The relationship between glandular kallikrein and growth factor-processing proteases of mouse submaxillary gland.

Three highly specific trypsin-like proteases from mouse submaxillary gland; nerve growth factor gamma subunit, beta nerve growth factor-endopeptidase, and epidermal growth factor-binding protein were tested for kallikrein activity. Low molecular weight kininogen was purified from mouse plasma and used as substrate for the three enzymes, and the kinin released by the enzymes was assayed by its ability to induce contraction of isolated rat uterus. All three enzymes were found to have significant kininogenase activity, and the most active enzyme, beta nerve growth factor-endopeptidase, has activity comparable to authentic kallikreins from other glandular sources. Essentially all of the kininogenase activity of submaxillary gland co-purifies with beta nerve growth factor-endopeptidase. Hence, beta nerve growth factor-endopeptidase appears to be identical with submaxillary gland kallikrein. Nerve growth factor gamma subunit, epidermal growth factor-binding protein, and beta nerve growth factor-endopeptidase have similar amino acid compositions and molecular weights, and are immunologically similar. Comparison of published partial primary sequence data confirms our conclusion that nerve growth factor gamma subunit, epidermal growth factor-binding protein, and kallikrein are very closely related enzymes. It is postulated that these three enzymes are members of a larger family of similar enzymes, all of which are involved in the processing of precursors to polypeptide hormones and growth factors.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

大鼠颌下腺及其离体培养细胞脱氢表雄酮（DHEA）的免疫组织化学定位

用免疫组织化学ABC法,研究了颌下腺及无血清培养的颌下腺上皮细胞DHEA的定位,结果显示,大鼠颌下腺的浆液性腺泡的上皮细胞及各级导管上皮细胞均呈DHEA免疫反应阳性,无血清培养腺上皮细胞也呈DHEA免疫反应阳性,阳性物质分布于胞质,胞核呈阴性反应,此结果提示：大鼠颌下腺能自身合成DHEA,DHEA对消化功能可能具有重要的调节作用。     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Tissue producing the serum factor stimulating the release of cathepsin D from lysosomes in vitro

Pancreatectomy as well as thyroparathyroidectomy resulted in the quick disappearance of a serum factor (stimulating cathepsin D release from lysosomes in vitro) from the rat or mouse blood. Extirpation of other organs such as duodenum, stomach, spleen, kidney, submaxillary gland, testis, adrenal gland or hypophysis, showed no effect on the serum factor level. Glucagon (but not insulin or thyroxine) given to the pancreatectomized animals restored the serum factor level in a dose-dependent manner. The serum factor-like activity was detected only in the parathyroids (but not thyroid), and the release of activity from parathyroid-slices was stimulated by glucagon, suggesting that the parathyroid may produce and/or secrete the serum factor under the influence of glucagon.     

In vitro synthesis of nerve growth factor related glioma C6 cell polypeptides.

Rat glioma C₆ cell polyribosomal preparations were tested in a heterologous $\text{in vitro}$ system for their ability to direct the synthesis of nerve growth factor related polypeptides. Two major polypeptides of MW ～ 21,000 and ～ 43,000 respectively were found, both of which were immunoprecipitable with specific anti-mouse 2.5S nerve growth factor serum. After incubation of $\text{in vitro}$ synthesized proteins with submaxillary gland extract the bulk of these protein species was converted into immunoprecipitable material of MW ～ 13,000, which comigrated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis with mouse 2.5S nerve growth factor.     

Biosynthesis of beta nerve growth factor in mouse submaxillary glands

The biosynthesis of beta nerve growth factor (betaNGF) was studied in isolated mouse submaxillary glands incubated with L-[35S]cystine. Sodium dodecyl sulfate gels of anti-betaNGF immunoprecipitates from labeled gland homogenates showed a single major peak of radioactivity, which comigrated with purified betaNGF. This species was nearly completely precipitated by the addition of equivalent amounts of anti-betaNGF, but was absent from immunoprecipitates obtained by the addition of ferritin plus anti-ferritin. The cystine-containing tryptic peptides of the labeled species appeared identical with those of purified betaNGF. In submaxillary glands from adult male mice, labeling of betaNGF represented approximately 0.2% of the trichloroacetic acid-precipitable radioactivity. Castration reduced this value to one-third, while testosterone treatment of castrated animals restored the relative betaNGF synthesis to normal or more. No betaNGF synthesis could be detected in glands from female animals. Several tissues were examined for their ability to synthesize betaNGF in culture. Only submaxillary gland incorporated detectable amounts of radioactivity into betaNGF. Labeling of betaNGF could also be obtained by direct injection of isotope into the submaxillary gland in vivo. The results are discussed in terms of the integration of betaNGF synthesis into neuronal development and maintenance.     

