Nikula H, Naor Z, Parvinen M, Huhtaniemi I
Mol Cell Endocrinol. 1987 Jan;49(1):39-49. doi: 10.1016/0303-7207(87)90062-1.
The distribution and role of the calcium-activated, phospholipid-dependent protein kinase C (PK-C) was studied in rat testis. When testis tissue was homogenized in the presence of 2 mmol/l EDTA and EGTA, the majority (greater than 70%) of the PK-C activity was soluble, the rest was released from the particulate fraction by solubilization with 0.3% Triton X-100. Without chelating agents the soluble PK-C activity was undetectable, and only partially recovered from solubilized membranes. Preincubation of the tissue with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-7) mol/l) translocated PK-C to the membranes, and the majority of this activity was recovered by solubilization. Mobility of testicular soluble PK-C activity in HPLC-DEAE cellulose chromatography was similar to that of the brain enzyme. This single step purified testicular PK-C activity 140-fold. The specific activity and subcellular distribution of PK-C was similar in whole testis tissue and separated seminiferous tubules (160-210 pmol 32P X mg protein-1 X min-1 in the soluble and particulate fractions), but 2- to 3-fold higher in purified Leydig cells. However, the majority of total testicular PK-C activity appeared to be of tubular origin. Unilateral cryptorchidism for 1 week reduced PK-C of the abdominal testis by 50%, and the activity of dissected seminiferous tubules varied according to the epithelial wave. Both findings suggest that the bulk of the activity resides in the seminiferous epithelium. Involvement of PK-C in Leydig cell function was demonstrated using the TPA, which at 10(-7) mol/l inhibited basal cAMP production by 50% (P less than 0.01) but increased that of testosterone by 2- to 3-fold (P less than 0.01). On the other hand, when incubated with hCG, TPA inhibited both cAMP and testosterone production; the ED50s of hCG stimulation increased 4- to 10-fold with both parameters. It is concluded that PK-C activity is present in both the seminiferous tubules and Leydig cells, and is involved in the regulation of these testicular compartments. Its total activity and subcellular distribution are at variance according to the functional state and endocrine milieu of the testis.
研究了钙激活的、磷脂依赖性蛋白激酶C（PK-C）在大鼠睾丸中的分布及作用。当睾丸组织在2 mmol/l乙二胺四乙酸（EDTA）和乙二醇双四乙酸（EGTA）存在下匀浆时，大部分（大于70%）PK-C活性是可溶的，其余部分通过用0.3% Triton X-100增溶从颗粒部分释放出来。在没有螯合剂的情况下，检测不到可溶性PK-C活性，且仅从增溶的膜中部分恢复。用促肿瘤佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA，10⁻⁷ mol/l）对组织进行预孵育可使PK-C转位至膜上，且大部分该活性可通过增溶恢复。睾丸可溶性PK-C活性在高效液相色谱-二乙氨基乙基纤维素色谱中的迁移率与脑酶相似。这一单步纯化使睾丸PK-C活性提高了140倍。全睾丸组织和分离的生精小管中PK-C的比活性和亚细胞分布相似（可溶性和颗粒部分中为160 - 210 pmol ³²P × mg蛋白⁻¹ × min⁻¹），但在纯化的睾丸间质细胞中高2至3倍。然而，睾丸总PK-C活性的大部分似乎起源于小管。单侧隐睾1周使腹腔内睾丸的PK-C降低50%，且解剖的生精小管的活性根据上皮波而变化。这两个发现均表明大部分活性存在于生精上皮中。使用TPA证明了PK-C参与睾丸间质细胞功能，TPA在10⁻⁷ mol/l时可使基础环磷酸腺苷（cAMP）生成抑制50%（P < 0.01），但使睾酮生成增加2至3倍（P < 0.01）。另一方面，当与绒毛膜促性腺激素（hCG）一起孵育时，TPA抑制cAMP和睾酮生成；hCG刺激的半数有效剂量在这两个参数上均增加4至10倍。结论是PK-C活性存在于生精小管和睾丸间质细胞中，并参与这些睾丸区室的调节。其总活性和亚细胞分布根据睾丸的功能状态和内分泌环境而有所不同。
