Modifications in Bowie's Staining of pepsinogen granules

Modifications of Bowie's technic for staining the pepsinogen granules of the body chief cells in the gastric glands, and a method of preparing the stomach of small rodents (e.g. the rat) for histo-topographical studies are described. The modifications involve counter-staining with aqueous alum hematoxylin, the use of the neutral synthetic mounting medium Permount, and changes in the timing of fixation, staining and differentiation.     

Isolation of pepsinogen granules from rabbit gastric mucosa

Pepsinogen granules were isolated from rabbit stomachs by a combination of differential and isopycnic gradient centrifugation. The isolation procedure utilized 1 M sucrose and alkaline pH to stabilize the granules. The isolated granules were shown to be 8.4-fold enriched in pepsinogen and free of mitochondria and microsome enzyme markers. In addition to pepsinogen, a cation-insensitive but anion-sensitive Mg2+-ATPase co-purified with the zymogen. The enzyme was unaffected by aurovertin, oligomycin, and ouabain, but inhibited by high concentrations of vanadate, N,N'-dicyclohexylcarbodiimide, and azide. The enzyme activity was stimulated by tetrachlorosalicylanilide and the combination of valinomycin and nigericin in K+-containing media. The similarities between this enzyme and other secretory granule ATPases are discussed.     

Association of glycoproteins and pepsinogen in the secretory granules of fundic epithelial cells isolated from guinea pig stomach

Epithelial cells were isolated from the fundic portion of the guinea pig stomach. Cells were separated by velocity sedimentation at unit gravity in a Ficoll 70 gradient and pooled in three fractions. By morphological and biochemical criteria, each fraction was characterized as a population highly enriched in one of the three main functional types: oxyntic cell; chief cell and mucus-secreting cell. Measure of the pepsinogen content and specific stainings of the secretory granules for light and electron microscopy led to the definition of two types of mucus-secreting cells in nearly equal quantity; mucous cells with smaller secretory granules entirely glycoproteic in nature and muco-peptic cells containing larger heterogeneous secretory granules. These granules were made of a proteic core containing pepsinogen surrounded by a thin membrane and a voluminous cap, both containing carbohydrates. The cap appeared as if built of orderly packed layers of glycoproteins. Secretory granules of chief cells were also surrounded by a membrane containing glycoproteins and occasionally a small glycoproteic cap. Pepsinogen content was estimated to be three times higher in a single chief cell than in a muco-peptic cell.     

Production of swine pepsinogen by protein-producing Bacillus brevis carrying swine pepsinogen cDNA

Summary An expression-secretion vector, pNU100, was constructed, utilizing the promoter and coding sequences for the signal peptide and nine amino-terminal amino acids of the middle wall protein, to produce foreign proteins by protein-producing Bacillus brevis. Expression of swine pepsinogen cDNA in B. brevis was examined with pNU100 as a vector. The recombinant swine pepsinogen synthesized by B. brevis was found to accumulate extracellularly in the form of a soluble protein and to have acid protease activity. The acid protease activity was completely inhibited by pepstatin. Furthermore, the recombinant pepsinogen was converted autocatalytically to pepsin under acidic conditions. This indicates that B. brevis produces a pepsinogen with the same conformation as authentic pepsinogen. Efficient production of the enzyme (11 mg/l) was achieved by regulating the pH of the medium. The enzyme produced by B. brevis remained stable on cultivation for a long period, up to 40 h. This is suggested to be due to a unique property of protein-producing B. brevis, i. e. a deficiency in extracellular protease production.     

N-terminal sequence of swine pepsinogen and pepsin. The site of pepsinogen activation

The sequence of 119 amino acids of swine pepsinogen comprising the fragment released during the zymogen activation as well as the N-terminal part of pepsin is established. The activation of swine pepsinogen is shown to be accompanied by specific cleavage of Leu-Ile bond in the sequence:

$\text{Ala}\text{41}\text{Ala Ala Leu Ile}\text{Gly}\text{46}$

where Ile-45 represents the N-terminal residue of pepsin. This sequence is attacked in the course of pepsinogen activation by external enzymes — neutral proteinases and elastase.     