Vasoactive intestinal peptide and substance P in salivary glands of the rat following denervation or duct ligation

Immunoreactive vasoactive intestinal peptide (VIP) and substance P (SP) were studied in parotid, submaxillary and sublingual glands of the rat. The concentration of VIP was highest in the submaxillary gland and lowest in the parotid gland. The concentration of SP was highest in the parotid gland; it was at, or below the limit of detection in the sublingual gland. In the parotid gland the total amounts of VIP and SP were reduced by 95% after parasympathetic denervation (section of the auriculo-temporal nerve). In the submaxillary gland the total amounts of the peptides were unchanged after parasympathetic decentralization (section of the chorda-lingual nerve). In this gland the total amount of SP was reduced by 92% and that of VIP by 50%, when the chorda tympani nerve fibres were cut deep into the hilum. Cutting the nerve fibres at the hilum left the total amounts of the peptides unchanged in the submaxillary gland, whereas in the sublingual gland the total amount of VIP was reduced by 70%. Sympathetic denervation did not reduce the total amounts of the peptides. Duct ligation caused gland atrophy. In the parotid gland the total amounts of VIP and SP were reduced by 40%. In the submaxillary gland the same percentage reduction occurred with regard to SP; however, the total amount of VIP was reduced by 99%. The VIP- and SP-containing nerve fibres reach the salivary glands by the parasympathetic nerves. In both submaxillary and sublingual glands a certain fraction of VIP originates within the glands.     

Rat epidermal growth factors: purification and tissue content

Rat epidermal growth factor (rEGF) was isolated from the submaxillary gland of male rat by reversed phase high performance liquid chromatography and ion exchange chromatography. The binding of purified rEGF to human carcinoma cells (A-431) and its tritiated thymidine uptake on rat epidermal fibroblast cells (FR) were almost the same as those of purified commercially available mouse EGF (mEGF). Antisera to rEGF was raised in rabbits and a radioimmunoassay (RIA) system was established. The assay range of the RIA was about 1.0 to 100 ng/ml. The within assay coefficients of variation were 5 to 10%, while the between assay coefficients of variation were 5 to 13%. The tissue content of rEGF of male rats (10 weeks old) was examined. As a result, the submaxillary gland was found to contain a very high concentration of rEGF (214 micrograms/g wet tissue) as predicted, and digestive tissues, stomach, intestine and duodenum contained 2.49, 3.57, 9.44 ng/g wet tissue, respectively. The amounts in prostate and seminal vesicle were relatively high, being 65.6 and 2,268 ng/g wet tissue, respectively. The amount in the submaxillary gland increased markedly after 7 weeks of age. These results suggest that EGF is an important factor in gonadal function.     

Cloning and mapping of 5' exons from the gene encoding chicken beta nerve growth factor.

An NGF cDNA containing the 5' exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5' portion.     

Highly sensitive enzyme immunoassay for beta-nerve growth factor (NGF): a tool for measurement of NGF level in rat serum

A sensitive two-site enzyme immunoassay (EIA) system was established for mouse beta nerve growth factor (NGF) isolated from mouse submaxillary gland. Our EIA system is based on the sandwiching of antigen between anti-mouse beta NGF antibody IgG coated on a polystyrene plate and biotinylated anti-mouse beta NGF antibody IgG. The bound antibody complex was quantified with streptavidin linked-beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). With this system NGF concentrations as low as 0.02 pg/well (corresponding to 8 x 10(-19) mol) could be measured reproducibly. The sensitivity of this EIA system permitted the quantification of endogenous immunoreactive beta NGF in rat serum. The mean level in serum of male rats (153.2 pg/ml) was found to be almost the same as that of female rats (127.6 pg/ml).     

