddish purple staining of the quail's heterochromatin seen adjacent to the nucleolus in the standard Feulgen stain. This new technique facilitates identification of quail cell types in the chick host and provides superior histology of the chick tissues by demonstrating cytoplasmic detail.     

Evaluation of different cell disruption processes on encysted cells of Haematococcus pluvialis: effects on astaxanthin recovery and implications for bio-availability

Although Haematococcus pluvialis is one of the most importantnatural sources of the carotenoid astaxanthin as a pigmentor for theaquaculture industry, the thick sporopollenin cell wall in the cysts hindersastaxanthin extraction and its subsequent bio-availability to fish. A rangeof physical and chemical processes were tested to promote the disruptionof the encysted cells. The efficacy of these processes was evaluated interms of astaxanthin recovery, which was assessed by determining theextent of leaching of astaxanthin into an organic solvent. The processestested were: autoclave 30 min, 121 ^°C, 1 atm; HCl 0.1 M, 15min and 30 min; NaOH 0.1 M, 15 min and 30 min; enzymatictreatment with a mixture of 0.1% protease K and 0.5% driselase in aphosphate buffer, pH 5.8, 30 ^°C, for one hour; spray drying, inlet180 ^°C, outlet 115 ^°C; and mechanical disruption, with acell homogeniser developed for this purpose. The mechanical(homogenisation) and autoclave treatments were the most effective in termsof extraction and availability.     

Extracellular alkaline proteases from alkalophilic Vibrio metschnikovii strain RH530

Summary Alkaline proteases, named VapT and VapK, from Gram-negative alkalophilic Vibrio metschnikovii strain RH530 were purified and characterized. Both enzymes had optimum pH and temperature of 10. 5 and 60 °C, respectively. VapT and VapK retained 40 % and 80 %, respectively, of their initial activities at pH 12 after 24-h incubation at 25 °C. The half-lives of VapT and VapK were 10 min and 24 min, respectively, at pH 8 and 60 °C. Addition of Ca²⁺ extended their half-lives more than 20 fold. VapT and VapK retained over 30 % and 90 %, respectively, of their activities in the presence of 5 % SDS and 8 M urea. Analysis of amino acid composition showed that VapT contained seven cysteine residues and VapK did two. The N-terminal amino acid sequences of the proteases were determined and compared with those of Bacillus licheniformis subtilisin Carlsberg, Vibrio alginolyticus exoprotease A, and Tritirachium album proteinase K.