The History of Staining the Development of Cytological Staining

Perhaps no other period has contributed more to our knowledge of the cell than the period 1875-1895. During these years most fundamental cytological phenomena were seen and described. Mitosis, maturation and fertilization, the great cornerstones of cytology, were firmly laid by the remarkable researches of Flemming, Strasburger, Van Beneden, Oscar and Richard Hertwig, Boveri and many others. Upon these researches experimental cytology developed and the significance of the morphological phenomena to inheritance and development was pointed out by such masters as W. Roux, Weismann, O. Hertwig, Boveri and E. B. Wilson.     

The History of Staining the Development of Cytological Staining

Perhaps no other period has contributed more to our knowledge of the cell than the period 1875-1895. During these years most fundamental cytological phenomena were seen and described. Mitosis, maturation and fertilization, the great cornerstones of cytology, were firmly laid by the remarkable researches of Flemming, Strasburger, Van Beneden, Oscar and Richard Hertwig, Boveri and many others. Upon these researches experimental cytology developed and the significance of the morphological phenomena to inheritance and development was pointed out by such masters as W. Roux, Weismann, O. Hertwig, Boveri and E. B. Wilson.     

Activation process of pepsinogen.

The activation process of pepsinogen was analyzed by a combination of computer simulation and experiment. In order to investigate in detail the behavior of the basic schemes proposed in the previous study, further computer simulations were conducted. Some experiments were performed based on the information obtained. The changes in the UV difference spectrum in the early stage was measured by the stopped-flow technique and the conversion of pepsinogen to pepsin [EC 3.4.23.1] was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, on the basis of the experimental results, the most reasonable scheme was selected and modified. As a result, a scheme for the activation process of pepsinogen was obtained (Scheme 8). On the basis of the above analyses, it was assumed that the first step and the third step are pH-dependent based on the change in the UV spectrum, that the second step is a nonlinear reaction containing a looped reaction with a dimeric intermediate (in this step, peptide fragments are released and pepsinogen is converted to a pepsin-like molecule), and that the third step is an equilibrium reaction involving proton binding.     

Immunocytochemical localization of pepsinogen in rat stomach

The localization of pepsinogen in rat stomachs was investigated by a postembedding immunoferritin method. When the preparations embedded in Epon were used, the secretory granules of chief cells were stained heavily and the granules of mucous neck cells were stained moderately. The secretory granules of cells intermediate between mucous neck cells and chief cells showed a bizonal staining; the electron dense parts were stained heavily and the electron lucent parts were stained moderately. The secretory granules of pyloric gland cells, on the other hand, were labeled faintly. However, the secretory granules of surface mucous cells, foveolar mucous cells, endocrine cells, cardiac mucous cells and cardiac serous cells were not stained by the method. The protein A-gold method showed a similar staining pattern of pepsinogen to that of the immunoferritin method. When the samples embedded in Lowicryl K4M were used to enhance the stainability of pepsinogen, essentially the same staining pattern as that of the samples embedded in Epon was obtained. In addition, the Golgi apparatus and the rough surfaced endoplasmic reticulum were more easily stained.     

Early events of pepsinogen activation

Stopped-flow measurements both with native pig pepsinogen and with a fluorescent derivative, labeled near the carboxyl terminus with a toluidinylnaphthalenesulfonyl (TNS) group at Lys364, show rapid fluorescence changes following acidification. The rate constants observed by intrinsic fluorescence of the native zymogen are distinctly greater than those exhibited by the TNS derivative in the pH range examined. The rate constants for two early events in the activation of the derivative increase as the pH decreases from pH 3 to pH 2. The fluorescent intensities of these two processes also vary with pH. Because the ratios of these amplitudes fit the Henderson-Hasselbalch equation, it is concluded that the two processes represent concurrent events, rather than sequential ones. It is proposed that a protonation separates two forms of the zymogen. The conjugate acid undergoes the slower event, whereas the conjugate base, which predominates at pH 3, undergoes the faster event. It is proposed that both these pathways result in activation.     

Immobilization of pepsinogen on Sepharose

Summary Porcine pepsinogen was attached to CNBr-activated-Sepharose 4B and self-activated to form Sepharose-pepsin. The maximum loading of pepsin on Sepharose was about 43 mg of pepsin per g of freeze-dried product. Sepharose-pepsin was stable on exposure to protein substrates and had a specific activity similar to that of the free enzyme.