Immunohistochemical demonstration of epidermal growth factor and nerve growth factor in experimental carcinogenesis in the mouse submandibular gland

Immunohistochemical demonstration of epidermal growth factor (EGF) and nerve growth factor (NGF) was made during chemical carcinogenesis in the mouse submandibular gland. The granular convoluted tubule cells in the normal male submandibular gland contained larger amounts of EGF and NGF than in the female. The initial phase and early stages in chemical carcinogenesis showed degranulation of the granular convoluted tubule cells with a marked decrease in EGF and NGF. Premalignant lesions such as duct-like structures and multicystic lesions showed variable staining for EGF and were usually negative for NGF. Material secreted into the luminal spaces revealed increased staining for EGF and NGF. Scattered tumor cells of the poorly differentiated squamous-cell carcinoma type and desquamated tumor cells contained abundant EGF, but not NGF. No positive reaction for EGF or NGF was found in the induced squamous-cell carcinoma cells.     

The sequence of a cDNA clone coding for a novel kallikrein from mouse submaxillary gland.

Mouse submaxillary gland contains many proteolytic enzymes, the most widely studied of which are the kallikreins. This gland also contains high levels of nerve growth factor (NGF), which is isolated as a complex of three subunits, alpha, beta, and gamma. We report here the cloning and sequence analysis of a novel kallikrein from mouse submaxillary gland. Antibodies directed against the alpha subunit precipitate the product of this clone, but do not precipitate the homologous gamma subunit. This new kallikrein is therefore closely related to alpha NGF, yet in contrast to the alpha subunit, its sequence suggests it has proteolytic activity.     

小鼠颌下腺上皮细胞的培养及表皮生长因子的鉴定

体外培养小鼠颌下腺细胞,形态学观察可见有上皮样细胞生长。免疫细胞化学及蛋白质印迹转移分析结果表明,体外培养的颌下腺上皮样细胞可合成并分泌表皮生长因子。     

Mouse nerve growth factor: a rapid isolation procedure for the alpha and gamma subunits.

A rapid method for isolating the α and γ subunits of mouse submaxillary gland nerve growth factor, as by-products of the commonly used Bocchini-Angeletti (2.5 S) procedure, has been devised. Approximately 40 mg of each subunit is obtained from 400 pairs of adult glands. The subunits isolated in this fashion are indistinguishable from those obtained from the homogeneous 7 S complex as judged by gel electrophoresis or their association-dissociation behavior.     

Demonstration of a considerable amount of mouse epidermal growth factor in aqueous humor

In a previous study we reported the presence of a considerable amount of mouse epidermal growth factor (mEGF) in abdominal effusion. Using our EIA system for mEGF, we identified a high level of EGF-like immunoreactive material(s) in mouse aqueous humor. This material(s) and mEGF from mouse submaxillary gland were virtually equivalent with respect to molecular weight and antigenicity. Also, on chromatofocusing analysis, the mEGF-like material(s) gave a major peak at pH 4.7 with a minor one at pH 4.2. These results demonstrate that the mEGF-like immunoreactive material(s) found in aqueous humor is a molecule identical to submaxillary gland EGF. Also, no clear difference was observed in the mEGF levels in aqueous humor between male and female. Further, sialoadenectomy did not change dramatically the EGF level in aqueous humor. From these results, it seems that mEGF found in aqueous humor may be synthesized by cells in the eyeball itself or be transported there from some site other than the submaxillary gland.     

Regulation of nerve growth factor synthesis in mouse submaxillary glands by testosterone.

—It has long been known that the activity of nerve growth factor (NGF) in extracts obtained from the male mouse submaxillary gland is higher than in extracts from the female gland, and that the activity present in female glands can be increased by testosterone treatment. This communication presents a study of the mechanism of the testosterone effect. Of several different steroids administered to female Swiss–Webster mice only testosterone propionate led to increased gland NGF activity. The increase did not appear to be due to an enhancement of the activity of pre-existing molecules on sympathetic nerve fiber outgrowth, or due to an altered affinity for the specific antibodies used in the estimation of NGF content, but appeared rather to be due to an accumulation of NFG molecules. The kinetics of change in the male gland NGF content upon castration and secondary testosterone propionate stimulation was analyzed by application of the plateau principle. The rate of loss of NGF from this organ was not measureably different between the castrate and testosterone propionate stimulated state. On the other hand, there was estimated to be a 10-fold difference in the rate of input between the basal and steroid stimulated state. Tracer amounts of radioiodine labelled NGF administered i.v. was not accumulated by the gland, and there is no evidence for uptake of this protein from the circulation. We, therefore, infer that the increased NGF concentration in male submaxillary glands is due to a 10-fold increase in the rate constant of synthesis.